Tutorial Lecture
Sunday, 16th September

Main Hall

15:30 － 16:15

Tutorial Lecture 1: Nico M M Nibbering
Chair:

Ryuichi Arakawa（Kansai University, Japan）

TL1-1530
15:30 － 16:15

Selected examples of gas-phase ion chemistry studies.

Nico M M Nibbering

Vrije Universiteit, Amsterdam, The Netherlands

Keywords:
tautomeric ions, time-resolved MS/MS, epimerization
Novel aspects:
Gas-phase infrared multiphoton dissociation spectroscopy and ion mobility have become in recent years new and invaluable tools
for gas-phase ion chemistry studies.
Abstract:
Gas-phase ion chemistry is an area in mass spectrometry that has received much research interest since the mid fifties of the lastcentury and there are still several groups in the world active in gas-phase ion chemistry of both positive and negative ions, either
unimolecularly and/or bimolecularly.
In this tutorial lecture the formation and determination of tautomeric ion structures and intra-ionic catalyzed tautomerization in
the gas phase will be discussed. In addition, an example of formation of different tautomeric structures in protic and aprotic
solvents under electrospray ionization conditions will be given, as established by gas-phase infrared multiphoton dissociation
spectroscopy. This will be followed by presenting an example of time-resolved MS/MS which enables to identify the structure of
an ion, generated at a particular molecular ion lifetime. At the end of the lecture the power of ion mobility will be shown inelucidating the mechanism of epimerization of bis-Tröger bases having chiral nitrogen centers.

Tutorial Lecture
Sunday, 16th September

Main Hall

16:15 － 17:00

Tutorial Lecture 2: Michael L Gross
Chair:

Yoko Ohashi（RIKEN, Japan）

TL2-1615
16:15 － 17:00“Can Mass Spectrometry Play a Role in Protein Biophysics and Structural
Biology?“

Michael L Gross, D Rempel, H Zhang, D Hambly, B Gau, J Chen, R Huang, J Zhang, L Jones, M Zhu
Washington University, St Louis, MO, USA

Keywords:
Protein footprinting, H/D exchange, fast photochemical oxidation of proteins
Novel aspects:
Mass spectrometry will play a role in structural biology by providing information of protein interaction interfaces, protein affinities,
and protein folding, ultimately to give coarse-grained structures.
Abstract:
There is a profound need in biological sciences to determine structure and properties of proteins that do not crystallize and are
not suitable for high-resolution NMR studies because the proteins are too large or they oligomerize at low concentration.
Furthermore, the relevant properties of a protein are manifest in unusual media（e.g., high concentrations of lipids and otherproteins）or in protein assemblies. Many low-resolution approaches exist（e.g., fluorescence, Raman, absorbance, circulardichroism, chromatography, calorimetry）, but most do not readily yield information at the peptide or amino-acid levels. Mass
spectrometry（MS）, on the other hand, is the basis for a middle-resolution method. In appropriate experiments, MS provides ameans of determining protein interactions, following folding and unfolding, and determining binding affinities and interaction sites
by using chemical footprinting. Driving this approach is the wide availability of mass spectrometers, separation methods, and dataprocessing software for analytical proteomics； these should also be immediately applicable to protein footprinting.
To this end, we are developing fast photochemical oxidation of proteins（FPOP）and implementing HD exchange（H/DX）, andother covalent modification approaches to interrogate protein interactions, interfaces, and dynamics of folding/unfolding. We are
implementing H/DX to map protein interfaces and developing strategies to measure protein affinities（we call the latter approach
PLIMSTEX）. For irreversible labeling, we use FPOP, which has a significant advantage of speed, owing to the use of free radicals
as footprinting reagents. Radical generation occurs in low nanoseconds, and the radical reactions are complete in 1 microsecond.
Only a single conformation of the protein exists during the footprinting because the distribution of oxidation products is Poisson.
Besides OH, other radicals can be used including the sulfate and carbonate radical anions and the iodide radical, which havedifferent selectivities for the amino-acid residues. FPOP can also be the“probe“ in a classic, two-laser“pump-probe“ experiment
whereby a temperature-jump perturbation is produced as a“pump“ by one laser and a second laser initiates FPOP as the“probe“ to afford residue-specific information. This experiment is capable of probing protein structural dynamics at the sub millisecond
level with improved sensitivity and more detailed structural resolution than any physical chemical method.
In the lecture, we will describe these approaches, provide context for them in terms of other MS-based methods, and outline
some applications in protein science. We will show that these approaches provide more information than fluorescence, UV
absorbance, Raman, IR, nuclear magnetic resonance, and circular dichroism and may ultimately be a source for coarse-grained
structures of proteins and protein assemblies.

Plenary Lecture
Sunday, 16th September

Main Hall

17:30 － 18:15

Plenary Lecture 1: Hiroyuki Hamada
Chair:

Koichi Tanaka（SHIMADZU Corp , Japan）

PL1-1730
17:30 － 18:15

Applied Conventional Technology- Look at Tradition for Our Future-

Hiroyuki Hamada

Kyoto Institute of Technology, Kyoto, Japan

Keywords:
Traditional technology, Highly cultural product, Implicit knowledge
Novel aspects:
Interdisciplinary engineering which develop new ideas based on implicit knowledge of the past
Abstract:
When we think a new material, a new processing and a new product； usually we have to develop technology, skill and soon.
Fortunately, we, human, have a long history, so that there are so many traditional technologies. For example, in Japan particularly
in Kyoto, there are many traditional craft works such as Urushi painting, roof tile named Kyo-gawara, and so on, because Kyoto
was capital of Japan for long time, for 1200 years.
Traditional craft products give customers some kind of satisfaction when customers see the products or when they have them intheir hands or use them. That is the reason why traditional craft products have been made and used for many years. We use aword“highly cultural products“ as the word that shows this kind of satisfaction. This word means the function of products andalso appeals to the sensitivity of customer.
In the modern industry and production at the moment this kinds of additional value should be required. Therefore it is nice idea
look at the past technologies to get some additional functions. There are appropriate words；
Learn a lesson from the past.
However traditional technologies have been brought down as implicit knowledge. Therefore it takes long time to understand thetechnologies and education system is not effective. In order to overcome this problem and easily understand traditional
technologies, we propose a new engineering field name as Applied Conventional Technology.
An example of applied conventional technology approach is to find secret and knack in the traditional craft works； it is sometime
material selection or sometimes fabrication method, by using scientific approach.
After this stage we can get explicit knowledge from implicit knowledge.
We can easily use the explicit knowledge to modern industry and products. Traditional craft works and processing exist in every
country and every area around the world. Therefore all of scientist and fabrication Engineers are interested in this new engineering
field.
The research topics undergoing are listed below；
1 ）Comparison of painting technique of Urushi products between expert and non-expert2 ）OBSERVATIONOF SURFACE STRUCTURE OF KYO-KAWARA（CERAMICMATERIAL FORROOFCOMPONENT）FABRICATED BY TRADITIONAL SKILLFUL TECHNIQUE
3 ）Effects of Processing Time and Years of Experience on Pre-Bending Process for Bamboo Beam in Kyo-Chouchin（Japanese
Lantern）Making Process4 ）Thermal Strain of the Soldering Product in Traditional Metalwork
5 ）Evaluation of Pre Bending Process of Bamboo Beam in Kyo-Chochin（Japanese Lantern）Making Process6 ）Motion Analysis of the“Temae“ in the Way of Tea Manufacturing movement analusis
7 ）Analysis of the bacterial community found in clay wall material used for the construction of Japanese traditional buildings
8 ）Quantification and Application of Implicit Knowledge of Bows made in Japan（Kyoto-bow）
9 ）Influence of proficiency in making“kanaami“Plenary Lecture
Monday, 17th September

Main Hall

08:00 － 08:45

Plenary Lecture 2: David E Clemmer
Chair:

Peter J Derrick（Massey University, New Zealand）

PL2-0800
08:00 － 08:45

Development of Next Generation Ion Mobility/Mass Spectrometry
TechniquesDavid E Clemmer

Indiana University Department of Chemistry/Bloomington, IN/United States

Keywords:
Ion mobility spectrometry
Novel aspects:
This is an invited lecture
Abstract:
When a pulse of ions is injected into a buffer gas and exposed to an electric field, different species will separate due todifferences in their mobilities through the gas. This separation is the basis of a widely used analytical method called ion mobility
spectrometry（IMS）. The approach has attracted considerable attention because： 1 ）it allows species with identical m/z values
but different shapes to be separated； 2 ）it can be coupled with high-speed mass spectrometry techniques to provide an
additional dimension of separation without influencing analysis times； and, 3 ）the combined approaches provide advantages forobserving low abundance ions as well as parallel fragmentation strategies. When combined with theoretical calculations themethod also complements mass spectrometry as a means of determining structure. At present the resolving power of ion mobility
instruments is relatively low. This talk will describe the early development of ion mobility/time-of-flight techniques and explore
current efforts to improve IMS resolving power. Specifically, progress in understanding ion separation by an overtone mobility
approach will be presented. This progress has driven the development of a new circular drift tube. By allowing ions to experience
multiple cycles we have taken advantage of very long effective drift regions（ions have now been moved through more than 100
meters）. Multipass designs facilitate a range of new experiments, including IMS n , as well as what we call mobility ringdown
experiments（where some ions are released for analysis after each cycle）. The ringdown studies have been used to follow thelevel of H/D exchange on specific conformations of ubiquitin. The work is at a very early stage and current limitations will bepresented.

Oral Session
Monday, 17th September

Main Hall

09:00 － 11:00

Session 1: Developments in Tandem Mass Spectrometry - Hybrid Instrumentation“Thewhole is greater than the sum of its parts”(Aristotle).
Chair:

Morio Ishihara（Osaka University, Japan）

S01-0900
09:00 － 09:40[Keynote Lecture] Orbitrap-based hybrid mass spectrometers: synergy ofanalyzers

Alexander A Makarov

ThermoFisher Scientific, Bremen, Germany

Keywords:
Orbitrap analyzer, high mass accuracy, ultra-high resolution, hybrid mass spectrometer
Novel aspects:
Overview of existing and future configurations of Orbitrap-based hybrid mass spectrometers and associated analytical methods
Abstract:
Orbitrap mass spectrometry has grown to become one of the most prominent techniques for high resolution, high mass accuracy
analysis. To large extent this is the consequence of intricate synergy between different combinations of analyzers employed in
Orbitrap-based hybrids.
Being the first high-performance mass analyzer that employs trapping of ions in electrostatic fields, Orbitrap analyzer can provide
high performance analytical characteristics only when highly integrated with the ion injection process. The advent of pulsed
injection from an external ion storage device has allowed the analyzer to become effectively de-coupled from previous stages ofmass analysis. Therefore such a storage device in the form of the C-trap became the crucial building block of all Orbitrap-based
hybrids which currently contain two major families： the original LTQ Orbitrap and the recent Q Exactive hybrids. First family
employs linear traps MSn analyzers for the first stages of mass analysis while the second one utilizes a quadrupole mass filter withhyperbolic electrodes. In both cases, preceding mass analyzer ensures optimum filling of the C-trap and thus stability of Orbitrap
operation in spite of significant ion loads.
Both of hybrid families have recently entered a new phase in their short but eventful history. For the Orbitrap Elite instrument, a
new construction of the analyzer with a higher field strength as well as new methods of data processing allowed to increase
drastically spectral acquisition rate, improve sensitivity and performance for proteins. For the Q Exactive instrument, a variety of
new methods of analysis became enabled by parallel isolation/detection as well as by multiplexing of quadrupole filtering.
Performance of new instruments is described in comparison to their predecessors and high-field FT-ICR instruments. Particularattention is given to recent improvements related to protein and top-down analysis.
In conclusion, future trends and perspectives of the Orbitrap mass spectrometry are discussed. Alternative hybrid configurations
are discussed from the point of view of their synergies, including ones with multiple Orbitrap analyzers operating in parallel.

Oral Session
Monday, 17th September

Main Hall

09:00 － 11:00

Session 1: Developments in Tandem Mass Spectrometry - Hybrid Instrumentation“Thewhole is greater than the sum of its parts”(Aristotle).
Chair:

Morio Ishihara（Osaka University, Japan）

S01-0940
09:40 － 10:00

High Mass Resolution MALDI TOF MS/MS With A Curved Field Reflectron(Or The CFR Comes Of Age)

Andrew R Bowdler, Ian Brookhouse
Kratos Analytical Ltd, Manchester, UK

Keywords:
Mass resolution, MALDI TOF MS/MS, High energy CID, CFR, TOF/TOF
Novel aspects:
State of the art mass resolution for MALDI TOF MS/MS. 20keV lab energy CID with high mass resolution.
Abstract:
The curved-field reflectron（CFR）has been in existence for nearly two decades since it was introduced by Cotter and Cornish［ 1 ］.
For several years the CFR was used in the only commercially available instruments capable of MALDI TOF MS/MS without
scanning the reflectron. Seamless post-source decay provided advantages in terms of both sensitivity and speed of acquisition.
However, the key advantage of the CFR over conventional linear field reflectrons, even those used in TOF/TOF instruments has
been the ability to produce MS/MS at the full extraction energy. This is because there is no need with the CFR to re-accelerate theions after the first TOF. So firstly, true high-energy CID is possible with lab collision energies of 20keV or more. Secondly, there isno need to discard any meta-stable ions（post-source decay）and thus very high efficiency is achieved for MS/MS.
However, even when used in MALDI TOF instruments with in excess of 20,000 mass resolution for MS, the mass resolution using
the CFR for MS/MS was typically limited to isotopic resolution up to about 1000Da. This has been known for a long time to be dueto the increase in the laser intensity required to produce the meta-stable ions required for MS/MS（even with high-energy CID）
which is much higher than the threshold for MALDI TOF MS. Until recently, this effect had not been understood or quantified.
More importantly, the reduced mass resolution has meant that the CFR instruments were not able to match the ultimate
performance of other TOF/TOF instruments.
In this paper, the theory and data for a method to produce high mass resolution MS/MS with a curved field reflectron will bepresented. Theoretical calculations and ion trajectory simulation data will be used to show how the reduction in mass resolution
when the laser intensity is increased is due to a large increase in the initial spatial distribution in the axial direction of ionsgenerated by the MALDI ion-source. This axial spatial distribution is passed on by the parent ions during meta-stable decay or CID
to the fragment ions. It will be shown that it is possible to gain MS levels of mass resolution for MS/MS if this axial spatial
distribution is re-focussed before all of the fragment ions reach the entrance to the curved-field reflectron. With this technique andin combination with the curved-field reflectron, fragment mass resolutions in excess of 10,000 can be achieved for MS/MS of metastable ions and CID at lab energy of 20keV. By using axial spatial distribution focusing（ASDF）, the curved-field reflectron is again
capable of defining the state of the art for MALDI TOF-MS/MS and can really be said to have come of age.
［ 1 ］Cornish TJ and Cotter RJ ’Non-linear field reflectron’ US patent 5464985

Oral Session
Monday, 17th September

Main Hall

09:00 － 11:00

Session 1: Developments in Tandem Mass Spectrometry - Hybrid Instrumentation“Thewhole is greater than the sum of its parts”(Aristotle).
Chair:

Morio Ishihara（Osaka University, Japan）

S01-1000
10:00 － 10:20

High resolution, high aperture, high sensitivity TOF mass-spectrometerswith expanded functionalities

Aldan A Sapargaliyev3, Igor F Spivak-Lavrov2, Yerbol A Sapargaliyev1,31

Scientific Center“REB“, 2Aktobe State University, Aktobe, Kazakhstan, 3Scientific Center REB, Almaty, Kazakhstan

Keywords:
TOF, instruments, parallel tandem, high sensitivity, high resolution
Novel aspects:
Novel designs of compact TOF analyzers with the longest flight-paths（TOF）are suggested. Ion-beam channels utilize paralleltandem analysis simultaneously by energy and mass in the“nested time“ mode.
Abstract:
The set of novel ion-optical（IO）designs of Time-of-Flight（TOF）analyzers with a loop ion trajectory formed by the Wedge-TypeTwo-Dimensional（WT）grid less mirrors is proposed［ 1 ］. These TOF analyzers possess record characteristics twice as muchovercoming their already known analogues by resolution and sensitivity. They can be used in mass-spectrometers of different
configurations（tandem, hybrid）for the preliminary selection of ions and/or for the final division of the flow by masses.
Furthermore, they feature extended functional capabilities enabling multi-object（simultaneous separate analysis of ion beams
from two and more different sources）and multi-parameter mass-spectrometry. Exceptional features of the TOF analyzers
proposed, including the high sensitivity, are due to the following innovations.
WT mirrors are made in the form of longish sheets which create two-dimensional electric field with the middle plane. Providing
the ion beam loop configuration, the disposition of mirrors in the analyzer allows reimbursing the transverse spatial dispersion byenergy, which appears at every reflection of the ion beam. The plane of the loop trajectories of the ion beam moves along thelongish sheets providing the increase of the unlooped path of ions. The use of various three-dimensional IO reflection elementsallows spatially to focus the ion beam in two transverse directions and thus to transport ion packages without significant loss.
Compared to axis symmetric IO elements, the middle plane IO elements have smaller aberrations and larger aperture, whichenables mass-spectrometers with high resolution and sensitivity. Meanwhile, the record-breaking characteristics can be achieved
while using the multilayer-multi-reflective TOF analyzers. The use of mass-spectrometers with TOF analyzers for parallel mass
spectrometry of two or more tract ion beams is stipulated, that provides the expansion of their functionality and also increases
the speed of receiving mass-spectrometry data and enhances the credibility of the results.
Some of the offered designs, including multi-channel, hybrid and tandem, provide possibility of simultaneous analysis of the ion
beam by energy and mass.
The proposed designs suggest the longest fly-distance（TOF）at the smallest sizes of ion channel. Resolution of massspectrometers may vary from tens of thousands（two-reflective）to several millions（multilayer-multi-reflective）. For example,
single-layer-multi-reflective TOF system at dimensions 0.4x0.2x0.05m 3 （layer width 0.05m）provides 32m path-of-flight（resolution
200,000）. Five-layer-multi-reflective system（dimensions 0.4x0.2x0.25m 3 ）provides 160m path-of-flight（resolution 1,000,000）. In
general, the TOF analyzer can contain any number（ten and more）of layers.
Suggested variants of ion-beam channels enable utilizing the parallel tandem TOF analysis in the“nested time“ mode, in the
better way than the ones suggested by other systems.
The general method of investigation and the corpuscular-optical theory of the spatial and temporal structure of charged particle
flows in systems including in elements with special points（mirrors and emission units）can be found in works［ 2 - 4 ］. The
methods and theory developed in these works are the basis for the theoretical substantiation of TOF analyzers proposed.
［ 1 ］Sapargaliev A.A., The method of mass-spectrometry（variants）and the device for its realization, International publication No.
PCT/KZ2011/000011, Pub. No.： WO/2012/005561, International Filing Date： 08.07.2011, Publication Date： 12.01.2012.
［ 2 ］Sapargaliev A.A., Yakushev E.M., Osnovyobshego metoda teorii fokussirovki zariazhennykh chastits,（Fundamentals of thegeneral method of the theory of focusing of charged particles）Izv.AN KazSSR, seria Phyz.mat. 1980, 2, pp. 22-26.
［ 3 ］ Yakushev E.M., Sapargaliev A.A., Elengeev A.K., Obshaya Teoria prostranstvennoy I vremiaproletnoi fokussirovki
zariazhennykhchastits v statsionarnykh electromagnitnykh poliakh（General theory of spatial and time-flight focusing of charged
particles in stationary electromagnetic fields）. ZhTF, 1985, V.55, 7, p.1291-1299.
［ 4 ］Bimurzaev S.B., Yakushev E.M., Electromagnetic mirror objective with removable spherical aberration, Nucl. Instr. Meth. in
Phys. Res., 2011, V. A 645, p. 33-34.

Oral Session
Monday, 17th September

Main Hall

09:00 － 11:00

Session 1: Developments in Tandem Mass Spectrometry - Hybrid Instrumentation“Thewhole is greater than the sum of its parts”(Aristotle).
Chair:

Morio Ishihara（Osaka University, Japan）

S01-1020
10:20 － 10:40

On the way to the holy grail of CID (20 keV and C12 precursor ion): fromMALDI-LinTOF/CF-RTOF- to MALDI-SpiralTOF/offset parabolic-RTOF-MS

Guenter Allmaier1, Ernst Pittenauer1, Pavel Rehulka2, Akihiko Kusai3

Vienna University of Technology, Vienna, Austria, 2University of Defence, Hradec Kralova, Czech Republic, 3JEOL(Europe), Croissy-sur-Seine, France

1

Keywords:
MALDI, High-energy CID, Spiral TOF/Parabolic RTOF, Triglycerides, Glycerophospholipids
Novel aspects:
A MALDI tandem MS（spiralTOF, HE（20 keV）-CID, parabolicRTOF）can be seen as reaching the holy grail of CID structural
analysis allowing C12 ion selection/true HE-CID of lipids out of mixtures.
Abstract:
With the introduction of MALDI-TOF/RTOF-technology during the turn of the 21 st century high-energy（beyond or at 1 keV）
collision-induced dissociation（HE-CID）of biomolecules（e.g. lipids, peptides, carbohydrates）for structural elucidation was backin business after the slow disappearance of classical high performance tandem four sector instruments due to sensitivity issues
and the complexity of the mass spectrometer compared to modern tandem or multi stage mass spectrometric instrumentation（IT,QqRTOF and QqQ）. Whereas tandem four sector instruments（for example EBEB（JEOL HX110/HX110）configurations）offeredmonoisotopic（C12）precursor ion selection and keV-collision regimes（rich in product ion formation across the whole m/z range））
at reasonable resolution the latter tandem mass spectrometric instrumental developments（IT, QqRTOF and QqQ）allowed only
eV-collisions frequently poor in formation of structure diagnostic product ion formation. Major draw-backs of recent HE-CID TOF/
RTOF-instrumentation particular for lipid analysis are mainly based on technical limitations in MS 1 and the associated ion gatetechnology for precursor ion-selection thus never enabling clear monoisotopic precursor ion selection. This is extremely importantas the smallest mass difference between two lipid species is often just 2 Da（i.e. one double bond）. Therefore monoisotopic
precursor ion selection（mass window 1 Da without any isotope interferences at suitable sensitivity）is urgently needed on
routine base for unambiguous lipid species characterization and identification under“true“ HE-CID（ELAB = 20 keV）conditions.
As recent advancement（i.e. coming close to the holy grail of CID structural analysis）the introduction of a MALDI tandem HE（20keV）-CID MS consisting of a spiralTOF as MS 1 and an offset parabolic RTOF as MS 2 can be seen allowing again C 12
（monoisotopic）precursor ion selection at good sensitivity. For evaluation of this latter, most advanced device samples of
selected triacylglycerols（TAGs from castor bean and linseed oil）and glycerophospholipids（GPL）from various classes（PC, PE,
PS, PI, etc.）were analyzed by means of the JMS-S300 SpiralTOF（JEOL）and including various other TOF/RTOF-instruments fromdifferent manufacturers（ABI 4700 and 4800 proteome analyzer（both： ABSciex）and Axima TOF2（Shimadzu Kratos Analytical）. As
precursor ions［M+H］+ and［M+nNa（n 1 ）H］+ ions（n = 1 - 3 ）were selected for positive ion and［MH］- for negative ion mode.
All product ion assignments were based on previously published product ion nomenclature for TAGs and GPLs. It turned out thatthe new device allowed clear monoisotopic precursor ion selection out of complex lipid mixtures at good sensitivity and 20 keV
HE CID fragmentation of these ions for detailed structure elucidation（i.e. double bond location）of lipids across the complete m/z
range of interest.

Oral Session
Monday, 17th September

Main Hall

09:00 － 11:00

Session 1: Developments in Tandem Mass Spectrometry - Hybrid Instrumentation“Thewhole is greater than the sum of its parts”(Aristotle).
Chair:

Morio Ishihara（Osaka University, Japan）

S01-1040
10:40 － 11:00

Identification of trace level process related impurities of small molecule
Irbesartan, an angiotensin II receptor antagonist through MSMS analysis

Saravanan Subramaniam, Raman Palvannanathan, Rampriya Uthayakumar,
Govindarajan Chandramohan, Mohan Kasi, Arvind Thyagarajan, Manohar Venkat
Indian Institute of Chromatography & Mass Spectrometry

Keywords:
Irbesartan, trace level impurities, SSSfunction
Novel aspects:
The novel features of the MS technology namely Synchronized Survey Scan（SSS）has been exploited to characterize process
related unknown impurities at a concentration of less than 0.10%.
Abstract:
Identification of impurities based on process parameters is one of the regulatory requirements to ensure the safety of the final
drug substance. If the impurities are not process related, but new ones, it is imperative that one has to establish the biological
toxicity of the unknown impurities, which in turn will be highly cost prohibitive. The MSMS technology available in triple
quadrupole mass spectrometer with multiple collision energy features becomes very much useful to identify these process related/ unknown impurities, which are present in trace level in drug substances. This paper presents the identification of five unknown
impurities of Irbesartan an angiotensin II receptor antagonist molecule. The MSMS analysis demonstrates that these five impurities
are process related only and not new unknown impurities.
Irbesartan was in-house synthesized at Indian Institute of chromatography and Mass Spectrometry（IICMS）, Chennai, India. The
purity of the compound was found to be 99.21%（Area normalization method）. The concentration of five impurities was found tobe varying from 0.04% to 0.32%（Area normalization method）.
The salient features of LCMS 8030 like Synchronized Survey Scan（SSS）provides the MS and MSMS data simultaneously in a single
run and paves a way for rapid identification of impurities. Further the simultaneous MS analysis in both positive and negativemodes, MSMS analysis with different collision energies and MSMS analysis with neutral loss function have enabled us tocharacterize the impurities of Irbesartan.
The molecular ion of the acive compound Irbesartan has been observed as m/z 429 in ESI positive mode, confirmed in thesimultaneous ESI negative mode analysis as m/z427. The MSMS analysis of the main compound irbesartan in ESI positive ionizationshows the charecteristic fragmentation ions such as m/z 429, m/z 207, m/z 195, m/z 180 and m/z 84.
It is intresting to note the MSMS features of LCMS 8030 shows the charecteristic neutral loss resulting to the presence of ion m/z
207. Simillar charecteristic ions of the impurities were also observed in the MSMS mode. The MSMS analysis have been carried out
at the collision energies same as that of the main compound and the data analysis has enabled us to observe the presence/
absence of methyl group leading to the formation ofimpurity- 2 and impurity-5. All the impurities except impurity- 3 have their
origin from the raw meterial spiroketones used in the synthesis of irbesartan. Impurity- 3 is already reported as USP impurity-A.
The entire analyses show how the MSMS architecture available in LCMS 8030 can effectively be employed to characterise theprocess related impurities. The unique feature of this capability is the identification of the molecular ion at very low concentration.

Oral Session
Monday, 17th SeptemberRoom A

09:00 － 11:00

Session 2: Advances in Methods and MS Instrumentation for Biomolecule Characterization
Chair:

Vicki H Wysocki（Ohio State University, USA）

S02-0900
09:00 － 09:40[Keynote Lecture] Chemical Cross-Linking and Mass Spectrometry: A
Fruitful Combination for Protein 3D-Structure Analysis

Andrea Sinz

Martin-Luther University Halle-Wittenberg, Halle, Germany

Keywords:
Chemical cross-linking, ESI-LTQ-Orbitrap-MS/MS, protein 3 D-structure
Novel aspects:
Combination of chemical cross-linking and MS for protein structure analysis.
Abstract:
Detailed knowledge of three-dimensional protein structures and the identifcation of protein-binding partners are critical forunderstanding cellular processes at the molecular level. However, applying conventional methods of structural biology ischallenging when analyzing membrane proteins, transient complexes or very large protein assemblies. Chemical cross-linking in
combination with mass spectrometry（MS）and computational modelling has emerged as an alternative strategy to obtain threedimensional structural information of proteins and protein complexes［ 1 - 3 ］. The chemical cross-linking/MS approach can beused in combination with other low-resolution structural methods to study proteins and protein assemblies, which are otherwise
not amenable to the high-resolution structural techniques of X-ray crystallography or NMR spectroscopy. Chemical cross-linking
relies on the introduction of a covalent bond between functional groups of amino acids within one protein, to gain insight into theconformation of a protein, or between interaction partners to elucidate interfaces in protein complexes. Based on the distance
restraints derived from the chemical cross-linking data, three-dimensional structural models of proteins and protein complexes canbe constructed. We employ the chemical cross-linking/MS strategy to study proteins and protein complexes with a wide range of
biological activities. The protein systems currently under investigation in our lab comprise the peroxisome proliferator-activatedreceptor alpha, the formate channel FocA, as well as calmodulin/Munc13 and laminin/nidogen complexes. Most commonly, homobifunctional amine-reactive cross-linkers, such as N-hydroxysuccinimide esters, are used for studying protein assemblies. Our aim
is to extend the arsenal of exisiting cross-linkers to obtain complementary structural information. We explore the incorporation of
unnatural photo-reactive amino acids into the proteins of interest, and we use specifically designed cross-linkers, which arecleaved by collisionally activated dissociation（CAD）conditions during MS analysis［ 4 ］. After the cross-linking reaction, thechemically modified proteins are enzymatically digested and the resulting highly complex peptide mixtures are analyzed by MS（nano-HPLC/MALDI-TOF/TOF-MS/MS and nano-HPLC/nano-ESI-LTQ-Orbitrap-MS/MS）. Mass spectra are screened for crosslinked products by the StavroX software［ 5 ］and the distance restraints we obtain serve as the basis for constructing structuralmodels of the proteins under investigation using Rosetta. With the continual improvements in mass spectrometric equipment andbioinformatics tools, the chemical cross-linking strategy can be expected to greatly facilitate the 3 D-structural analysis of proteins.
Literature References：［ 1 ］Sinz, A., Mass Spectrom. Rev. 2006, 25, 663.［ 2 ］Leitner, A. et al., Mol. Cell. Proteomics. 2010, 9, 1634.［ 3 ］Rappsilber, J., J.
Struct. Biol. 2011,173, 663.［ 4 ］Muller, M.Q. et al., Anal. Chem. 2010, 82, 6958.［ 5 ］Gotze, M. et al., J. Am. Soc. Mass
Spectrom.2012, 23, 76.

Oral Session
Monday, 17th SeptemberRoom A

09:00 － 11:00

Session 2: Advances in Methods and MS Instrumentation for Biomolecule Characterization
Chair:

Vicki H Wysocki（Ohio State University, USA）

S02-0940
09:40 － 10:00

Applications of online- bioaffinity- mass spectrometry to structure andaffinity determination of neurodegenerative proteins from brain material

Stefan Slamnoiu1, Camelia Vlad1,2, Adrian Moise1, Mihaela Stumbaum1,2, Thomas Gronewold2, Markus
Perpeet2, Michael Przybylski1

1
Laboratory of Analytical Chemistry and Biopolymer Structure Analysis, Department of Chemistry, University ofKonstanz, Germany 2SAW Instruments GmbH, Bonn, Germany

Keywords:
affinty mass spectrometry, SAW-ESI-MS, biosensor, neurodegenerative proteins, alpha synuclein
Novel aspects:
The new affinity-MS method based on the online coupling of SAW-biosensor to ESI-MS provides information of biopolymer
interactions at molecular level, enabling affinity detection and quantification, and ligand structure identification.
Abstract:
Introduction:
In the analysis of affinity interactions biosensors are a key analytical tool, being able to detect and quantify bioaffinity bindings.
Nonetheless, biosensors do not provide the chemical structure identification of ligands, for which mass spectrometry is a powerfultool [1]. Here we show that the online combination biosensor- mass spectrometry provides simultaneous chemical structure
identification and quantification of bioaffinity interactions.
Methods:
In this study we report affinity interaction and structural studies of antigen-antibody and peptide-protein complexes, as well as of
biological samples: mouse brain homogenate containing α-Synuclein and human CSF containing Aß peptides. The SAW-biosensor
was directly coupled to (i) ESI ion trap, and (ii) ESI FT-ICR-MS. The analysed complexes included Aß (1-16) peptide / anti- Aß (116) antibody; Aß(12-40) / anti- Aß (17-28) antibody; Tau protein (isoform 2N/4R) / anti-Tauspecific antibody (clone TAU5); melitin/ calmodulin, and α-Synuclein / anti- α-Synuclein antibodies. An anti- α-Synuclein antibody was used for the affinity characterisa­
tion of mouse brain homogenate, while anti- Aß antibodies were employed in the analysis of human CSF. Antibodies / proteins
were immobilised via a thiol linker on the surface of a gold chip inside the biosensor, and their interactions with the specificpeptide or protein were monitored with the SAW biosensor. Using the online interface developed in our laboratory, the boundpeptide or protein was eluted at acidic conditions from the gold chip and analysed by ESI mass spectrometry, which provided thestructural characterisation of the ligands [1].
Results:
The online combination of SAW biosensor and mass spectrometry allowed the structural identification and affinity determinationof affinity-bound Tau, Aß and α-Synuclein. The mass spectrometric characterisation provided details on post translational
structure modifications of proteins and peptides present in biological samples, while the analysis of the binding kinetics delivered
the KD values for the antibody-antigen interactions. The key achievement of the method consists in the ability to structurally
characterize specific biopolymer molecules (Aß, α-Synuclein), occurring at low concentrations in biological material. Using thisnew hybrid tool, screening for biomarkers of neurodegenerative diseases will become more efficient and accurate, leading toeasily quantifiable and interpretable results.
(Supported by grant KF2026662 from the Bundesministerium für Wirtschaft und Technologie, Berlin Germany)[1] Dragusanu, M. et al. J. Am. Soc.Mass Spectrom. 21, 2010, 1643-1648.

Oral Session
Monday, 17th SeptemberRoom A

09:00 － 11:00

Session 2: Advances in Methods and MS Instrumentation for Biomolecule Characterization
Chair:

Vicki H Wysocki（Ohio State University, USA）

S02-1000
10:00 － 10:20

Pinpointing individual sites that change conformation in large protein
complexes by H/D exchange and ETD-MS

Song Hongjian1, Jeff M Brown2, Steven D Pringle2, Michael Morris2, Kasper D Rand1

Laboratory for Biomacromolecular Analysis, Advanced Drug Analysis Section, Department of Pharmacy, University of
Copenhagen, Denmark, 2Waters MS Technologies Centre, Manchester, UK.

1

Keywords:
Hydrogen/deuterium exchange； protein conformation, conformational changes, protein-protein complexes, electron transfer
dissociation
Novel aspects:
The ability of mass spectrometry to obtain single-residue structural information even for large and challenging protein complexesof therapeutic interest.
Abstract:
Introduction
Hydrogen/deuterium exchange（HDX）analysis of individual residues in proteins by electrontransfer dissociation（ETD）has paved the way for MS analysis of protein structure at an
unprecedented level of detail［1,2］. HDX-ETD experiments on large protein systems［ 3 ］however
remain a challenge as these yield highly complex peptide mixtures for which optimal ETD
spectra can be difficult to acquire on a chromatographic timescale. Here we combine optimized
ETD with chromatographic separation in an online HDX-ETD workflow to successfully obtainsite-specific HDX information on the therapeutically important 75KDa protein complex formedby Coagulation Factor VIIa（FVIIa）and Tissue Factor（TF）during blood coagulation［ 4 ］. Our
results demonstrate the ability of mass spectrometry to obtain single-residue structural
information even for large and challenging protein complexes of biopharmaceutical interest.
Methods
Deuterium labeled samples of recombinant FVIIa and TF were prepared by an automated liquidhandling robot（LEAP technologies）. Labeled protein samples were loaded onto a refrigerated
nanoACQUITY UPLC system（Waters Inc.）interfaced with a Synapt G 2 mass spectrometer（Waters Inc.）for pepsin digestion, chromatography and mass spectrometry. The mass
spectrometer was equipped with an ESI source operated at predefined settings for minimal H/Dscrambling as described previously［5,6］. ETD was performed in the trap T-wave by reacting
radical 1,4 dicyanobenzene anions with quadrupole-selected deuterium labeled peptide ions.
Results
Pepsin proteolysis of FVIIa-TF samples yielded a complex mixture of overlapping peptides and
57 unique peptides covering 95% of the sequence of FVIIa were identified. HDX-MS
experiments performed without ETD on free and TF-bound FVIIa revealed a subset of 11
representative FVIIa peptides that displayed reduced deuterium uptake upon TF binding andthus contained residues whose conformation was affected upon TF-induced activation of FVIIa.
HDX-ETD experiments on the hybrid Q-TOF platform were performed by use of a datadependent method to optimize precursor selection and ETD acquisition parameters for the 11
FVIIa peptides of interest based on their respective elution time and most abundant charge
states observed. Further, mild supplemental activation of ETD product ions performed in adata-dependent manner in the transfer T-wave was found to improve ETD efficiency for 2 +peptide ions. Using the tailored HX-ETD workflow, we have extracted site-specific HDX
information for FVIIa when bound to TF which has allowed us to pinpoint, for the first time, a
network of residues in FVIIa that relay a concerted structural change from the TF binding site of
FVIIa to the active site region of the protease. This provides detailed molecular insight into theallosteric regulation of this enzyme and key information for the design of variants of FVIIa withimproved activity and biopharmaceutical efficacy. Our results demonstrate that ETD can be
performed online on a chromatographic timescale using a hybrid Q-TOF platform in an
improved HX-ETD type workflow that is poised to open up for high-resolution HDX analysis oflarge and challenging protein systems of key biological and pharmaceutical interest.

References［ 1 ］Rand, K. D., Zehl, M., Jensen, O. N. & Jorgensen, T. J. D. Anal. Chem. 2009, 81, 55775584［ 2 ］Zehl, M., Rand, K. D., Jensen, O. N. & Jorgensen, T. J. D. J. Am. Chem. Soc. 2008, 130,
17453-17459［ 3 ］Huang, R. Y. C., Garai, K., Frieden, C. & Gross, M. L. Biochemistry 2011, 50, 9273-9282［ 4 ］Furie, B. & Furie, B. C. Cell. 1988, 53, 505-518［ 5 ］Rand, K. D., Adams, C. M., Zubarev, R. A. & Jorgensen, T. J. D. J. Am. Chem. Soc.
2008, 130, 1341-1349［ 6 ］Rand, K. D., Pringle, S. D., Morris, M., Engen, J. R. & Brown, J. M. J. Am. Soc. Mass
Spectrom. 2011, 22, 1784-1793

Oral Session
Monday, 17th SeptemberRoom A

09:00 － 11:00

Session 2: Advances in Methods and MS Instrumentation for Biomolecule Characterization
Chair:

Vicki H Wysocki（Ohio State University, USA）

S02-1020
10:20 － 10:40

What happens on the histone multimers in the gas phase?

Kazumi Saikusa1, Sotaro Fuchigami1, Kyohei Takahashi1, Yuuki Asano1, Aritaka Nagadoi1,
Hiroaki Tachiwana2, Hitoshi Kurumizaka2, Mitsunori Ikeguchi1, Yoshifumi Nishimura1, Satoko Akashi1
1

Yokohama City University, Yokohama, Japan, 2Waseda University, Tokyo, Japan

Keywords:
intrinsically disordered protein, histone H 2 A/H 2 B dimer, histone H 3 /H 4 tetramer, electrospray ionization ion mobility mass
spectrometry, molecular dynamics simulation.
Novel aspects:
First application of IM-MS in combination with MD simulation to characterize the structural varieties of histone multimers withdisordered tail regions
Abstract:
Gas-phase behaviors of various protein complex ions have been characterized by electrospray ionization mass spectrometry（ESIMS）in combination with ion mobility（IM）separation. In the present study, we have investigated gas-phase structures of histone
multimers, which have a folded core structure with disordered tail regions, by ESI-IM-MS and molecular dynamics（MD）simulation.
Histone proteins are chief components of nucleosome core particle（NCP）, the basic structural unit of eukaryotic chromatins.
NCP consists of approximately 147 base pairs of DNA wrapping around a histone octamer,（H 2 A/H 2 B/H 3 /H 4 ）2, and its assemblyand disassembly is a dynamic process that regulates DNA transcription, replication, and repair. In order to understand themechanism of the NCP assembly and disassembly, structural characterization of the histone multimers, H 2 A/H 2 B dimer and H 3 /H 4 tetramer, should be of great help. However, their atomic-level structures have not been determined because of the highflexibility of the N-terminal disordered tail region of each histone protein. Thus we have characterized these histone multimers by
MS and MD simulation.
Human histone H 2 A/H 2 B dimer and H 3 /H 4 tetramer were prepared by refolding of the recombinant proteins. When the sample
solutions were prepared in an extremely high concentration of ammonium acetate and subjected to ESI-MS, the intact histone
multimer ions were successfully observed without dissociation into each monomer. In the IM-MS analysis, experimentally obtained
arrival times of these histone multimer ions presented rather wide distributions, suggesting that they were in various
conformations.
In order to characterize the structures of these histone multimers observed by IM-MS, consecutive MD simulation for these histone
multimers was carried out first in aqueous environment then in vacuo. MD simulation in aqueous solutions containing high salt
concentrations for 10 ns revealed the structures of histone multimers with stable folded core parts and disordered tail regions. The
10 simulated structures every 1 ns in solution were then subjected to the gas-phase simulations. The behaviors of the histone
multimers in vacuo were analyzed by MD simulation with attaching 11+（H 2 A/H 2 B dimer）or 16+（H 3 /H 4 tetramer）charges,
which were the dominant charge states observed in the ESI mass spectra, to the initial solution structures. When the distributionof the calculated CCS values for 150 simulated structures of each histone multimer was analyzed, it was found that the MD
simulated structures at the ESI-charge state exhibited a relatively narrow CCS distribution, which was not completely consistent
with the results obtained by IM-MS. Therefore, another set of MD simulation of the histone multimers at the Native-charge state,i.e., the charge state that was employed in the MD simulation in aqueous environment, was also carried out. It was found that theMD simulated structures at the Native-charge state demonstrated a wide CCS distribution, which was similar to that observed in
the IM-MS experiments. In addition, detailed analysis of the simulated structures revealed that the wide CCS distribution wasderived from structural variety of the tail regions in the gas phase.
This study indicates that the combination of IM-MS and MD simulation enables comprehensive characterization of the gas-phasestructures of biomolecular complexes containing disordered regions. Further discussion on the transition of the structures during
ES ionization process will be carried out based on the results of IM-MS and MD simulation.

Oral Session
Monday, 17th SeptemberRoom A

09:00 － 11:00

Session 2: Advances in Methods and MS Instrumentation for Biomolecule Characterization
Chair:

Vicki H Wysocki（Ohio State University, USA）

S02-1040
10:40 － 11:00

Dibasic Site-Specific Proteolysis for Improved Bottom-Up and MiddleDown Proteomics

Unige A Laskay1, Upir Oxana1, Luca Fornelli1, Kozhinov N Anton1, Michel Monod2, Goran Mitulovic3,
Yury O Tsybin1
1
Ecole Polytechnique Federale de Lausanne Switzerland, 2Centre Hospitalier Universitaire Vaudois, Lausanne,
Switzerland, 3Medical University Vienna, Austria

Keywords:
Middle-down proteomics, proteomics profiling, supercharging, high resolution
Novel aspects:
We present a summary of our efforts focusing on different steps of the proteome analysis pipeline, starting from proteasecharacterization, sample preparation, LC separation, ionization to data acquisition and analysis.
Abstract:
In-depth bottom-up mass spectrometry（MS）-based proteomics profiling is currently limited by the effective dynamic range of theproteins present and the under-sampling of the peptides generated by proteolysis. Nonetheless, it allows for identification ofseveral hundred, or even thousands of proteins in a chromatographic run, while top-down proteomics is successful in detecting
and analyzing multiple isoforms and modification sites on the same protein. It can be envisioned, that by decreasing the frequencyof cleavage sites of a protease, the average size of the generated peptides will increase, and consequently, the sample complexity
decreases. Middle-down proteomics（MDP）is a recently emerged approach that targets exactly this hypothesis by combining thebenefits of bottom-up and top-down techniques and it is fully compatible with the scan speed of the state-of-the-art FTMS
instrumentation operating in LC-MS/MS mode. Here, we advance bottom-up and middle-down proteomics by judicious selectionof endoproteases capable of providing increased sequence coverage and LC-MS/MS method tailoring to the novel proteolytic
peptide properties to deliver the required improvement in protein identification and characterization.
Trypsin and LysC are the two most commonly used proteases in bottom up proteomics； the benefits of having a basic residue on
the C-terminus for the quality of the MS/MS data have been extensively studied. Much less studied, dibasic site-specific proteases
cleave at adjacent K and/or R sites, where cleavage may occur before, after or between the two basic residues, therefore
resulting in longer peptides carrying multiple charges at one or both termini. To evaluate the applicability of dibasic proteases for
MDP, peptides mimicking these theoretical protease products were synthesized by Fmoc solid state peptide synthesis and their
HCD and ETD fragmentation behavior was compared. We have determined optimal fragmentation conditions using direct infusionof these synthetic peptides by comparing the sequence coverage obtained at different HCD collision energies or ETD reaction
times for the different charge states. All MS/MS experiments were performed on a high-field compact Orbitrap Elite mass
spectrometer（Thermo Scientific）, with resolution setting of 60k（at m/z 400）for precursor ion scanning and 15k for product ionsusing the eFT function enabled.
Another practical requirement for dibasic proteases is the availability, ease and cost of large scale production. We have found
two proteases, Sap 9 and Sap10 that are specific to dibasic sites and cleave after two adjacent basic residues； these wereexpressed in Pichia pastoris and His-tag purified； a green fluorescent enzyme assay activity kit was used to determine the optimal
working conditions. We have found that the enzymes are most active at acidic pH conditions, at short incubation times.
Nano-LC separation was performed on a Dionex UPLC 3000 system（Thermo Scientific）on a 25 cm C18 column. Solvent andgradient optimization was performed on the synthetic peptides. We have found that the presence of methanol in the mobile
phase causes an even distribution of the peptides over the LC gradient resulting in peak broadening； this is expected to allow for
more sampling time for co-eluting peptides and increased MS/MS efficiency. To increase the intensity of the highly charged
precursor ions we have used an in-house built dual-spray microchip obtained through collaboration. Importantly, ion supercharging
occurs in-spray；［ 1 ］therefore it has no detrimental effect on the chromatographic separation.
Our preliminary data indicates that commercial bioinformatics software designed for bottom-up experiments cannot be readily
implemented due to the maximum m/z constraint for the peptide precursor ion and because the advantage of high resolution MS/
MS data is not fully exploited. We thus implement software tailored for top-down data analysis for interpretation of the MDP data.
［ 1 ］Miladinovic S.M., et al., Analytical Chemistry（2012）, in print

Oral Session
Monday, 17th SeptemberRoom B-1

09:00 － 11:00

Session 3: Structures and Dynamics of Atomic and Molecular Clusters
Chair:

Fuminori Misaizu（Tohoku University, Japan）

S03-0900
09:00 － 09:40[Keynote Lecture] Recent Advances in the Vibrational Spectroscopy of
Mass-Selected Gas Phase Cluster Ions

Knut R Asmis

Fritz-Haber-Institut der MPG, Berlin, Germany

Keywords:
infrared photodissociation, clusters, ion trap, messenger-tagging, isomer-selective
Novel aspects:（ 1 ）Measurement of infrared photodissociation spectra of thermalized mass-selected cluster ions.（ 2 ）Isomer-selective
photodissociation spectra using population-labeling IR/IR double resonance spectroscopy combined with a triple mass
spectrometer.
Abstract:
There is an ongoing effort, spanning many branches of the physical sciences, to characterize the structure of gas phase ions,
ranging from simple polyatomic species all the way to large biomolecules containing many thousand atoms. Several techniques
have been refined over the past decade, of which infrared photodissociation（IRPD）spectroscopy has evolved to be one of themost powerful and widely applicable ones. I will give an overview of IRPD experiments performed in our group, focusing on the
structural characterization of mass-selected cluster ions, and how these results can help in our fundamental understanding ofheterogeneous catalysis as well as ion solvation.
Due to their structural variability and redox activity transition metal oxides are widely used in heterogeneous catalysis. However,in most supported catalysts, neither the size and distribution of the active particles on the support surface, nor their structure issufficiently known. Therefore it proves helpful to study the size-dependent properties of such particles in the gas phase, i.e., in theabsence of any interactions with a medium or support. Our ongoing IRPD experiments on transition metal oxide clusters are
aimed at gaining a molecular level understanding of their structure-reactivity relationship and ultimately unravelling the adsorbate/
active site/support interaction important in heterogeneous catalysis.
Understanding how protons are hydrated is another important and challenging research area. The anomalously high proton+andmobility of water can be explained by a periodic isomerization between the Eigen and Zundel binding motifs, H 3 O（aq）+
respectively, even though the detailed mechanism is considerably more complex and still not fully understood. These
H5O2（aq）,
rapidly interconverting structures from the condensed phase can be stabilized, isolated and studied in the gas phase in the formof protonated water clusters. The smallest protonated water cluster that exhibits structural isomers related to both motifs（Eigen+
and Zundel）experimentally is the protonated water hexamer H（H2 O）
6 . Here, results on isomer-selective double resonance+experiments on H（H2O）6 are presented. Protonated water clusters are formed by electrospray ionization, mass-selected, cooled
to cryogenic temperatures, and messenger-tagged with H2 in a buffer gas filled ion trap. Isomer-selective photodissociation spectra
are measured from 300-4000 cm -1 using population-labeling IR/IR double resonance spectroscopy combined with a triple mass
spectrometer.

Oral Session
Monday, 17th SeptemberRoom B-1

09:00 － 11:00

Session 3: Structures and Dynamics of Atomic and Molecular Clusters
Chair:

Fuminori Misaizu（Tohoku University, Japan）

S03-0940
09:40 － 10:00

Laser desorption supersonic jet spectroscopy of neurotransmittermolecules and partial peptides of their receptors

Shun-ichi Ishiuchi, Masaaki Fujii

Tokyo Institute of Technology, Yokohama, Japan

Keywords:
laser desorption, supersonic jet, IR spectroscopy, neurotransmitter, peptide
Novel aspects:
Specifically small numer of conformers of catecholamines in gas phase is found by uniquely improved laser desorption supersonicjet spectroscopy.
Abstract:
The neural signal is transmitted by specific molecules known as neurotransmitters, which bind to receptor protein, from one
neuronal cell to the next, through the accurate molecular recognition process. The same signalization process is utilized for the
regulation of various vital functions by hormones. One of the well-known and typical neurotransmitters is catecholamine, such as
dopamine and adrenaline etc., which has catechol ring and amine chain. Investigation of the conformational geometries of thesemolecules is important as fundamental knowledge to understanding the molecular recognition process of the neurotransmitters.
In this work, we applied laser desorption supersonic jet laser spectroscopy, which enables the gas phase spectroscopy of
biomolecules with distinguishing each conformers as isomers, to catecholamines and the irrelevant molecules. From the results, it
was found that catecholamines have specifically small number, 1 or 2, of conformers in gas phase, while the relevant molecules
which do not have catechol ring have many conformers, more than 10. This drastic reduction of number of conformers observed in
catecholamines is attributed to neighboring phenolic OH group of catechol ring.
In addition, we turned our eyes to another lead role, receptor protein of catecholamines. However, since the receptors are hugemolecules, whose molecular weights are larger than 40,000, it will be unreal to apply the gas phase spectroscopy to themselves.
Fortunately, it was suggested that the binding site of the catecholamines is a small number of residues, so we projected thegasphase spectroscopy of the synthesized partial peptide of the binding site. We took notice of 5 -recidue sequence of adrenergic
beta 2 -receptor, Ser-Ile-Val-Ser-Phe, which behaves as the binding site of catecholic OHs, and applied the laser desorption
supersonic jet spectroscopy. By measuring IR spectrum and comparing it with results of quantum chemical calculations, we
determined a geometrical structure of the 5 -recidue peptide. For the next step, we are planning to generate a complex of
5 -residue peptide and adrenaline in gas phase and determine its structure by IR spectroscopy and theoretical calculations.

Oral Session
Monday, 17th SeptemberRoom B-1

09:00 － 11:00

Session 3: Structures and Dynamics of Atomic and Molecular Clusters
Chair:

Fuminori Misaizu（Tohoku University, Japan）

S03-1000
10:00 － 10:20

The ’sticky business’ of cleaning gas-phase membrane proteins: adetergent oriented perspective

Antoni J Borysik, Carol V Robinson
University of Oxford

Keywords:
ClustersMSMSion-mobilityCIDdetergents
Novel aspects:
Detergent cluster characterisation using ion mobility mass spectrometry. Mechanism of cluster dissociation
Abstract:
Recent advances in the field of biological mass spectrometry have shown that it is possible to use this technique to obtain crucial
insights into the subunit stoichiometry, topology and lipid binding of membrane protein complexes. Central to this approach is theuse of collision induced dissociation（CID）to remove protein-bound detergents prior to mass analysis. However, this ’clean-up’
strategy employs brute force to thermally agitate the ions to levels than can often result in the disruption of protein structure, thus
abrogating any downstream structural characterisation. In order to improve these strategies there now exists a need tounderstand the mechanism/s by which detergent clusters protect protein complexes during CID. This may then lead to morerational selection criteria for detergents that are best suited for the gas-phase characterisation of membrane protein complexes.
In order to understand these processes we have characterised a range of different detergent clusters using electrospray ionisation
mass spectrometry（ESI-MS）in tandem with ion mobility mass spectrometry（IMS）and tandem mass spectrometry（MSMS）.
These important initial studies have been conducted in the absence of proteins which tend to occlude all of the detailed
information that can be gained when studying the cluster in isolation.
Aside from having packing densities to within a few percent of micelles in solution we show that detergent clusters are unique tothe gas-phase. Detergent clusters have topological features that are not representative of micelles in solution. Furthermore, theproperties of these clusters do not depend on amphiphile orientation prior to the electrospray process. We also show that thewell-known relationship between aggregate size and alkyl chain length in solution is actually reversed in the gas-phase. Thus, ourdata suggest that detergent clusters are formed non-specifically during the ESI process. The distribution of different aggregate
sizes and shapes favours a system explained by weak forces governed by the cluster surface tensions.
In order to deduce the mechanism by which detergent clusters protect membrane proteins during CID we then investigated theproperties of the clusters during thermal agitation by MSMS. We show that detergent clusters dissociate by mechanisms that are
predominately described by charge fission processes and the loss of neutrals by evaporation. The latter of these two processes
can be extensive and probably explains the high persistence of residual clusters even at maximum CID thresholds. Using thesedata we propose that evaporative cooling mechanisms protect membrane protein complexes during CID. Future directions
designed to confirm this proposal are also discussed.

Oral Session
Monday, 17th SeptemberRoom B-1

09:00 － 11:00

Session 3: Structures and Dynamics of Atomic and Molecular Clusters
Chair:

Fuminori Misaizu（Tohoku University, Japan）

S03-1020
10:20 － 10:40

Adsorption, Activation and Reaction of Molecules on Size-Selected Metal
Cluster Ions

Masahiko Ichihashi1, Shinichi Hirabayashi2
1

Toyota Technological Institute, Ichikawa, Japan, 2Genesis Research Institute, Inc. Ichikawa, Japan

Keywords:
copper clusters, CO oxidation
Novel aspects:
A carbon monoxide molecule reacts with an oxygen atom on copper cluster anions. This CO oxidation proceeds efficiently on
copper pentamer and nonamer anions.
Abstract:
In a chemical reaction of molecules on a metal cluster composed of several to tens of atoms, its constituent metal atoms
contribute cooperatively in some cases to the chemical reaction. The electronic and the geometric structures change sensitively ina reaction pathway, and the fundamental features of the reaction pathway vary characteristically with cluster size. One of ourgoals is to elucidate the relation of the reactivity with the electronic and geometric structures. This relation will provide a recipe
for developing catalysts of a high utility. To this end, we studied reactions of molecules on metal cluster ions by use of a tandem
mass-spectrometer with reaction cells. For instance, we investigated the reaction of CO and O2 on size-selected copper cluster+, was observed in the reaction of CunO2 + with CO,
ions, Cun +（n = 4 -18）and Cun -（n = 4 -11）. A coadsorbing product, CunO（CO）
2++
and it was found that CO adsorbs onto Cu nO2 more efficiently than onto Cu n . This shows that CO adsorbs cooperatively with O 2
onto Cun +. On the other hand, in the reaction of CunO2 - with CO, a reduced product, CunO-, was given instead of the coadsorbing. In particular, Cu 5 O 2 - and Cu 9 O 2 - have relatively high efficiency for the production of Cu n O - . This result
product, Cu n O（CO）
2suggests that CO is oxidized and CO2 is produced on Cun -. Further, we found that Cu5O4 - is reduced sequentially and Cu5Om -（m= 0 - 3 ）are observed in higher CO pressures.
-> Cu5O（CO2）- is only 0.79 eV while that
The DFT calculation indicates that the activation energy in the reaction of Cu5O（CO）
2of the corresponding cation is 1.79 eV. The structure of Cu n is more flexible than that of Cu n + probably because of its excess
electron. It is concluded that the stabilization of the transition state and the decrease of the activation energy make the COoxidation proceed on Cun -.

Oral Session
Monday, 17th SeptemberRoom B-1

09:00 － 11:00

Session 3: Structures and Dynamics of Atomic and Molecular Clusters
Chair:

Fuminori Misaizu（Tohoku University, Japan）

S03-1040
10:40 － 11:00

Formation, Characterization and Reactivity of a Gaseous Adduct ofCarbon Dioxide to Magnesium(I), HOMgCO2-

Héloïse Dossmann (Soldi-Lose)2, Carlos Afonso3, Denis Lesage2, Jean-Claude Tabet2, Einar Uggerud1
1

University of Oslo, 2Pierre and Marie Curie University (Paris VI), 3University of Rouen

Keywords:
Carbon dioxide fixation · gas phase reactions · computational chemistry · nucleophilic substitution · photosynthesis
Novel aspects:
We report a key intermediate of high relevance to natural and synthetic activation and fixation of carbon dioxide.
Abstract:
Activated forms of carbon dioxide are of great interest due to the relevance to photosynthetic CO2 uptake and to sequestrationof CO 2 from flue gases formed upon the burning of fossil fuels. One efficient fixation of carbondioxide by covalent C-C bondformation is schematically described as RX +CO2 + 2 e-® RCO2 - + X-. In plant cells, electrons are provided from chlorophyll via
NADPH and formation of a C-C bond between CO2 and another substrate occurs at the magnesium-centered active site of the
enzyme RuBisCO. In this context, we report here on the gas-phase characterization of a novel and fragile structural form ofcarbondioxide bonded to magnesium（I）, HOMgCO2 - showing interesting reactivity features
HOMgCO 2 - ions were formed via electrospray ionization of a mixture of oxalic acid and magnesium salts MgCl 2 and
MgBr2dissolved in CH3OH/H2O（v/v 90/10）. Analysis of the ions was done using a homemade modified Quattro II（Micromass,
Manchester, U.K）and a hybrid quadrupole Fourier transform ion cyclotron resonance（hQh-FT/ICR, Solarix, Bruker Daltonics,
Billerica, USA）mass spectrometer. Different types of activation were performed on the complexes, including collision-induced
dissociation（CID）, sustained off-resonanceirradiation collision-induced dissociations（SORI-CID）and ion-molecule reactions（with
H2O and CH3Cl）.
In parallel to experiments, quantum chemical calculations were carried out using the program system GAUSSIAN 09. Geometry
optimization and single point energies were obtained using the composite G 4 method.
Electrospray ionization was applied to mixtures of oxalic acid and magnesium salts MgCl2 and MgBr2dissolved in CH3OH/H2O（v/v
90/10）. From the recorded mass spectra, signal corresponding to the MgC2O5H-ion was observed. Upon collisional activation, thision dissociates by losing CO2, 2 CO2 and MgC2O4 leading to the formation respectively of HOMgCO2 -, HOMg-and HO-. Different
structures are probed theoretically for the ion of interest, HOMgCO2 - as well as its mechanism of formation from the MgC2O5Hion. Results show that two structures are conceivable for this ion, HOMg（η2-O2C）-and HOMg（η2-OCO）-, separated from eachother by 31kJ/mol. Furthermore, the complex shows a strong reactivity upon water which is present as a trace in the mass
spectrometer. This reaction leads to the formation of an unique ion, Mg（OH）3 -. Theoretical study of the mechanism of formationof this ion reveals a very exothermic pathway from HOMg（η2-O2C）- but not from HOMg（η2-OCO）-.As will be discussed, this
supports the hypothesis of the presence of only one structure for HOMgCO2 -, i.e. HOMg（ η2-O2C）-.Finally, the reaction of thecomplex with CH3Cl is investigated. Several product ions are observed, in particular Cl - and CH3CO2 -that are products from an
SN 2 -type reaction.

Oral Session
Monday, 17th SeptemberRoom D

09:00 － 11:00

Session 4: Imaging-I
Chair:

Mitsutoshi Setou（Hamamatsu University School of Medicine, Japan）

S04-0900
09:00 － 09:40[Keynote Lecture] Imaging molecular signals with multimodal imaging
mass spectrometry

Ron M Heeren

FOM-Institute AMOLF

Keywords:
Imaging mass spectrometry, high resolution, microscope mode, detector technology, quantitative proteomics
Novel aspects:
A new multimodal MS imaging approach reveals changing signaling pathways in disease
Abstract:
The study of molecular signaling processes related to diseases requires not only the detection and analysis of the molecules
involved but also the evaluation of their spatial organization. This allows the detailed chemotyping of cells under duress and canprovide critical insight in the activated pathways related to the progression of the disease. Imaging mass spectrometry is ideallysuited for this task as it allows the generation of high resolution molecular images of a wide variety of biological compounds fromcomplex surfaces without the need for chemical labels or antibodies. High fidelity molecular ion images can be generated using
modern desorption and ionization techniques combined with high end mass spectrometric systems. The distribution of several
hundreds of peptides and proteins on the surface of histological tissue sections can be determined in a single imaging MS
experiment. This enables molecular pathway analysis and the role of the different molecular signals and their behavior under adrastically changing chemical or biological environment in these pathways.
The complexity of these pathways in diseases prohibits the detailed chemical characterization by a single technique. We havedeveloped an approach that combines a number of innovative mass spectrometric imaging approaches with bright field imaging,magnetic resonance imaging and targeted IHC images to refine our understanding of disease progression in human tissue samples.
In addition we have integrated state-of-the-art quantitative proteomics and metabolomic studies of selected tissues in ourmolecular imaging workflow. The common histological tools typically only provide generic morphological information unlessimmunohistochemistry is used to determine the distribution one specific known protein. Imaging mass spectrometry has evolved
to bring together these two disciplines, mass spectrometry and histology. This approach, sometimes referred to as molecular
histology, can take great benefit from the use of high resolution mass spectrometry, gas-phase ion mobility separation and smart
surface preparation protocols. Combined they provide new molecular visualization tools for biomedical researchers. These
innovative developments towards high resolution imaging mass spectrometry can reveal new tissue details that remain hiddenwith conventional molecular imaging approaches. In this contribution we will discuss applications of new chemical microscopes inbiomedical tissue analysis. This lecture will discuss how imaging MS can elucidate the way in which local environments caninfluence signaling pathways in diseases.

Oral Session
Monday, 17th SeptemberRoom D

09:00 － 11:00

Session 4: Imaging-I
Chair:

Mitsutoshi Setou（Hamamatsu University School of Medicine, Japan）

S04-0940
09:40 － 10:00

Single cell level mass spectrometry imaging for plant metabolites

Young-Jin Lee1,21

Iowa State University, Ames, USA/Iowa, 2Ames Laboratory-USDOE, Ames, USA/Iowa

Keywords:
MALDI, mass spectrometry imaging, plant, metabolites, single cell
Novel aspects:
single cell level imaging on plant metabolites； multiplex MS imaging technology to save data acquisition time while achieving richchemical information.
Abstract:
We develop mass spectrometric（MS）imaging techniques to map metabolite distributions within plant tissues, particularly insingle cell level high spatial resolutions. In this presentation, I will present a few of our recent work that demonstrate sometechnological advancements and applications to plant metabolic biology.
Technology development 1 -Single cell level MS imaging of a whole flower： Chemical images of epiculticular wax lipids wereacquired on the surface of Arabidopsis flower in 12um ultrahigh spatial resolution. Optical fiber with 25um core diameter was used
to narrow the laser beam size and modified oscillating capillary nebulizer was used to homogeneously spray colloidal silver as a
MALDI matrix. With this approach, single cell level very fine chemical images could be obtained including visualization of single
pollen grains and fine structures of anthers.
Technology development 2 -Multiplex MS imaging： Multiplex MS imaging technology has been developed to reduce the dataacquisition time while obtaining diverse chemical information in a single experiment. In this methodology, each raster step is split
into a few spiral steps. While orbitrap mass spectrometer is scanning dataset from the first spiral position, ion trap is scanningnext few dataset from the following few spiral steps. Using this approach, we could simultaneously obtain high mass resolution
imaging in low spatial resolution and high-spatial resolution imaging in low mass resolution, in much less data acquisition time. In
addition, we could also incorporate a few MS/MS and MSn imaging at the same. We successfully applied this technique to obtainasymmetric distribution of structural isomers of flavonoids. This methodology is also applied to latent finger printing,simultaneously obtaining MS imaging and MS/MS imaging, and further extended to obtain positive and negative ion MS imaging ina single experiment.
Application 1 -MS imaging as a tool for high-resolution functional genomics： Metabolite images from two genetically mutatedArabidopsis plants were studied using MS imaging and compared to those of wild-type. In MS imaging of CER 1 mutant, systematic
decrease of alkane, ketone, and C26 fatty acids were monitored on various plant tissues including flower, leaf, and stem, incontrast to slight increase of C30 fatty acids. High resolution MS imaging could distinguish C28 aldehyde from C29 alkane andrevealed the accumulation of aldehyde in CER 1 mutant, suggesting the involvement of CER 1 gene in decarbonylation ofaldehyde. Flavonoid imaging of the Arabidopsis flower showed an asymmetric distribution of kaempferol and its glycosides on the
distal region of petals, and quercetin, isohamnetin, and their glycosides on the proximal region of petals. In contrast, ahomogeneous distribution of kaempferol and its glycosides is observed in the mutant（tt 7 ）, suggesting asymmetric gene
expression of TT7.
Application 2 -Cellular level lipid distribution in cottonseeds： MS imaging of cryosections of cotton embryos revealed a distinct,
heterogeneous distribution of molecular species of triacylglycerols（TAG）and phosphatidylcholines（PC）, the major storage andmembrane lipid classes in cotton embryos. Other lipids were imaged, including phosphatidylethanolamines, phosphatidic acids,
sterols, and gossypol, indicating the broad range of metabolites and applications for this chemical visualization approach.
Comprehensive lipidomics images generated by MALDI-MSI report accurate, relative amounts of lipid species in plant tissues andreveal previously unseen differences in spatial distributions providing for a new level of understanding in cellular biochemistry. For
example, TAG species with palmitic and linoleic acids（c.f., TAG 50： 2 （PLL）or TAG 52： 4 （LLL））have a higher abundance in
cotyledon than in the embryonic axis. The similar distribution in PC（c.f., PC 34： 2 （PL）or PC 36： 4 （LL））supports the synthesis of
TAG via membrane lipids as an intermediate.

Oral Session
Monday, 17th SeptemberRoom D

09:00 － 11:00

Session 4: Imaging-I
Chair:

Mitsutoshi Setou（Hamamatsu University School of Medicine, Japan）

S04-1000
10:00 － 10:20

Molecular Imaging of Cells and Tissues with Continuous Cluster Ion
Beams

Jiro Matsuo1,2, Takaaki Aoki1,2, Toshio Seki1,2
1

Kyoto University, Kyoto, Japan, 2JST, CREST

Keywords:
Mass Imaging, SIMS, Lipid, Cluster ions
Novel aspects:
Advanced mass imaging system employing with Ar cluster ion beams. Mass images of cells and tissues will be presented.
Abstract:
Various mass spectroscopic techniques, such as secondary ion mass spectrometry（SIMS）, matrix-assisted laser desorption
ionization（MALDI）and desorption electrospray ionization（DESI）, have been examined intensively during the last decade,because molecular, structural and chemical state information is considered invaluable in life science. The SIMS technique isconsidered to have the highest spatial resolution, and submicron resolution has been reported for elemental analysis of inorganic
materials. Energetic ion irradiation also leads to emission of molecular ions representing the chemical composition of biologicalmaterials. However, secondary molecular ion yields are usually quite low, because of decomposition of molecules and low-density
excitation of surface. Novel ion beams, such as cluster ion beams and swift heavy ion beams, are utilized to enhance secondary
molecular yields. Cluster ion beams have been reported to enhance the yields of secondary ions, because of the high-density
energy deposition and multiple collisions near surfaces. SF5, C60, Au3 and Bi3 were found to be quite useful for SIMS of organic
materials［ 1 ］. Because these primary ion beams cause significant damage on organic surfaces, the primary ion dose is limited toa certain threshold value（known as“the static-limit“, ~1012 ions/cm2）. Therefore, the intensity of small molecular ions（>500 Da）
is too low to obtain high-resolution mass images.
We proposed the use of a large Ar cluster beam for SIMS analysis of organic materials. This beam presents a number ofadvantages over other cluster beams for molecular depth profiling of various organic materials, and provides new opportunities
for sputtering molecules without inducing significant damage［2, 3 ］. Biomolecules are also very fragile and thus difficult to
sputter with conventional ion beams； therefore, a large Ar cluster beam would be quite suitable for biological material analysis.
We have developed a new Ar cluster ion gun aiming to obtain a fine-focused beam, and Ar cluster ion beam with several mm
diameters was obtained.
The beam current density is too low to measure mass spectra with the conventional time-of-flight（TOF）method, when the
primary ion beam is pulsed. An orthogonal acceleration time-of-flight（oa-TOF）mass spectrometer, which allows the use of acontinuous beam, was employed in a new bioimaging system. There was no need to use the ion-bunching technique in thissystem, and therefore there was no need for tradeoff between beam diameter and mass resolution, which is a problem in massimaging of biological samples with conventional SIMS. This is another advantage of our new molecular imaging system over theconventional TOF instrument. Because of the complexity of chemical compositions in natural samples, such as cells and tissues, it
is very important to maintain high mass resolution（>10000）to separate between different molecules with similar mass.
The latest results of this system and its performance in molecular imaging of cells and tissues will be presented and discussed.
Acknowledgements
This work is supported by the Core Research of Evolutional Science and Technology（CREST）of Japan Science and Technology
Agency（JST）.
References［ 1 ］N. Winograd, Anal. Chem. 77（2005）, 142A
［ 2 ］J. Matsuo, S. Ninomiya, Y. Nakata, Y. Honda, K. Ichiki, Appl. Surf. Sci. 255（2008）1235［ 3 ］S. Ninomiya, K. Ichiki, H. Yamada, Y. Nakata, T. Seki, T. Aoki, J. Matsuo, Rapid. Commun. Mass Spectrom., 23（2009）, 1601.
［ 4 ］J. Matsuo, S. Ninomiya, H. Yamada, K. Ichiki, Y. Wakamatsu, M. Hada, T. Seki, T. Aoki, Surf. Interface Anal. 42（2011）, 1612

Oral Session
Monday, 17th SeptemberRoom D

09:00 － 11:00

Session 4: Imaging-I
Chair:

Mitsutoshi Setou（Hamamatsu University School of Medicine, Japan）

S04-1020
10:20 － 10:40

Spatial Resolution of Metabolites during Barley Grain Development by
Imaging Mass Spectrometric Analysis

Manuela Peukert1, Andrea Matros1, Ales Svatos2, Hans-Peter Mock1
1

IPK Gatersleben, Germany, 2MPI Jena, Germany

Keywords:
MALDI MSI, metabolites, barley grain
Novel aspects:
Application of MALDI MSI to plant tissues such as grains for elucidation of metabolite distributions during development and direct
identification of compounds from the tissue section
Abstract:
Spatially resolved analysis of metabolites and proteins is essential for elucidating compartmentalized cellular processes. During
the last decade enormous progress has been achieved in MALDI MS based imaging（MALDI MSI）and application of this techniqueto animal and human tissues has revealed a number of novel clinical markers for better diagnosis or for pharmacological studies.
Investigation of plant tissues with MALDI MSI is more recent and adaption of protocols is needed as plant organs comprisecomplex tissue types. For example, visualization of specific classes of compounds relies on the right conditions for ionization. We
are interested in the elucidation of components involved in barley grain development. An understanding of developmentally andecologically regulated processes affecting agronomical traits such as final grain weight, seed quality and stress tolerance is ofoutmost importance, as barley provides one of the staple foods.
The presentation will introduce an untargeted MALDI MSI approach to the analysis of metabolite patterns during barley grain
development. We analyzed longitudinal and cross sections from developing barley grains（3, 7, 10 and 14 days after pollination）.
A number of candidate m/z values showing high tissue as well as developmental specificity were detected and selected examples
will be shown. Challenging aspects regarding spatial resolution, sensitivity and identification of unknown compounds will also be
discussed. First analytical efforts allowed for assignment of a number of the candidate m/z values to various metabolite classes,
such as sugars, phospholipids and chlorophyll, directly from tissue sections. However, identification from tissue sections failed formany of the candidate m/z values due to their low abundance in the individual MALDI MS spectra. Thus, current work includes
fractionated extraction of differentially distributed compounds out of manually dissected barley grain tissues and identification ofrespective molecular ions by GC-MS and LC-MS approaches. Finally, further approaches for quantification of selected metabolites
by LC-MS and GC-MS as well as approaches for relative quantification of selected metabolites by DESI-MS will be presented. The
future task will be to relate the patterns to distinct cellular and physiological events. First results revealed particular metabolite
distributions indicative for nutrient transport into the developing endosperm and for grain hardness（e.g. dough and germination
properties）.

Oral Session
Monday, 17th SeptemberRoom D

09:00 － 11:00

Session 4: Imaging-I
Chair:

Mitsutoshi Setou（Hamamatsu University School of Medicine, Japan）

S04-1040
10:40 － 11:00

Mass spectrometry imaging (MALDI-TOF/TOF) of drugs in a brain mousemodel of Parkinson disease

David Touboul1, Hanane Kadar1, Gael Le Douaron2, Majid Amar3, Bruno Figadère2, Laurent Ferrié2,
Rita Raisman-Vozani3, Alain Brunelle1
1
3

ICSN CNRS, Gif-sur-Yvette, France, 2UMR-8076 (BioCIS) Laboratoire de Pharmacognosie, Châtenay -Malabry, France,
INSERM UMR-S 975, CNRS UMR 7225, UPMC, CRIMC, Paris, France

Keywords:
mass spectrometry imaging, Parkinson disease
Novel aspects:
Drugs localization and lipid composition of brain from mice models of Parkinson disease
Abstract:
Introduction
Neurodegenerative disorders, such as Parkinson and Alzheimer diseases, constitute a major public health issue affecting morethan thirty millions of people worldwide. Patients affected by Parkinson disease suffer from different symptoms, motor impairments
as muscular rigidity, slowness of movement, trembling at rest. Although the etiology of the disease is not clearly understood,
death of dopaminergicneurons in substancia nigra pars compacta（SNpc）has been found to be one of the main features of thedisease. Therapies correcting the loss of dopamine only allow a decrease of some of the symptoms, but don’t alter theneurodegenerative process. The goal of our experiments is to localize the drug（ 1 -methyl- 4 -phenylpyridinium, MPP+）inducing
the Parkinsonism in mouse model and to survey the modifications of lipid profiles in the different parts of the brain by MALDITOF/TOF imaging. A new synthetic active molecule（SF41）, providing both neuroprotective and neuritogenic activities for
dopaminergicneurons, was also localized by mass spectrometry imaging（MSI）.
Methods
Experiments were performed using a MALDI-TOF/TOF（4800 and 5800 AB-Sciex）instrument. For each mouse brain（control, treatedby MPTP or SF41）, images of 5 sections at different depths were recorded. A major advantage of this technique is theopportunity to obtain a chemical mapping of a wide range of substances in a single run, particularly lipids. Then 9 -aminoacridine
andalpha-cyano- 4 -hydroxycinnamic acid matrices were deposited on the tissue sections with a TM-Sprayer for negative andpositive modes, respectively. All instrumental parameters were optimized for the best sensitivity. Lateral resolution was fixed to 70µm.
Results
MPP+ was detected in the positive ion mode in mouse brain treated by MPTP, which is the precursor of the active drug. Clear
accumulations in the SNpc and the striatum, which represent the two areas rich in dopaminergic neurons and fiber, were
demonstrated. Lipid profiles in both positive and negative ion modes were compared between control and MPTP-treated mouse.
The process consists in the comparison of mass spectra of the relevant regions of interest（ROIs）so as to discriminate differencesin lipid composition and to highlight some potential biomarkers for Parkinson disease. Significant modifications will be discussed.
SF41 was also detected in the positive ion mode in treated mouse brain. This indicates that SF41efficiently crosses the blood-brain
barrier as confirmed by LC-MS/MS. SF41 was detected in different anatomical brain areas, especially the striatum and the SNpc.
Ion images were associated to immunohistochemistry TH+ and Nissl preparation of adjacent brain sections to precisely localize
the altered areas. It emphasizes that the compound reaches its target and can have an in vivo neuroprotective effect on
dopaminergic neurons loss. EasyMSI processing software was used for this purpose. Lipid profiles were also extracted from the
MSI data and compared to the ones from the control or MPTP-treated mouse brain.

Oral Session
Monday, 17th SeptemberRoom E

09:00 － 11:00

Session 5: Advances in Spray Ionization Techniques
Chair:Charles N McEwen（University of the Sciences, USA）

S05-0900
09:00 － 09:40[Keynote Lecture] Cold-Spray Ionization Mass Spectrometry

Kentaro Yamaguchi

Tokushima Bunri University, Kagawa, Japan

Keywords:
CSI-MS, non-covalent-complex, charged probe
Novel aspects:
Cold-spray ionization method was presented. Application to labile organic species as well as non-covalent complexes are shown.
Metal-complex-based ionization probes exhibited effective ionization by using cold-spray ionization method.
Abstract:
Electrospray ionization（ESI）MS is generally used for the characterization of labile supramolecules in which non-covalent bonding
interactions are predominant. However, molecular ions are not detected in many cases because of their instability, and even if themolecular ions are detected, thermal decomposition generates fragment ions whose peaks also appear in the mass spectrum. In
conventional ESI, the heat produced by the desolvation chamber was thought to be necessary for the ionization in the gas phase；
however, it might be responsible for the thermal decomposition.
  
is designed for the MS detection of labile organic species. It may be an appropriate method to analyze
Cold-spray ionization（CSI）
the structures of biomolecular complexes and labile organic species in solution. The method, a variant of ESI-MS, operates at low
temperatures and can simply and precisely characterize labile non-covalent complexes that are difficult or impossible to observeby conventional MS.
CSI is particularly suitable for elucidating the structures of labile organometallic compounds in solution. This method provides ameans to investigate the dynamic behavior of unstable molecules and/or labile clusters in solution.In the first example, thecomposition of Grignard reagent in solution was investigated. CSI-MS revealed that RMg（m-Cl
23 ）is the structure of RMgCl in THF
solution. CSI-MS is also a powerful tool to analyze the equilibria of multiply linked self-assembling catenanes in solution. Itsapplication to unstable and complex supramolecules will be shown.
The CSI-MS can be used to investigate labile solution structures of various biomolecules. In the case of DNA, for example, various
oligodeoxynucleotide complexes can be observed by CSI-MS； examples include such unstable species as low Tm DNA duplexes.
CSI shows its superiority to other methods by effectively elucidating the interactions of DNA complexes. In the case of amino acidanalysis, aggregation to form corresponding clusters is widely observed. Large-scale aggregated chain structures of simple
biomolecules, such as amino acids, can be introduced to the gas phase. A clear chain structure can be seen for amino acids,
including glycine, L-valine, L-serine, L-methionine, and L-phenylalanine, by means of CSI-MS. A difference, diffusion-based NMR
technique and CSI-MS can be employed as a solution-based approach to identify a ligand binding to a protein receptor. The
difference, diffusion-based NMR technique, called difference NOE pumping, can directly detect a ligand binding to a protein
receptor. The CSI method can also detect the ligand-receptor complex. The efficiency of these techniques for identifying binding
ligands is demonstrated with a human serum albumin HAS-drug system.
Various labile organic compounds are analyzed by the CSI method in organic chemistry. Investigations of real solution behavior oflabile organic molecules by MS are difficult. The nature of hydrogen-bonding interactions remains unclear even for simple watermolecules, to say nothing of the precise behavior of complex biomolecules in solution. This is because higher-order structures arelinked by weak intermolecular interactions that are readily disrupted. CSI-MS was used to investigate the solution behavior of
aggregated steroid compounds, bisguanidinobenzene-benzoicacid complexes, and cavitand cages.
Recently, we developed an effective ionization method that uses metal-complex-based ionization probes containing 2,6-bis（oxazolinyl）pyridine（pybox）ligands. By using these probes, we were able to obtain multiply charged ions of target molecules.
This method effectively ionized large complex molecules, including biomolecules and various supramolecules, as well as carbon
clusters, such as fullerenes. Moreover, isotope-labeled pybox-La complexes were used to clearly detect isotopic labeling shifts.
Their applications to multiply charged ionization, including isotope labeling of biomolecules and carbon clusters using CSI-MS, will
be shown.

Oral Session
Monday, 17th SeptemberRoom E

09:00 － 11:00

Session 5: Advances in Spray Ionization Techniques
Chair:Charles N McEwen（University of the Sciences, USA）

S05-0940
09:40 － 10:00

Mass Spectrometric Analyses of Isomeric Sugars, Lipids and their
Conjugates: Introduction to“Charge-Localization Isomers“

Yoko Ohashi1,2, Masayuki Kubota3, Takashi Hirano2

1
RIKEN, Wako, Japan, 2The Univ. of Electro.-Comm., Chofu, Tokyo, Japan, 3ThermoFisher Sci. Co., Yokohama, Kanagawa,Japan

Keywords:
charge-localization isomers； ESI-MS/MS； glycosaminoglycan
Novel aspects:
ESI-MS/MS is not only capable of distinguishing conventional isomers but also has shown existence of a new class of isomers.
Abstract:
With an unexpected success in distinguishing positional isomers of phospholipids in the early ’80s, we used FAB-MS for isomer
distinctions of sphingoid isomers, ganglioside series and sugar linkages. Afterwards, ESI-MS was found to be more promising inaccuracy with much less sample consumption. Recently, we have come to think that we have covered isomer distinctions ofessentially all categories of similar biological isomers by mass spectrometry when stereoisomers such as epimers and anomers
were finally successfully distinguished by ESI-MS/MS. 1, 2
However, an extensive examination of an unsolved problem left in the elucidation of one minor fragmentation path has led us toconclude that there is a new class of isomers, which we call“charge-localization isomers“. This specific class of isomers may exist
transiently as the negative ion of strong dibasic acids, exemplified here as a glycosaminoglycan disaccharide
N-acetyllactosamine-6, 6 ’-disulfate disodium salt（M’’W 587, where M’’ stands for the disodium salt）. 3 As shown in ref. 3, a
negative precursor ion［M’’-Na］- at m/z 564, originating from this dibasic acid, is capable of fragmenting by two different routes innegative ion ESI-MS/MS. Two possibilities ought to be considered：（ 1 ）There is only one common precursor ion for the tworoutes.（ 2 ）There are two different precursor ions individually leading to two different fragmentation routes. In the former case,such precursor ion structure may represent a doubly dissociated negative ion weakly bridged by a Na+, which compensates one
negative charge. This situation will be conceivable in the solution phase. However, at the spray ionization, Na+ must localize ateither one of the two sulfate anions in the gas phase, eventually leading to the latter case. Evidence is provided as follows.3 The
negative ion ESI-MS full scan spectrum of N-acetyllactosamine-6, 6 ’-disulfate disodium salt showed a doubly negative ion［M’’- 2 Na］2- at m/z 270.5 in higher abundance than［M’’-Na］- at m/z 564. This means that the two negative groups are well separated
owing to the Coulomb repulsion. Since a preliminary optimization of the structure of the dihydrogensulfate by semi-empiricalmolecular orbital calculations（PM 5 ）indicated that the two hydrogen sulfate groups were geometrically far apart, 1 themechanism including two different precursor ions is more probable. Molecular structure optimization by DFT calculations is now
in progress.
We assume that either one of the two sulfate groups is available for the negative center. Thus, the two formed individual negativeions（charge-localization isomers）proceed along different routes of decomposition, even though some product ions apparently
show isobaric mass numbers. Thus, we claim that the entity of［M’’-Na］- is not a Na+-bridged doubly negative ion but a mixture oftwo separate singly negative ions, which we call“charge-localization isomers“.
Experimental：
Galb 1 - 4 GlcNAc-6,6’-disulfate disodium salt（M’’W 587）was obtained from shark fin keratan sulfate by keratanase II digestion,and dissolved in H2O/CH3OH（ 1 ： 1 ）at a concentration of 10 ng/mL. A ThermoFisher Scientific mass spectrometer LTQ FT wasused in the accurate mass measurement mode with the spray voltage 1 kV and the resolving power 100,000. The CID energy
parameter for MS2 and MS3 was 30%.
References：
1 ）Yoko Ohashi, Yoshiyuki Itoh, Masayuki Kubota, Kensaku Hamada, Mamoru Ohashi, Takashi Hirano and Haruki Niwa, Eur. J.
Mass Spectrom., 10, 269-278（2004）.
2 ）Yoko Ohashi, Masayuki Kubota, Hiroshi Hatase, Mitsuhiro Nakamura, Takashi Hirano, Haruki Niwa, and Yoshitaka Nagai, J. Am.
Soc. Mass Spectrom., 20, 394-397（2009）.
3 ）Yoko Ohashi, Masayuki Kubota, Hiroshi Hatase, Takashi Hirano, Shojiro Maki, Haruki Niwa, Int. J. Mass Spectrom. 312, 179-184（2012）.

Oral Session
Monday, 17th SeptemberRoom E

09:00 － 11:00

Session 5: Advances in Spray Ionization Techniques
Chair:Charles N McEwen（University of the Sciences, USA）

S05-1000
10:00 － 10:20“Supercharging“ and“Subcharging“ Proteins and Protein Complexes to
Explore Electrospray Ionization

Rachel R Loo, Rajeswari Lakshmanan, Jiang Zhang, Joseph A Loo
University of California-Los Angeles, Los Angeles, CA/USA

Keywords:
supercharging, conformation, charge state distributions, charged residue model, electrospray mechanism
Novel aspects:
The role of various solution additives in altering charge distributions forms a basis to explore the mechanism of electrosprayionization.
Abstract:
Introduction
Increasing charge deposited on analytes by electrospray ionization is desirable, because higher charged molecules are more
effectively dissociated by tandem MS and because they reduce analyzer m/z requirements. Supercharging ability is especiallyvaluable when examining protein-protein and protein-ligand associations where, for many non-covalent complexes, a sufficiently
broad reagent concentration window exists to elevate charge without altering stoichiometry； e.g., with the 28 subunit, 600-kDa 20S
proteasome and with 14 subunit, 800-kDa GroEL. Regardless of supercharging’s utility, the mechanism responsible for that charge
increase is under debate, as is the question of whether separate mechanisms are required to describe the charge elevation
observed from non-denaturing and denaturing solutions. Here we consider experimental observations of protein charging withregard to an ESI mechanism.
Methods
Positive ion electrospray ionization mass spectra were acquired with three different systems： a hybrid quadrupole time-of-flight（QTOF）mass spectrometer with Triwave ion mobility（IM）separator（QTOF/IM； Waters Synapt HDMS, Manchester, UK）, an
LTQ-FT Ultra mass spectrometer（Thermo Fisher Scientific）, and an LTQ ion trap mass spectrometer（Thermo Fisher）. The
nanoESI source using borosilicate glass capillaries with Au/Pd coatings（Proxeon Biosystems, Odense, Denmark）was operated at
low analyte flow conditions（50 nL/min）. Protein samples were desalted with centrifugal filter devices using 20 mM ammonium
acetate prior to analysis. The final protein concentration for ESI-MS measurements from non-denaturing solutions wasapproximately 5 uM in 20 mM ammonium acetate, pH 6.8. Reagents for altering charge included m-NBA, sulfolane, DMSO,
hexafluoroacetone, and others.
Results
Supercharging of denatured solutions has been suggested to arise from the increased surface tension imparted to late-stage ESI
droplets by low-volatility additives. Since that proposal, complicated boiling point/evaporation arguments have sometimes been
applied to rationalize or to question how surface tension would vary during an organic/water/acid-containing droplet’s lifetime.
We return to these considerations, and also to the charged residue model（CRM）, essential to justifying a relationship between
charge and surface tension.
It is notable that proponents of the CRM assert that native proteins and compact molecules ionize by the classic CRM, whereas
denatured proteins must instead be described by a modified CRM or ion evaporation. Modifications are needed to rationalize
CRM’s poor fit to denatured proteins’ predicted and observed charge distributions. Modified CRM argues that non-spherical
droplets deform without exploding, stabilized by the polymer backbone to attain charge densities beyond the limit imposed by
the solvent surface tension. However, the need to invoke“stabilization“ to rationalize the observed charge distributions ofdenatured proteins invalidates the Rayleigh limit relationship to surface tension. Notably, the results most often used to support
surface tension dependence in supercharging are obtained from denaturing solutions.
Reagents increasing or decreasing charge in electrospray ionization for native and denaturing solutions, independent ofconformational changes, can be divided into several classes and assessed for volatility, basicity, acidity, and ability to alter charge
in positive and negative ionization modes. These classes provide hints about relevant considerations in electrospray ionization
charge state distributions and to the mechanism of ionization.
Impact on charge state distributions of gas versus solution phase denaturation and gas versus solution phase charge transfer are
also considered, as is the presence of supercharging reagent adducts that localize primarily to higher charge ions.

Oral Session
Monday, 17th SeptemberRoom E

09:00 － 11:00

Session 5: Advances in Spray Ionization Techniques
Chair:Charles N McEwen（University of the Sciences, USA）

S05-1020
10:20 － 10:40

Ionization Methods Requiring as little as the vacuum of the Mass
Spectrometer to Produce Highly-Charged Ions from Surfaces or Solutions

Sarah Trimpin

Wayne State University, Detroit, MI

Keywords:
vacuum ionization, spray ionization, imaging, instrumentation, highly charged ions
Novel aspects:
Novel ionization methods at atmospheric pressure and vacuum not requiring voltage, laser, or heat to produce highly charged ionsdirectly from native, complex samples such as tissue
Abstract:
We recently introduced a new mass spectrometric method applicable to proteins which uses laser ablation of a matrix-analyte
mixture similar to atmospheric pressure MALDI but produces mass spectra nearly identical to ESI. This new technique called
laserspray ionization（LSI）（[1]）has advantages of speed of analysis, high spatial resolution for imaging, mass range extension, andimproved fragmentation common with multiply charged ions. This approach was extended to producing highly charged ions fromproteins by laser ablation using vacuum MALDI sources（[2]）. We demonstrate that proteins as large as BSA（~66 kDa）produce
ESI-like multiply charged ions from certain matrices when introduced to vacuum from AP. Crucial for the production of highly
charged ions are desolvation of the matrix from charged matrix/analyte clusters by thermal or vacuum assistance and the use of alaser is just a sophisticated means of transferring the clusters into the gas-phase.
Applying this concept, we show first examples of highly charged ions produced directly from a solid matrix-analyte material using
a vacuum MALDI source without need of a laser. Charge states up to 44 are observed for BSA. Protein, peptide, and lipid ions areobserved directly from mouse brain tissue and analyzed using IMS and MS（SYNAPT G 2 ）separations without the need of harshconditions imposed by laser ablation. Initial results for protein complexes directly from surfaces will be shown. Other uses are
rapid and automated analyses of small molecules such as drugs and body fluids including blood spots, saliva, and urine. Producing
ions under vacuum conditions in a mass spectrometer enhances sensitivity and the multiply charged ions offer the potential to usehigh performance mass analyzers for advanced structural characterization. Such a simple ionization method, requiring only thevacuum necessary for the proper functioning of the mass spectrometer, should prove useful in clinical settings and in field portable
instruments.
We show that all of the newly developed AP and vacuum methods as well as ESI produce ubiquitin ions of similar structures as
determined by IMS-MS. Further, multiply charged ESI-like ions are also produced from solvent solutions allowing us to interface
this method with a NanoAcquity UPLC for separation prior to high sensitivity mass analysis of, e.g., drugs and protein digests（[3]）.
The multiply charged ions from any of these methods produce nearly complete backbone fragmentation using electron transfer
dissociation. Applications, besides high sensitivity LC/MS, include tissue imaging and characterization of fragile ganglioside lipids
and neuropeptides directly from mouse brain tissue（[4]）.
（[1]）Inutan, E.D., Richards, A.L., Wager-Miller, J., Mackie, K., McEwen, C.N., Trimpin, S. Laserspray Ionization - A New Method forProtein Analysis Directly from Tissue at Atmospheric Pressure with Ultrahigh Mass Resolution and Electron Transfer Dissociation.
Mol. Cell. Proteomics, 10（ 2 ）： 1 -8, 2011.
（[2]）Trimpin, S., Ren, Y., Wang, B., Lietz, C.B., Richards, A.L., Marshall, D.D., Inutan, E.D. Extending the Laserspray Ionization
Concept to Produce Highly Charged Ions at High Vacuum on a Time-of-Flight Mass Analyzer. Anal. Chem. 83（14）： 5469-75, 2011.
（[3]）Wang, B., Inutan, E.D., Trimpin, S. A New Approach to High Sensitivity Liquid Chromatography-Mass Spectrometry ofPeptides using Nanoflow Solvent Assisted Inlet Ionization. J. Am. Soc. Mass Spectrom. 23（ 3 ）, 442-445, 2012.
（[4]）Richards, A.L., Lietz, C.B., Wager-Miller, J., Mackie, K., Trimpin, S. J. Lipid Res. 2012, doi：10.1194/jlr.D019711.

Oral Session
Monday, 17th SeptemberRoom E

09:00 － 11:00

Session 5: Advances in Spray Ionization Techniques
Chair:Charles N McEwen（University of the Sciences, USA）

S05-1040
10:40 － 11:00

Rapid Characterization of Chemicals in Plant via Direct Electrospray Probe
Coupling with Mass Spectrometry

Hung Su, Chu-Nian Cheng, Min-Zong Huang, Jentaie Shiea
National Sun Yat-Sen Univrsity, Kaohsiung, Taiwan

Keywords:
Direct electrospray probe；plant tissue； Steviarebaudiana
Novel aspects:
Direct electrospray probe mass spectrometry was used to rapid characterization of chemicals in plant.
Abstract:
Electrospray ionization（ESI）, an atmospheric ionization method, can directly ionize analytes in liquid and provide an effectivemeans of analyzing large biomolecules as well as small organic and inorganic compounds. In ESI, however, the sample solution
subjected to ESI/MS analysis must be carefully cleaned to prevent from capillary clog. Direct electrospray probe（DEP）has beendeveloped as a direct sampling ionization method for mass spectrometric analysis of complex solution without the need ofa
sample introduction capillary. Ions of analyte are directly generated by applying a high voltage to the probe wetted with a small
volume of solution. The DEP technique provides a number of unique analytical features including：（a）sample consumption is low
and sample switching is quick；（b）the capillary and pump are non-required； and（c）the probe is low cost and easy to beconstructed and cleaned. In this study, a modified DEP probe is successfully applied to characterize the chemicals from planttissues（e.g. leaf, stem, and root）for disease diagnosis.
In DEP, the analyte molecules are first dissolved while the extract solvent is pre-loaded onto the plant tissues. Next, analyte ions
are generated from the tip of probe via electrospray ionization. In this study, we demonstrated that the main components of planttissues were successfully determined through the DEP analysis under ambient conditions. For example, both positive and negativeions of the organic compounds such as steviolbioside, rubusoside, stevioside, dulcoside A, and rebaudioside E in the plant tissuesof Steviarebaudiana were detected directly via DEP technique. Furthermore, the distribution of organic compounds was obtainedby using DEP to directly examine different areas of the same plant tissue. For example, different molecular ions were acquired
from the green- and white-color surface of the Erythrina indica var. picta tissue. In consequence, we demonstrated that DEP
technique can be applied to directly and rapidly characterize and identify major compounds in plant tissue and its great potential
for the diagnosis of plant diseases.

Oral Session
Monday, 17th September

Main Hall

15:00 － 17:00

Session 6: Novel Approaches in Proteomics Analysis
Chair:

Roman A Zubarev（Karolinska Institutet, Sweden）

S06-1500
15:00 － 15:40[Keynote Lecture] Instant Spectral Assignment for Rapid, High-Throughput
Targeted Proteomics

Derek J Bailey, Amelia C Peterson, Christopher M Rose, MIchael S Westphall, Joshua J Coon
University of Wisconsin-Madison/Madison, Wisconsin/USA

Keywords:
Selected Reaction Monitoring, Multiple Reaction Monitoring, Targeted Proteomics, Shotgun Proteomics, Orbitrap
Novel aspects:
Use of FT-MS for large-scale targeted proteomics
Abstract:
The most widespread technique in proteomics is the shotgun method. Proteins are digested into peptides, chromatographicallyseparated, and measured by mass spectrometry（MS）. Many types of mass spectrometers are used quadrupole ion traps（QIT）,
QIT hybrids such as the Orbitrap or LTQ-FT-ICR, and quadrupole time-of-flight （Q-TOF） but the experiments from MS
measurements onward are basically the same. Eluting peptide cations have their mass-to-charge（m/z）values measured in theMS1 scan. The most abundant precursors are then selected for a series of sequential tandem MSevents（MS2）. The number ofthese events depends on the acquisition rate of the device, but generally ranges from 3 to 15. Following a second MS1 scan, a
new group of targets is selected. The process, called data-dependent acquisition（DDA）continues for the duration of thechromatographic separation. Because this method has not changed over the past fifteen years, advancements have come fromthe constant evolution of the MS hardware. Significant improvements in key Figures of Merit such as sensitivity, scan rate, massaccuracy, and resolution have evolved over this period. Constant operation generates hundreds of thousands of spectra in days.
These spectra are then mapped to peptide or protein sequence using highly evolved database search algorithms. Successful
results can be obtained within just a few days of instrument analysis, and are nothing short of spectacular： tens of thousands ofunique peptide spectral matches mapping to several thousand unique protein isoforms have become the norm.
Doubtless the shotgun approach achieves high throughput, it does, however lack sensitivity and reproducibility. Specifically,
complete coverage of specific pathways or functional groups is not typical（i.e., all 500 kinases, 1,400 transcription factors, etc.）.
Likewise, overlapping IDs in replicate experiments are low（35-60 %）. This lack of completeness and reproducibility limit thequantity and quality of biological conclusions that can be drawn from a proteomic experiment. These limitations have propelled
recent fervor in target-based methods, namely selected reaction monitoring（SRM）. SRM achieves the reproducibility that the
shotgun approach lacks and can determine absoluteabundance, but suffers from low throughput and low resolution, as it isprimarily restricted to the triple-quadrupole（QqQ）MS platform. Here we describe a new data acquisition paradigm thatharnesses the mass accuracy and speed of new generation of high resolution hybrid MS systems to develop to achieve thesensitivity and reproducibility of SRM methods with the high-throughput capacities of the shotgun technique. Our approach is to a
new targeting concept, parallel reaction monitoring（PRM）, that is uniquely enabled by the high-resolution mass analyzers. PRM
stands to remedy the extremely low throughput problem of SRM by exploiting high resolving power to readily distinguish target
product ions from contaminants. To test this, we analyzed a test-set of fourteen synthetic peptides, present in a biological matrixof yeast tryptic peptides, using either the PRM method（Q-Exactive）or traditional SRM（QqQ）. These data demonstrated lowattomole level detection limits, high measurement reproducibility, and good linearity over 5 orders-of-magnitude for PRM. PRM
analysis, on average, was significantly more sensitive than QqQ SRM, quantifying peptides over a greater number of concentration
orders-of-magnitude（3.9 versus 3.2 average orders quantified, p = 2.8E-3, n = 14）. The linearity of response between the two
methods was the same（0.93 versus 0.87 average logged adjusted % RSDs, p = 0.51,n = 14）. These data suggest that the PRM
method, with very little assay development time（about 1 hr）can target analytes in matrix with the same or better fidelity than ahighly optimized QqQ SRM method（extensively developed over several days）.

Oral Session
Monday, 17th September

Main Hall

15:00 － 17:00

Session 6: Novel Approaches in Proteomics Analysis
Chair:

Roman A Zubarev（Karolinska Institutet, Sweden）

S06-1540
15:40 － 16:00

Miniaturised Chemical Proteomics to Profile Clinically-relevant Kinase
Inhibitors in Tumour Core Biopsies

Ivo Chamrad2, Uwe Rix3, Manuela Gridling1, Katja Parapatics1, Andre C Mueller1, Alexey Stukalov1,
Giulio Superti-Furga1, Eric B Haura3, Keiryn L Bennett1

CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, 2Palacký University, Olomouc,Czech Republic, 3H. Lee Moffitt Cancer Center and Research Institute, Tampa, Florida, USA

1

Keywords:
kinase inhibitor, chemical proteomics, core biopsy, Orbitrap
Novel aspects:
The optimisation and miniaturisation of a robust method to study the kinase inhibitor profile of human tumour core biopsies
Abstract:
Specific anticancer agents have been undeniably successful in the treatment of the disease, however, several issues still remain.
Even when enormous efforts have been made to design and develop compounds affecting only given molecular nodes in cancer
circuits, strictly-selective agents are rare. The majority of novel, potential therapeutics display broader inhibitory profiles thanoriginally envisaged. Affecting unexpected targets can lead to unwanted toxicity and cause adverse side effects. Alternatively,
incidental drug promiscuity is not necessarily always harmful. Hence, for every drug, determining the specificity profile anddelineating a plausible mechanism-of-action should be an integral part of the modern drug-discovery pipeline.
Chemical proteomics is a compound-centric affinity approach that utilises immobilised drugs to isolate protein interactors fromprotein mixtures. Identification of specific protein sets is achieved via modern high-end mass spectrometry（MS）. Research in ourlaboratory has previously downscaled the affinity procedure to an input of 500µg total protein.1 The study presented here, entailed
a further miniaturisation of the protocol to 100µg - the range of material that can be obtained from a core biopsy. The possibility
to perform systematic chemical proteomics with material from core biopsies taken from patient tumours will provide newopportunities for personalised medicine.
An analogue of the broad-spectrum tyrosine kinase-inhibitor bosutinib was coupled to sepharose beads. 2 Cell lysate was
generated from the human K562 cell line（derived from chronic myelogenousleukaemia）. Drug pulldowns were performed
according to Fernbach et al. 1 except that the total quantity of input material was lowered to 100µg protein. Experimental
conditions such as protein concentration, bead bed-volume, total quantity of drug, drug concentration on the beads, incubation
time, agitation speed, elution volume, and quantity of trypsin were assessed as a multi-dimensional matrix. Samples were analysed
byLCMS on hybrid LTQ Orbitrap instruments and based on the number of kinases identified, the experimental conditions were
optimised for drug pulldowns from low quantities of protein.
In the first phase of this study, the published protocol1 was assessed with 100µg protein input. Approximately 100 protein groups（98 and 120）were unambiguously identified in two replicates, with the target kinases accounting for ~20 % of all protein
identifications. For specific interaction partners（~30 kinases）, many known major targets of bosutinib such as SRC kinases（LYN
and YES）, Bcr-Abl, CSK, BTK, GAK, EPHB 4 and PTK 2 were present. Functional annotation analysis of the proteins revealed anover-representation of gene ontology terms associated with kinase activity. These results were highly-encouraging, as lowering theprotein input by 5 -fold still resulted in the identification of a high proportion of kinases. Only 10 fewer kinases were apparent ascompared to the data obtained with 500µg protein input. Phase two of the study involved a systematic assessment of several
experimental metrics that can influence the result of the chemical proteomic experiment. To monitor the various permutations offactors investigated in optimising the protocol, normalised sequence abundance factor（NSAF）was utilised. Several blocks ofexperimental parameters were designed and evaluated, prior to selecting the ideal conditions to design the next experiments.
After several rounds, the following conditions were selected： protein concentration（0.5mg/mL）； bead volume（50µL）； total
quantity of drug（50nmol）； drug concentration（ 1 nmol/µL）； incubation time（ 2 h）； agitation speed（10r.p.m.）； acid elution
volume（250µL）； and trypsin quantity（1.25µg）. To ensure that our optimised approach was not tuned specifically for bosutinib,
the down-scaled methodology will be assessed with additional tyrosine kinase inhibitors. Ultimately, the approach will be applied
to lysates of core biopsies from tumour samples and the protein profile determined from clinically-relevant tyrosine kinase
inhibitors.
1
2

Fernbach, N., et al.（2009）J Proteome Res 8, 4753-4765
Remsing Rix, L., etal.（2009）Leukaemia 23, 477-485

Oral Session
Monday, 17th September

Main Hall

15:00 － 17:00

Session 6: Novel Approaches in Proteomics Analysis
Chair:

Roman A Zubarev（Karolinska Institutet, Sweden）

S06-1600
16:00 － 16:20

Combining ribosome profiling (ribosome captured mRNA sequencing) andmass spectrometry into an unprecedentedly comprehensive protein
identification strategy.

Gerben Menschaert1, Petra Van Damme1,2, Jeroen Crappe1, Geert Baggerman3,4, Wim Van Criekinge1

Ghent University, Ghent, Belgium, 2VIB (Flemish Institute for Biotechnology), Ghent, Belgium, 3Antwerp University CFP(Center For Proteomics), Antwerp, Belgium, 4VITO, Mol, Belgium

1

Keywords:
ribosome profiling, COFRADIC, shotgun proteomics, alternative TIS
Novel aspects:
This study combines state of the art mass spectrometry techniques（i.e. shotgun proteomics and COFRADIC）and very recently
described deep sequencing techniques（i.e. ribosome profiling： sequencing of ribosome captured mRNA）.
Abstract:
Tandem mass spectrometry（MS/MS）-based shotgun proteomics or specific COFRADIC（COmbined FRActional DIagonal
Chromatography）technologies are very effective for large-scale proteomics or chromatographically isolated peptide class
identification. Protein sequence database searching is the most commonly used technique for the identification of peptides
following the acquisition of MS/MS spectra. Although convenient for routine use, these public databases are collections of all
known and predicted proteins in a species and may not closely represent the real protein pool in a specific sample. In contrast,
mRNA-seq derived translation products give a more representative expression state of the sample under investigation. Also,
databases employed in proteomics searches are usually incomplete with respect to sequence variation information, such as single
nucleotide variations（SNVs）and RNA-splice and -editing variants. Without taking them into account, proteomic studies may failto detect novel, important protein forms. Surely, usage of mRNA-seq derived protein databases in the identification process has
several advantages（as mentioned）and will become mainstream in due time.
Ribosome profiling（the deep sequencing of ribosome-protected mRNA fragments）is a new technique developed by Ingolia et al.,that allows precise and quantitative analysis of genome-wide in vivo translation. Ribosome profiling measures expression at thelevel of translation, opening new windows on in vivo translation. Recently, Ingolia et al. also described a strategy exploiting thepropensity of harringtonine to cause ribosomes to accumulate at sites of translation initiation. Combination of the ribosome
profiling mRNA-seq data with a machine-learning algorithm can define protein products systematically. On the other hand,
Guttman et al. state that ribosome profiling has indeed provided us with a strategy for identifying ribosome occupancy on RNA,
serving as a method to distinguish between coding and non-coding transcripts. However, this first needs to be tested because
non-coding transcripts that show an association with the ribosome have not been shown to have a protein product.
To verify the potential of ribosome profiling in providing us with a true snapshot of the translational landscape, we generated a
database of translation products based on the ribosome profiling experiments from the Ingolia et al. study on mouse embryonicstem cells. Also, we in silico derived a human version of this data. Several shotgun-proteomics experiments and N-terminal
COFRADIC（isolating N-terminal peptides in the chromatography step）MS/MS result sets were searches against these compiledtranslation product databases. Extra MS experiments, trying to isolate small endogenous peptides in mouse embryo samples arestill ongoing.
We were able to validate several findings of the initial ribosome profiling data. Several alternative translation start sites wereidentified, of which many with near-cognate start codon. We could also reaffirm N-terminal truncation and elongation products.
Furthermore, ongoing experiments could possibly reveal true uORF（upstream open reading frame）and/or sORF（small ORF）
translation products.
As deep sequencing techniques are becoming more standard, inexpensive, and widespread, the ribosome profiling methodology
will in the future probably serve as an alternative to MS-based protein identification, moreover since its large dynamic range,
sensitivity, and comprehensive nature. On the other hand, enrichment techniques preceding MS experiments（such as for example
N-terminal COFRADIC）will still be indispensible to lower the complexity of the peptide mixture and enrich for selected peptideclasses. Furthermore, MS-techniques in general, provide us with true evidence of the in vivo proteins/peptides and not to forget
their post-translational modification status. Anyhow, combination of aforementioned deep sequencing and mass spectrometry
techniques will definitely become mainstream in the future in revealing translation levels.

The first two authors contributed equally to this work.

Oral Session
Monday, 17th September

Main Hall

15:00 － 17:00

Session 6: Novel Approaches in Proteomics Analysis
Chair:

Roman A Zubarev（Karolinska Institutet, Sweden）

S06-1620
16:20 － 16:40

Top-down mass spectrometry for comprehensive quantitative proteomics

Ljiljana Pasa-Tolic, Shawna M Hengel, Si Wu, Zhixin Tian, David Stenoien, Rui Zhao, Nikola Tolic,Joshua T Aldrich, Da Meng, Ronald J Moore, Errol W Robbinson
Pacific Northwest National Laboratory

Keywords:
top-down quantitative proteomics PTMs
Novel aspects:
High throughput quantitative top-down LCMS for global and targeted analyses
Abstract:
Combinatorial post-translational modifications（PTMs）, signal peptide cleavages, proteolytic processing and site mutations are allimportant biological processes that largely go undetected in traditional bottom-up proteomic analyses. While several PTMs are
successfully identified using bottom-up methods, information including stoichiometry of modifications on a single protein, orpresence of a combination of multiple modifications on a single protein isoform are impossible to infer from peptide-level data.
Because most proteins in a typical global proteomic study are not identified with 100 % sequence coverage, it is not known
whether the lacking sequence coverage is due to biologically relevant proteolytic processing events, sample preparation, or MS
duty cycle. The potential information gleaned from top-down proteomic studies, or through integration of top-down and bottomup approaches, is vast and, combined with recent improvements in MS instrumentation, is rapidly becoming an important avenue
for proteomic studies. The work presented here will discuss specific examples demonstrating why top-down analysis is preferredover traditional bottom-up studies.
Typically PTMs are not identified in global bottom-up quantitative studies, despite the fact that a large number of proteins are
known to contain biologically relevant PTMs. While top-down analyses provide this information, throughput and sensitivity havebeen limiting factors due to extensive pre-fractionation efforts required for adequate proteome coverage. To tackle thesechallenges, we have optimized commercially available, LCMS platforms for high-throughput, comprehensive and sensitive topdown quantitative analysis using an E. coli lysate. Of the 502 proteins confidently identified from a combination of ETD and HCD
MS/MS experiments searched, over half contained PTMs. This approach has been successfully applied to other systems, including
thermobacteria, cynobacteria, and Salmonella.
However, complications at both the experimental and data-analyses levels remain, particularly in the case of a large number of cooccurring PTMs and other modifications, demonstrating a need for further improvement in the top-down proteomics field. Histones
contain a large number of combinatorial modifications, providing a significant analytical challenge. While many modifications are
localized to the N-terminus, modifications span across the entire sequence emphasizing the need for top-down studies toaccurately reflect the number of relevant isoforms. To this end, we have developed a histone specific two dimensional LC-MS/MS
platform that enabled identification of over 700 histone isoforms from 7.5 µg of starting material in a single 24-hour analysis.
Bottom-up quantitative studies can provide information on several thousand proteins, however, depending on activated biologicalprocesses, data can be difficult to interpret when going from a peptide to protein abundance. For example, iTRAQ analysis of ahuman skin cell model was used to identify abundance changes and translocation in response to exposure to ionizing radiation.
The global study identified several hundred proteins changing with statistical significance. However, proteins that underwent
processing or modification as a result of radiation exposure required a more complex analysis. Filaggrin consists of several
domains that are cleaved and translocate in response to specific stimuli. Because filaggrin subunits could be considered uniquespecies, we broke down the observed peptides into the N-terminal nuclear targeting domain, internal domains or C-terminal
domains. Quantitative analysis indicated that the N-terminal domain localized to the nucleus as expected, and decreased inabundance following radiation exposure. Additionally, peptide rollup to proteins that underwent extensive radiation dependent
phosphorylation were analyzed separately. Differences in fold-change were observed when the peptides containing known
phosphorylation sites were included/excluded. These extra steps required previous knowledge of biology involved, and would nothave been required if a top-down analysis was employed. Examples featured here highlight the complexity of comparing peptide
abundance values in the context of protein abundance, and suggest that future top-down studies may be required forcomprehensive analysis of biological processes.

Oral Session
Monday, 17th September

Main Hall

15:00 － 17:00

Session 6: Novel Approaches in Proteomics Analysis
Chair:

Roman A Zubarev（Karolinska Institutet, Sweden）

S06-1640
16:40 － 17:00

In silico Compensation of Instrumental Response Fluctuations Improves
Label-free Quantification of High-Resolution Proteomics Data

Yaroslav Lyutvinskiy1, Hongqian Yang1, Dorothea Rutishauser1, Hilkka Soininen2, Roman Zubarev1
1

Karolinska Institutet, Stockholm, Sweden, 2University of Eastern Finland, Kuopio, Finland

Keywords:
label-free software accuracy Alzheimer
Novel aspects:
In silico method of instrumental response compensation improves accuracy of label-free quantification by post-acquisition dataprocessing. The generality of the technique promises its widespread application in mass-spectrometry based quantitative
methods.
Abstract:
Introduction:
In the analysis of human biopsies, particular bodily liquids, the biological variations of interest are often small（~10%）, which poses
high requirements on the accuracy of proteomics analysis, especially when it is performed by the label-free method. One of themain contributors to the inaccuracy of label-free LC/MS experiment is the variability in the instrumental response, which includes
fluctuations in the electrospray current, instrument transmission and detection sensitivity. We developed in silico method of
reducing this variation, and dramatically improve the precision of label-free proteomics analysis by post-processing.
For abundant proteins, precision of ca. 1 %（CV）is achieved in relative quantification of blood plasma proteins of ageing
population, which allowed clear gender differentiation of pooled samples and 86% accurate differentiation of individual samples.
Method:The compensation method is based on the observation that in many proteomics experiments, most of the proteome stays
unchanged and only a relatively small number of proteins（10-30 %）change their abundance as a biological response to thestimulus.
Since the fluctuations of instrumental response affect all simultaneously eluting peptides to the same degree, peptides from the“unchanged“ part of the proteome can be used as references for abundance alignment of other peptides eluting in a different
LC/MS run within the same narrow time window（ 1 min）. By statistical analysis of the multitude of simultaneously eluting
peptide species in both runs, for any given retention time the average ratio is calculated between the two runs for the“unchanged“ peptides. Thus ratio is used as a correction factor that compensates such time-varying artifacts as ESI current
fluctuations.
Implementation:
The above compensation method was implemented in Visual C# 2010 as a part of Quanti, a label-free quantification workflow.
First, MS/MS data from the whole experiment（ 2 -200 individual Orbitrap LC/MS .raw files）are merged together with elimination of
duplicates, and searched by Mascot. Then Quanti aligns retention times of peptides by minimizing the changes in the elution
order in different runs. In each LC-MS run, chromatographic peak of every Mascot-identified peptide is integrated after performing
charge and isotope deconvolution. Relative abundances of peptides for each pair of LC-MS run are corrected as described above.
Final relative abundances are inferred by statistical analysis of the matrix of pairwise comparisons for all possible pairs of LC-MS
runs.
Preliminary data:
Label-free quantification was applied to proteomic blood plasma analysis of 8 samples pooled from 218 age-matched persons
according to gender and the stage of Alzheimer’s disease（AD）（Control； Mild Cognitive Impairment, MCI； Progressive MCI；
AD）. Protein extraction and digestion was repeated three times for each sample. Each prepared peptide mixture was analyzed
twice on Orbitrap Velos with Easy-nLC（both Thermo）using 90 min LC gradient. Altogether, 1441 peptides were identified with 1 %FDR. Label-free quantification was performed by Quanti. When technical replicates were compared, the average R 2 factor
between peptide abundances was 0.98 for the linear abundance scale and 0.96 for the log 10 scale. Application of instrumental
correction improved the R 2 factor to 0.995 and 0.98 for the linear and logarithmic scales, respectively. For abundant proteins,
precision of ca. 1 %（CV）is achieved.

Significantly, measured protein abundances after correction correlated better with known biological aspects of the samples. 100%
accurate sex differentiation is achieved on pooled blood plasma samples. When the same proteins as in the pooled samples wereused for sex differentiation in individual samples, correct sex identification from a single LC/MS analysis of each sample wasachieved in 31 out of 35 cases（88% accuracy）, as opposed to 28 cases out of 35（80% accuracy）without the correction.

Oral Session
Monday, 17th SeptemberRoom A

15:00 － 17:00

Session 7: New Ionization Methods and Related Topics for the Next Generation
Chair:

Kenzo Hiraoka（University of Yamanashi, Japan）

S07-1500
15:00 － 15:40[Keynote Lecture] What is the Opposite of Pandora’s Box? Direct
Analysis, Ambient Ionization, and a New Generation of AtmosphericPressure Ion Sources.

Robert B Cody
JEOL USA, Inc.

Keywords:
DART, ambient, ionization, direct
Novel aspects:
Ambient ionization has led to development of many new ion sources. Direct analysis relies on the mass spectrometer selectivity,
but chemistry and complementary methods can provide major benefits.
Abstract:
Ion sources have changed dramatically over the decades since chemical ionization was first introduced as an alternative toelectron ionization. Game-changing developments such as FAB and plasma desorption were followed by the development of
atmospheric pressure ion sources. Two revolutionary new ion sources became widely available in the early 1990’s： ESI and MALDI
suddenly made the analysis of large biomolecules possible for the first time.
The first ambient ion sources, DART and DESI, were first publicly presented in adjacent talks at the ASMS Sanibel Conference inJanuary 2005. Both were new atmospheric pressure ion sources .The surprising aspect of what is now termed“ambient ionization“
was that it was much easier to generate ions than we thought! Mass spectrometry could now analyze samples ionized in open air
in the laboratory environment, with little or no sample preparation. This led to the development of a new generation of
atmospheric pressure ion sources based on plasma techniques like DART, spray techniques like DESI, or hybrid methods
combining multiple ionization mechanisms.
Our work with DART has led us to press the limits of direct ionization to see how far we can go in analyzing a wide range ofsimple and complex samples with minimal sample preparation. In some cases, the mass spectrometer provides enoughinformation through high resolution and/or MS/MS to identify complete unknowns, even in complex mixtures.“Fingerprinting“ ofcomplex mixtures by DART has been used to identify biological species and to characterize commercial products.
However, no single method is ever sufficient to solve all analytical problems by itself. One must appreciate the limitations of atechnique as well as its advantages and try to decide where improvements can be made. Taking advantage of simple chemical“tricks“ such as in-situ derivatization, rapid sample cleanup methods, and in-situ H/D exchange, we have been able to ionize,
and obtain more information for, a larger set of compounds than by DART alone. A better understanding of the ionization
mechanism has allowed us to control the selectivity of DART for some analyses. After trying to find out how far we can go to
analyze complex samples without chromatography, we found that reintroducing separations in combination with ambient
ionization can provide some unique benefits.
A recent example has been the use of DART to identify new designer drugs that are suddenly appearing on the market inattempts to circumvent the laws governing drugs of abuse. In some cases, we have been able to identify specific designer drugs in
so-called“herbal incense“ or“herbal smoking mixtures“ by DART alone. Exact mass measurements and isotopic abundances incombination with fragmentation may suffice for many known target compounds. However, complementary techniques including
HPLC and NMR are needed to separate isomers and conclusively determine structure for new drugs for which standards are notavailable.

Oral Session
Monday, 17th SeptemberRoom A

15:00 － 17:00

Session 7: New Ionization Methods and Related Topics for the Next Generation
Chair:

Kenzo Hiraoka（University of Yamanashi, Japan）

S07-1540
15:40 － 16:00

Temperature Dependence of Proton Transfer for Biomolecular Ions with
Electrospray Ionization

Shinji Nonose, Kazuki Yamashita, Ayako Sudo, Keishi Machida, Kanako Yokoyama, Minami Kawashima,
Takashige Mori, Yuto Ohshima
Yokohama City University, Yokohama, Japan

Keywords:
temperature dependence, proton transfer
Novel aspects:
Temperature dependence of proton transfer for isolated multiply-charged protein and peptide ions with gaseous molecules wasinvestigated. Dramatic change was observed for distribution of product ions and absolute reaction rate.
Abstract:
The structures and reactions of gas-phase biological molecules bridge the gap between the gas phase and the real living system.
In order to investigate the intra- and intermolecular interactions, nonvolatile molecules should be introduced into the gas phase asions. These studies are expected to provide a deeper understanding of the intra- and intermolecular interactions that determine
the conformations. In the present work, temperature dependence ofproton transfer for isolated multiply-charged protein andpeptide ions with gaseous molecules of large proton affinity（PA）was investigated with mass spectrometry, which elucidates thesynchronized processes in biomolecular systems.
A home-made tandem mass spectrometer with electrospray ionization（ESI）was used for measurements. Multiply-charged
protein and peptide ions were produced by ESI of a dilute solution of proteins in methanol-water mixture including acetic acid.
We choose lysozyme, cytochrome c, myoglobin, ubiquitin, insulin, hemoglobin and albumin as sample proteins, and choose
angiotensin I, angiotensin II, bradykinin, substance P, somatostatin, neurotensin, melittin, insulin chain B, ACTH 1 -24, RKRARKE, KKK,KKKK and KKKKK as sample peptides. The ions produced by ESI are admitted into the vacuum chamber through stainless capillary.
The charge-selected protein and peptide ions emerging from a quadrupole mass spectrometer are admitted into a collision cellwith octapole ion trap. The collision cell is filled with He including gaseous molecules of large PA. We choose 1 -propylamine,
1 -butylamine, 1 -pentylamine, pyridine, tert-butylamine, diethylamine, dipropylamine, 2,6-dimethylpyridine, or triethylamine astarget molecules. Temperature dependence of reaction rate and branching fractions for proton transfer from multiply-charged
protein ions to the target molecules was measured, by changing temperature of collision cell. The parent and product ions are
mass-analyzed by a time-of-flight mass spectrometer equipped with reflectron.
Proton transfer from the protein and peptide ions to the target molecules was occurred by collisions in the cell. Absolute reaction
rate for proton transfer was estimated with intensity of ions in the mass spectra. In any proteins, the reaction rate increased
rapidly with increasing number of the charges. By changing temperature of collision cell in region from 280 to 470 K, temperaturedependence of reaction rate and branching fractions for proton transfer from multiply-charged protein and peptide ions to target
molecules was measured. Dramatic change was observed for distribution of product ions and reaction rate. These results would
correlate with conformation change of protein and peptide ions with change of temperature, which originates in self-solvation of
the proton by hydrophilic residues in polypeptide chains, delocalization of charges with self-solvation, and Coulomb interaction
between charges.

Oral Session
Monday, 17th SeptemberRoom A

15:00 － 17:00

Session 7: New Ionization Methods and Related Topics for the Next Generation
Chair:

Kenzo Hiraoka（University of Yamanashi, Japan）

S07-1600
16:00 － 16:20

Design and Development of A Novel Nuclear Magnetic Resonance
Detection for the Mass-selected Gas-Phase Ions by“Magnetic ResonanceAcceleration“ Technique

Kiyokazu Fuke1,2, Masahide Tona1, Akimasa Fujihara2, Haruki Ishikawa3
1

Kobe University, Kobe, Japan, 2Osaka Prefecture University, Osaka, Japan, 3Kitasato University, Kanagawa, Japan

Keywords:
NMR, mass-selected ion, ICR
Novel aspects:
This work is the first arttempt to detect nuclear magnetic resonace for mass-selected gas-phase molecular ions with a mass
spectroscopic sensitivity.
Abstract:
Nuclear Magnetic Resonance（NMR）technique is a powerful tool to study the physical and chemical properties of materials inwide area. However, this technique is limited for the materials in condensed phase. Although NMR is also highly expected to use
for mass-selected gas-phase ions in both fundamental and applied sciences, the method to extend to the gas-phase ions is notreported yet. In the present paper, we report a principle of the detection of NMR for the gas phase ions in a gradient magnetic
field based on a“magnetic resonance acceleration“ technique and describe the design and the construction of an apparatus,
which we are developing. This technique is a new extension of a molecular-beam magnetic resonance method and is readily
coupled with an ion cyclotron resonance mass spectroscopy（ICR）. Thus, in near furture, it may allow us to use for chemical
analysis of mass-selected ions such as an ICR-NMR. We also present the experimental techniques and the results on the formationand manipulation of ultra cold ion packets in a strong magnetic field, which are the key techniques to detect NMR signal using thepresent method.

Oral Session
Monday, 17th SeptemberRoom A

15:00 － 17:00

Session 7: New Ionization Methods and Related Topics for the Next Generation
Chair:

Kenzo Hiraoka（University of Yamanashi, Japan）

S07-1620
16:20 － 16:40

New prospects for post-ionization mass spectrometry using ultrafast laser
pulsesAlessia Longobardo2, Alisdair Macpherson2, John Vickerman1, Nick Lockyer1,2Manchester Interdisciplinary Biocentre, University of Manchester, Manchester, UK, 2Photon Science Institute, Universityof Manchester, Manchester, UK

1

Keywords:
Sputtered neutral mass spectrometry, photofragmentation, photoionization.
Novel aspects:
There are very few studies of molecular photoionization using femtosecond lasers in the IR wavelength range. Systematic study ofthis type is required to optimise post-ionization techniques.
Abstract:
Mass spectrometric techniques including secondary ion mass spectrometry and matrix assisted laser desorption ionisation havebeen very successfully applied in applications involving surface chemical analysis and imaging. Powerful though these approaches
are, they rely on the detection of analytes ionized as a direct consequence of the desorption process. However, with very few
exceptions, the dominant proportion of desorbed analyte species is neutral. The issue of enhanced ionization efficiency remains
central to further developments in performance.
With regard to secondary ion mass spectrometry（SIMS）, the introduction over the last decade polyatomic and cluster ion
sources have improved the molecular sensitivity significantly, opening up new areas of application, particularly in the biosciences.
However, the fraction of ejected species that are ionized in the SIMS process remains very small（typically 10-7-10-3）and is subject
to large matrix ionization effects which limit routine quantification. Detecting the secondary neutral flux by post-ionizing analytes
subsequent to desorption therefore offers potentially further increases in sensitivity and in ease of quantification. This isparticularly important in imaging applications.
Laser post-ionization of sputtered neutrals has been very successfully applied for elemental analytes. For molecules the situationis complicated by photofragmentation and a detailed understanding of the ionization-dissociation characteristics of analytes isrequired to optimize the experimental parameters［ 1 ］. Recent work has shown that using femtosecond lasers with mid-IR
wavelengths and/or ultrahigh intensity can overcome the traditional trade-off between molecular ionization efficiency andfragmentation［ 2 ］.
Here we report on the efficiency of molecular ion and fragment ion production of a range of molecules as a function of laser
wavelength and intensity. A clear transition in behaviour is observed favouring molecular ion production. This will be discussed in
the context of the underlying mechanisms.
［ 1 ］N.P. Lockyer“Laser Post-Ionisation for Elemental and Molecular Surface Analysis“, Ed by J.C. Vickerman and D.Briggs, Surface Spectra（Manchester）and IM Publications（Chichester）（in press）［ 2 ］D.Willingham, A. Kucher and N. Winograd,“Strong-field ionization of sputtered molecules for biomolecular imaging“, Chem.
Phys. Lett., 468,264（2009）.

Oral Session
Monday, 17th SeptemberRoom A

15:00 － 17:00

Session 7: New Ionization Methods and Related Topics for the Next Generation
Chair:

Kenzo Hiraoka（University of Yamanashi, Japan）

S07-1640
16:40 － 17:00

Desorption/ionization induced by neutral clusters as matrix-free, soft, andefficient ion source for ion-trap mass spectrometry of biomolecules

Markus Baur1, Bong-Jun Lee1, Christoph R Gebhardt2, Michael Durr1
1

University of Applied Sciences Esslingen, Esslingen, Germany, 2Bruker Daltonik GmbH, Bremen, Germany

Keywords:
cluster, desorption/ionization, matrix-free, oligopeptides, ionization efficiency
Novel aspects:
Desorption/ionization induced by neutral clusters is a soft and matrix-free ionization source with high absolute ionization
efficiency. Combination with ion-trap MS allows for high sensitivity and batch-type analysis of biomolecules.
Abstract:
Desorption and ionization induced by neutral clusters（DINeC）can be employed as a very soft and matrix-free method fortransferring surface-adsorbed biomolecules into the gas phase. Using neutral clusters with polar constituents such as SO2 or H2O,
the method makes use of the dipole moment of the cluster molecules which allows both for solvation and charge transfer
processes in the cluster. Furthermore, shattering of the cluster during its impact on the surface leads to a rapid redistribution of
the system’s energy and an efficient cooling of the desorbed molecules. Thus the impacting cluster provides not only the energy
for the desorption process but also serves as a transient matrix. As a consequence, desorption and ionization of oligopeptides
and smaller proteins proceeds without any fragmentation of the biomolecules at comparably low energies of the impacting
clusters［ 1 ］.
In combination with ion trap mass spectrometry, desorption and ionization induced by neutral cluster impact can be utilized forsoft and matrix-free, batch-type analysis of biosamples. Especially when the cluster beam is produced by a pulsed nozzle withpulse duration in the sub-millisecond regime, all ions generated during one pulse can be collected in the ion trap. In combination
with an absolute ionization efficiency of the process on the percent level, this leads to a very low consumption of analyte material.
E.g., for oligopeptides such as angiotensin II or bradykinin, the ionization efficiency was determined to be 3 to 4 % and femtomol
sensitivity was achieved in the case of these oligopeptides. Furthermore, as no primary ions but neutral molecular clusters areused for the desorption and ionization process, the background intensity in the spectra is comparably low.
Due to the soft nature of the cluster-induced desorption process, the majority of the peaks in the initial spectrum can be directly
associated with intact sample molecules； in combination with full MS n capabilities, structural analysis and straight-forward
spectrum analysis is possible as illustrated for a set of rhodamine dyes.
The presented experimental setup is relatively simple and allows for easy combination of a DINeC source with standard mass
spectrometers； the process can be efficiently applied for a wide range of surface concentrations and configurations, i.e. frommicrometer-thick films down to submonolayer surface concentration of biomolecules［ 2 ］. Samples are prepared by means of
directly drop-casting the respective solution on a millimeter-size spot； neither special sample preparation nor addition of matrix isneeded. Multiple sample arrays and low sampling time then allows for batch-type analysis.
［ 1 ］C. R. Gebhardt, A. Tomsic, H. Schroder, M. Durr, and K.L. Kompa, Angew. Chem. Int. Ed. 48, 4162（2009）.
［ 2 ］M. Baur, B.-J. Lee, C. R. Gebhardt, and M. Durr, Appl. Phys. Lett. 99, 234103（2011）.

Oral Session
Monday, 17th SeptemberRoom B-1

15:00 － 17:00

Session 8: Collision Dynamics and Spectroscopy Using Ion Storage Rings and Traps
Chair:

Haruo Shiromaru（Tokyo Metropolitan University, Japan）

S08-1500
15:00 － 15:40[Keynote Lecture] Spectroscopy of firefly luciferin and oxyluciferin anions
in vacuo. Color tuning by a micro-environment?

Steen Brøndsted Nielsen

Aarhus University, Aarhus, Denmark

Keywords:
Ion spectroscopy, micro-environment, fireflies, luciferin
Novel aspects:
First gas-phase absorption spectra of luciferin and oxylucerin anions isolated in vacuo and the role of single water molecules
Abstract:
Luciferin is the substrate for the enzymatic reaction responsible for the characteristic yellow light emission from fireflies. In
aqueous solution at physiological pH, the molecule is present in its anionic form. It undergoes an Mg2+- mediated reaction with
ATP in the active site of the luciferase enzyme. The resulting luciferyl adenylate compound reacts with O 2 to give the productanion, oxyluciferin, in a singlet excited state, which emits a photon of light as it relaxes to the ground state. Even though different
species of bioluminescent beetles all use the same luciferin, the emission spectrum varies greatly, e.g., the light emitted from thefirefly Photuris pennsylvanica is green while that emitted by the railroad worm Phrixotrix hirtus is red. The most studied species,
the North American firefly P. pyralis, produces a yellow-green light. The reason for this variability is up for much current debate. To
better understand the bioluminescence phenomenon and environmental influences, my group has carried out gas-phase
spectroscopy experiments on the bare luciferin and oxyluciferin anions to establish their electronic properties when isolated invacuo. We have also studied the impact of single water molecules. Absorption spectra were obtained at the electrostatic ion
storage ring in Aarhus and a sector instrument where ionic dissociation is monitored on a long time scale（up to milliseconds）andon a short time scale（few microseconds）, respectively. I will discuss the implications of our gas-phase results on theunderstanding of firefly bioluminescence. Finally, but not least, our data serve to benchmark theoretical models used to calculate
excited states.

Oral Session
Monday, 17th SeptemberRoom B-1

15:00 － 17:00

Session 8: Collision Dynamics and Spectroscopy Using Ion Storage Rings and Traps
Chair:

Haruo Shiromaru（Tokyo Metropolitan University, Japan）

S08-1540
15:40 － 16:00

Electronic and photonic collision experiments on molecular ions with anelectrostatic storage ring at KEK

Tetsumi Tanabe1,3, Manabu Saito2, Koji Noda3, Evgeni B Starikov4
1
3

High Energy Accelerator Research Organization (KEK), Tsukuba, Japan, 2Kyoto Prefectural University, Kyoto, Japan,
National Institute of Radiological Sciences, Chiba, Japan, 4Chalmers University of Technology, Gothenburg, Sweden

Keywords:
storage ring, electron-ion collisions, biomolecule, photodissociation, interconversion
Novel aspects:
Electron-capture dissociation of S-S bonded peptide cations at high energies. Interconversion of tautomeric anions in vacuo.
Abstract:
The electrostatic storage ring at KEK is equipped with an electrospray ion source, an ion-trap, a merged electron beam device［ 1 ］
and an OPO laser（wavelength： 410-600 nm）. The ring is also characterized by its ultra-high vacuum（10 -11 Torr）, which realized
long beam lifetimes resulting from less frequent collisions of stored ion beams with residual gas molecules. This makes it possible
to perform electronic and photonic collision experiments on various molecular ions including biomolecules in an extreme
environment. Here, we introduce some of the recent experimental studies using this ring.
It is well known that the electron-capture dissociation（ECD）is efficient for thermal electrons and the cross sections rapidly
decrease with an increase of electron energies. Meanwhile, the ECD rate for peptide cations was also found to be significant forhigh energy（ 3 -13 eV）electrons and this phenomenon is called ’hot electron capture dissociation’（HECD）［ 2 ］. Still, HECD
reported so far are for peptides without disulfide（S-S）linkage［ 3 ］. Now we report on collisions of high-energy electrons withvarious protonated peptide monocations with disulfide bonds. Resonant neutral particle emissions at the energies of 6 - 7 eVwere observed, as well as a rise towards zero-energy, which are typical electron-capture dissociation profiles. The presence of
disulfide bonds tends to enhance the resonant bump heights. Chemical nature of the amino-acid residues adjacent to cysteines
appears to correlate with the bump strength［ 4 ］.
Fluorescein and its derivatives are the most widely recognized dyes owing to their unique bright fluorescence. The monoanions
are considered to adopt two basically possible tautomeric forms, i. e., the monoanionic carboxylate（MAC）and the monoanionicphenolate（MAF）forms. Whereas the photoabsorption properties of MAC have been extensively studied, there is a dearth of
information regarding MAF. The photodissociation of fluorescein monoanions was studied by varying the storage times of the ionsin the ion trap and the electrostatic storage ring, before laser irradiation. The photodissociation neutral spectra as a function oftime consist of components with short and long lifetimes, and vary in a manner dependent on the storage time as well as the laser
wavelength. By comparing the neutral yields as a function of wavelength obtained herein with the photoabsorption spectra offluorescein reported recently for the solution and gas phases, it was deduced that the spectra originated from various tautomersof fluorescein monoanions. Moreover, interconversion of these tautomers occurs during long-term storage in the storage ring invacuo. The wavelength spectra of the converted ions depend strongly on the storage time in the ring. In addition, we have also
performed detailed semiempirical quantum-chemical computations, to try revealing what kind of monoanion tautomers we are
dealing with［ 5 ］.
References［ 1 ］T. Tanabe, K. Noda, Nucl. Instrum. Methods A 496, 233-237（2003）.
［ 2 ］F. Kjeldsen, K. F. Haselmann, B. A. Budnik, F.Jensen, R. A. Zubarev, Chem. Phys. Lett. 356, 201-206（2002）.
［ 3 ］T. Tanabe, K. Noda, M. Saito, S. Lee, Y. Ito, H. Takagi, Phys. Rev. Lett. 90, 193201（2003）.
［ 4 ］T. Tanabe, K. Noda, S. Miyagi, N. Kurita, S. Tanaka, J. Setzler, W. Wenzel, E. B. Starikov, G. Cuniberti, Chem. Phys. Lett. 504, 8387（2011）.
［ 5 ］T.Tanabe, M. Saito, K. Noda, E. B.Starikov, Eur. Phys. J. D（2012）in press.

Oral Session
Monday, 17th SeptemberRoom B-1

15:00 － 17:00

Session 8: Collision Dynamics and Spectroscopy Using Ion Storage Rings and Traps
Chair:

Haruo Shiromaru（Tokyo Metropolitan University, Japan）

S08-1600
16:00 － 16:20

Cold chemical reactions between sympathetically cooled molecular ionsand slow polar molecules

Kunihiro OKADA1, Takahiro FURUKAWA1, Masanari ICHIKAWA1, Michiharu WADA2, Hans SCHUESSLER3
1

Sophia University, Tokyo, Japan, 2RIKEN, Saitama, Japan, 3Texas A&M University, Texas, USA

Keywords:
ion trap, ion Coulomb crystal, Stark velocity filter, ion-molecule reaction, polar molecules
Novel aspects:
We developed a novel method to directly measure cold ion-polar molecule reactions by using a Stark velocity filter and a
sympathetic laser-cooling method, and applied to the measurement.
Abstract:
During the recent decades several techniques for producing cold atoms, molecules, and their ions have been developed, and they
are now being applied to the study of cold atomic and molecular processes, such as cold ion-molecule reactions. In fact, amearsurement of the reaction rate between a laser-cooled Ca+ Coulomb crystal and slow CH3CF molecules, produced by a Stark
velocity filter［ 1 ］, was demonstrated at about 1 K［ 2 ］. The method can be extended to reaction rate measurements of cold
molecular ion-polar molecule reactions, which play an important role in the chemical evolution of dark interstellar clouds［ 3 ］.
Here we report on this extension and describe our apparatus and results to directly measure cold molecular ion-polar molecule
reactions. The setup consists of a Stark velocity filter and a detection vacuum chamber enclosing a cryogenic linear rf ion trap. We
have produced slow ND 3 , CH 2 O, and CH 3 CN beams by using the Stark velocity filter. A time-of-flight method was applied todetermine the velocity distribution of these slow polar beams. The typical peak velocity is 30 - 40m/s, and the number density ofthe polar molecules is determined to be n = 10 4 -10 6 cm -3 in the ion trapping region. Both these quantities are importantparameters to determine the reaction rate constant of the ion-molecule reactions of interest. For preparing the cold molecularions, sympathetic cooling by laser-cooled Ca + ions is applied in the cryogenic linear trap. In doing so we have successfully
produced various Ca+ Coulomb crystals including two-species Coulomb crystals, and in addition characterized them by molecular
dynamics simulations［4, 5 ］. Recently, cold CaH+, N2 + and N2H+ ions embedded in a Ca+ Coulomb crystal have already been
produced in our experiments. Before starting the reaction experiment with sympathetically cooled molecular ions, the reaction
rate measurements between a Ca+ Coulomb crystal and slow polar molecules were performed to check the reactivity of the slow
polar molecules with the Ca+ Coulomb crystal itself. As a result, the reaction rates are of the order of 10-6- 10-5 s-1, which are very
slow compared to typical reaction rates of molecular ion-polar molecule reactions at very low temperatures. This fact shows that
Ca + is well suited as a coolant when sympathetically cooling molecular ions, which are targeted for the reaction-rate
measurements. Finally, we have performed initial reaction rate measurements between some of the above sympathetically cooled
molecular ions and velocity-selected CH3CN molecules. Preliminary results will be presented and discussed.
［ 1 ］S. A. Rangwala et al., Phys. Rev. A67, 043406（2003）.
［ 2 ］S.Willitsch et al., Phys. Rev. Lett. 100, 043203（2008）.
［ 3 ］V. Wakelam et al., Space Sci. Rev. 156, 13-72（2010）.
［ 4 ］K. Okada et al., Phys. Rev. A81, 013420（2010）.
［ 5 ］N. Kimura et al., Phys. Rev. A83, 033422（2011）.

Oral Session
Monday, 17th SeptemberRoom B-1

15:00 － 17:00

Session 8: Collision Dynamics and Spectroscopy Using Ion Storage Rings and Traps
Chair:

Haruo Shiromaru（Tokyo Metropolitan University, Japan）

S08-1620
16:20 － 16:40

Development of a portable electrostatic ion storage ring in TMU

Jun Matsumoto, Haruo Shiromaru

Tokyo Metropolitan University, Tokyo, Japan

Keywords:
Electrostatic ion storage ring, large molecular ions
Novel aspects:
A portable electrostatic ion storage ring is being developed. It complements one-pass experiment apparatus and the conventional
electrostatic ion storage rings in terms of an observation time range.
Abstract:
An electrostatic ion storage ring is a powerful device for investigation of slow reactions such as meta-stable decay and radiative
cooling of large molecular ions including clusters and bio-molecules. We are developing a portable electrostatic ion storage ring
（μE-ring）reducing ten times from the existing ring at Tokyo Metropolitan University（TMU）, keeping the basic idea for beam
manipulation unchanged. That is, the μE-ring consists of two 160 degree deflectors, four 10 degree deflectors and four focusing
and defocusing electrostatic quadrupole doublets. It will store positive or negative ions with the energy range from several to 20
keV with the storage time up to several seconds under expected base pressure. Compact size of the ring has great advantages. A
short period of revolution provides higher time resolution of observations to be made at the straight section of the ring. The
portability enables us to bring it to various ion beam facilities. In addition, use of the ring in a synchrotron radiation facility willdefinitely expand the field explored by the ion-storage ring.
In the present study, first, we reconsidered transport property of an ion beam in order to check the effect of the miniaturization.
Based on the equation of motion of ions in the ring, transfer matrices were produced, and the Twiss parameters were calculated.
Then the stable conditions of betatron oscillations were derived by changing voltages of the quadrupole doublets. Second, actual
electric fields generated by the electrodes were calculated by a simulation code for electric fields and the trajectories of charged
particles, SIMION. The simulated ion beam trajectories show considerable deviation from ideal electric field. Therefore, the ion
optics was refined with field clamps.
The dimensions of each electrode were determined with taking care of accuracies of machining and assemblage due to theminiaturization. In the final design, the circumference of an ion beam trajectory is about 0.8 m, and all electrodes are mounted ona single rectangle plate of 480 mm x 200 mm, for precise alignment of the electrodes and efficient bake-out of the ring. Now, thering is under mounting operation. In the presentation, we will show the detailed design and the status of the μE-ring.

Oral Session
Monday, 17th SeptemberRoom B-1

15:00 － 17:00

Session 8: Collision Dynamics and Spectroscopy Using Ion Storage Rings and Traps
Chair:

Haruo Shiromaru（Tokyo Metropolitan University, Japan）

S08-1640
16:40 － 17:00

Investigation of a novel apparatus which combines ion trap - laser coolingtechnique with ICP-MS for trace isotope analysis

Masanori Kitaoka, Yuta Yamamoto, Takuma Yoshida, Kyunghun Jung, Shuichi Hasegawa
The University of Tokyo, Tokyo, Japan

Keywords:
Isotope Analysis, Ion Trap, Laser Cooling, ICP-MS, Calcium
Novel aspects:
A novel apparatus which combines ion trap - laser cooling technique with ICP-MS is developed to be applied to trace isotope
analysis.
Abstract:
Analysis of rare isotopes has applications in biomedicine, archeology and planetary science［ 1 ］. For instance, calcium has six
stable isotopes, 40,42,43,44,46,48Ca（abundance：96.9%, 0.647%, 0.135%, 2.09%, 0.004%, 0.187%, respectively）and a long-lived radioactive
ultra-trace isotope, 41 Ca（ 41 Ca/Ca = 10 -15 - 10 -14 ）. Today only AMS（Accelerator Mass Spectrometry）is utilized for practical
purposes. In general, isobaric interference is an unavoidable issue for mass spectrometry. Therefore, collision/reaction cell, which
is utilized to remove the isobaric ions by means of gas collisions or chemical reactions, has to be built in ICP-MS（Inductively
Coupled Plasma Mass Spectrometry）. However, the chemical reactions in a cell produce other isobaric ions. In the case of 41Ca+,
for instance, 40 CaH + is produced when NH 3 is introduced to the cell for removing 40 Ar + or 40 ArH + . Alternatively, laser-based
methods, such as RIMS（Resonance Ionization Mass Spectrometry）and ATTA（Atom Trap Trace Analysis）, have been developed
for laboratory application［ 2 ］. We have developed a novel method which combines ion trap - laser cooling technique［ 3 - 5 ］
with ICP-MS. Before using 41 Ca, we will demonstrate the principle of the method and evaluate the performance of ourexperimental system with the stable isotopes of Ca.
Our experimental system consists of ICP-MS, ion transport system, linear ion trap, laser system and observation system. Liquidsample is continuously injected to the ICP-MS and ionized by the Ar+ plasma. Ions are transported through the collision/reaction
cell and the quadruple mass separator. Mass-selected ions are extracted to the transport system, deflected 90 degrees anddecelerated by collisions with buffer gas. Then the ions are loaded to a linear ion trap and laser-cooled. We employ External
Cavity Diode Laser（ECDL）systems for generating 397 nm and 866 nm wavelengths to laser-cool Ca+ . To observe Ca + , Laser
Induced Fluorescence（LIF）is collected by an objective lens and projected to an Intensified Charge-Coupled Device（ICCD）.
Isotope selective observation can be achieved by means of the isotope shifts of the transition wavelengths.
We investigated the transport and trapping efficiency as functions of the flow rate of cell gas, the buffer gas pressure and theisotope species. We found that the cell gas worked not only for removing interference ions, but also improving the beam quality.
Therefore, there was an optimum flow rate to maximize the number of ions extracted from ICP-MS. The dependence on the buffer
gas pressure was investigated between 10-6 and 10-3 Torr. The loading and loss rate of trapped ions increased as the buffer gas
pressure increased and 10-4 Torr was the optimum pressure to maximize the LIF. After the optimization of these parameters, all thestable isotopes of Ca including 46 Ca + , whose abundance is only 0.004 % , could be observed. To observe 43 Ca + , the hyperfine
structure should be considered because of its nuclear spin. To create closed cycle, two 397 nm lasers were employed and a
relatively high power 866 nm laser was utilized to excite the metastable levels. We will discuss the details of these properties of
our new apparatus and the observation of all the stable isotopes of Ca.
References［ 1 ］D. Elmore et al., Nucl. Instrum. Methods Phys. Res., Sect B52, 531（1990）.
［ 2 ］I. D. Moore et al., Phys. Rev. Lett.92, 153002（2004）.
［ 3 ］S. Hasegawa et al., J. Nucl. Sci. Tech.43, 300（2006）.
［ 4 ］Y. Hashimoto et al., Spectrochimica Acta B 63, 645（2008）.
［ 5 ］Y. Hashimoto et al., Int. J. Mass Spectrom.279, 163（2009）.

Oral Session
Monday, 17th SeptemberRoom D

15:00 － 17:00

Session 9: Imaging-II
Chair:Jiro Matsuo（Kyoto University, Japan）

S09-1500
15:00 － 15:40[Keynote Lecture] Nanoscale Chemical Imaging of Biomaterials with
Cluster SIMS

Nicholas Winograd
Penn State University

Keywords:
C60, Argon clusters, imaging, single cells, depth profiling
Novel aspects:
Imaging with mass spectrometry with 250 nm spacial resolution and 5 nm depth resolution. Metabolite distributions in single cells.
Abstract:
Bombardment of molecular solids with polyatomic projectiles allows interrogation of the sample with reduced chemical damage
accumulation. Hence, it is now possible to perform depth profiling experiments with a depth resolution of less than 5 nm. In ourhands, the projectiles of choice are C60 and argon gas cluster ions such as Ar2500. To retain molecular information during beaminduced erosion, any damage accumulation must be removed at least as rapidly as it is formed. Here we discuss a number offundamental descriptions associated with molecular depth profiling. These descriptions, which include both analytical models
valuable in parameterizing the acquired signals and a molecular dynamics approach important for visualizing the action on amolecular level, point towards experimental conditions that optimize the quality of a depth profile. For example, the size andkinetic energy of the polyatomic projectile, the angle of incidence and the temperature all have significant influence on whether
the important molecular ion signals are retained. Atomic force microscopy（AFM）is shown to be an essential technique forquantitative characterization of any molecular profile.
An important application of this mode of mass spectrometry is to provide chemical information for a wide variety of molecules inbiological tissue and single cells. Although early attempts at implementing this application focused on low molecular weightfragment ions, polyatomic primary ion sources have opened detection schemes to a much wider range of molecules withmolecular weights extending to greater than 1000 Da. In this regard, a number of workers have reported the distribution of drug
molecules and of metabolites under various conditions. Here we discuss a number of challenges facing this field given the newmeasurement paradigms. Very different sample preparation measurement considerations emerge as the desired spatial resolution
approaches 1 micron. At values above 1 micron, freeze drying methods appear to be successful and there are enough moleculesin the probe area to detect higher mass species directly. For example, we have detected the molecular ion of more than 50
different lipids in brain tissue and in a lawn of cells, using ms/ms techniques to assign structure. Characterization of single cells
becomes much more challenging, however, since there are many fewer molecules available for detection, and sample
pretreatment to enhance ionization can move molecules around, reducing the effective lateral resolution. For these systems,
preparation of frozen hydrated samples appears to be the best way to avoid artifacts. Moreover, since polyatomic projectiles now
allow molecular depth profiling, use of frozen hydrated samples is the only way to preserve 3 -dimensional structure of cells.
Currently, using a conventional high performance imaging TOF-SIMS system, the sensitivity is on the limit of being able to detect
the higher mass molecular ions. Several examples will be given to illustrate each of the above issues. The prospects for improving
the sensitivity of this type of imaging will also be discussed.

Oral Session
Monday, 17th SeptemberRoom D

15:00 － 17:00

Session 9: Imaging-II
Chair:Jiro Matsuo（Kyoto University, Japan）

S09-1540
15:40 － 16:00

TOF-SIMS imaging analysis of atherosclerotic aortic sinuses and gastriccancer tissues

Eun-Soo Lee1,2, Ji-Won Park1,2, Se-Hwa Kim1, Hyun Kyong Shon1, Sohee Yoon1, JuYeon Oh1,
Tae Geol Lee1,2, Hark Kyun Kim3, Daehee Hwang4, DaeWon Moon1,2

Center for Nano-Bio Technology, Korea Research Institute of Standards and Science, Daejeon, 305-340, Korea,
Department of Nano and Bio Surface Science, University of Science and Technology, Daejeon, 305-350, Korea,
3
National Cancer Center, Goyang, Republic of Korea, 4School of Interdisciplinary Bioscience and Bioengineering,
POSTECH, Pohang, Republic of Korea
1
2

Keywords:
TOF-SIMS, Atherosclerosis, Gastric Cancer, Spectroscopic Mass Imaging, Ar gas cluster ion beam
Novel aspects:
1 ）. TOF-SIMS imaging applications to biomedical tissues such as atherosclerotic aortic sinuses and to gastric cancer tissues 2 ）Development of SIMS/MALDI microscopic mass imaging system for high throughput imaging.
Abstract:
Time-of-flight secondary ion mass spectrometry（TOF-SIMS）has been a useful tool to profile secondary ions from the near surface
region of specimens with its high molecular specificity and submicrometer spatial resolution. We have been trying to develop
TOF-SIMS into a molecular histology tool, which can be a practically useful biomedical diagnostic for clinical applications in
cardiovascular and oncology areas. Summaries of TOF-SIMS applications to map the distribution of cholesterol ester inatherosclerotic aortic sinuses and to discriminate gastric cancer tissues from normal tissues with identification of biomarker
candidates associated with the amino acid metabolism
Changes in cholesterol ester（CE）content regulate the progression of atherosclerosis. However, the spatial dynamics of CE
subsets and their quantitative changes during lesion progression are not well understood due to a lack of appropriate imaging
techniques. In this study, we developed an imaging based analysis method to map the distribution of CE subsets using ToF-SIMS.
Serial sections of atherosclerotic aortic sinuses from apolipoprotein E knock-out mice（n=15）fed a 0.15 % high-fat diet for 1220
weeks were examined by ToF-SIMS. We found that the ratio of cholesteryl palmitate（Ch-PA）to cholesteryl oleate（Ch-OA）
increased by approximately 99%（p=0.02）as atherosclerosis progressed, whereas the ratios of cholesteryl linoleate（p=0.09）andcholesteryl stearate（p=0.22）to Ch-OA did not change significantly. In advanced atherosclerotic plaques, in situ cell death assays
showed that local Ch-PA densities were highly correlated with an increase in the number of apoptotic cells. These results suggestthat increased Ch-PA may contribute to the formation of a necrotic core by increasing cell death. Our results indicate that theregional ratio of CEs as measured by ToF-SIMS might be a valuable new marker of atherosclerotic progression.
The TOF-SIMS analysis of even a moderately large size of samples has been hampered due to the lack of tools for automaticallyanalyzing the huge amount of TOF-SIMS data. Here, we present a computational platform to automatically identify and alignpeaks, find discriminatory ions, build a classifier, and construct networks describing differential metabolic pathways. To
demonstrate the utility of the platform, we analyzed 43 data sets generated from seven gastric cancer and eight normal tissues
using TOF-SIMS. A total of 87 138 ions were detected from the 43 data sets by TOF-SIMS. We selected and then aligned 1286 ions.
Among them, we found the 66 ions discriminating gastric cancer tissues from normal ones. Using these 66 ions, we then built apartial least square-discriminant analysis（PLS-DA）model resulting in a misclassification error rate of 0.024. Finally, network analysis of the 66 ions showed disregulation ofamino acid metabolism in the gastric cancer tissues. The results show that the proposed framework was effective in analyzing
TOFSIMS
data from a moderately large size of samples, resulting in discrimination of gastric cancer tissues from normal tissues andidentification of biomarker candidates associated with the amino acid metabolism.
Finally, TOF-SIMS for biomedical applications is still suffered from insufficient molecular ion yields, which may be overcome withan Ar gas clusters ion beam. For clinical applications, high throughput is critical to make the diagnosis technique inexpensive.
KRISS has been tackling these two issues by developing a TOF-SIMS/MALDI microscopic mass analysis system based on an Ar gas
cluster ion beam and a broad laser beam. Recent preliminary progresses will be reported and discussed together.

Oral Session
Monday, 17th SeptemberRoom D

15:00 － 17:00

Session 9: Imaging-II
Chair:Jiro Matsuo（Kyoto University, Japan）

S09-1600
16:00 － 16:20

Diagnostic application of Imaging Mass Spectrometry

Mitsutoshi Setou1, Kiyoshi Ogawa2, Akiko Kubo3, Ikuko Yao4, Masaaki Matsuura5

Hamamatsu University School of Medicine, 2Shimadzu, 3Keio Universiy, 4Kansai Medical University, 5Japanesefoundation for cancer research

1

Keywords:
imaging diagnosis biopsy pathology
Novel aspects:
meta analysis of histopathological lipidomics with high resolution imaging mass spectrometry
Abstract:
We developed high resolution imaging mass spectrometry system and determined quantitative and qualitative alterations in lipids.
Molecular species of phosphatidylcholine（PC）on thin slices were determined during the occurrence and progression of diseases
in patients and model animals to identify potential clinical indicators of pathology. In addition, species of phospholipids were
quantified by liquid chromatography-electrospray ionization-tandem mass spectrometry（LC-ESI-MS/MS）of lipid extracts fromcontrol/patients. Unique distribution patterns were observed for phospholipids with different fatty acid compositions, and distinct
dynamic changes were seen in both their amounts and their distributions in tissue as tissue damage resulting from diseases. The
analysis was applied for various diseases such as colon cancer, gastric cancer, inflammatory bowel diseases, Alzheimer disease,schizophrenia, atherosclerosis, and soon. The specificity and generality of the lipid changes in human pathology revealed by the meta-analysis will be presented anddiscussed.

Oral Session
Monday, 17th SeptemberRoom D

15:00 － 17:00

Session 9: Imaging-II
Chair:Jiro Matsuo（Kyoto University, Japan）

S09-1620
16:20 － 16:40

High-throughput analysis for metabolic dynamics and in situ metabolite
imaging by MALDI mass spectrometry

Daisuke Miura1, Yoshinori Fujimura1, Shin-ichi Yamaguchi2, Noriyuki Ojima2, Mitsuru Shindo1,
Hiroyuki Wariishi1
1

Kyushu University, 2Shimdzu Co., Kyoto, Japan

Keywords:
MALDI-MS, High-throughput, Metabolite imaging, Synthesized matrix
Novel aspects:
Our developed MALDI-MS techniques combined with newly synthesized matrices may be useful for the advances in systems
biology approach and biological or biomarker discovery.
Abstract:
Introduction
Mass spectrometry（MS）-based techniques such as LC-MS or GC-MS have been known to be a conventional strategy for lowmolecular-weight metabolite analysis. However, these methods have drawbacks in analysis of tissue samples due to a requirementof metabolite extraction that causes loss of information on spatial localization. Moreover, total analysis time is still on the order ofseveral minutes for a sample. Matrix-assisted laser desorption ionization（MALDI）has recently been reported to be applicable forlow-molecular-weight metabolite analysis. Direct MALDI-MS analysis dose not require pre-separation step, and then the analysis
time is within a minute for a sample. In addition, MALDI system enables us to analyze tissue sample directly（mass spectrometry
imaging； MSI）. It is generally known that the detectable molecules in MALDI-MS are completely dependent on the matrix used.
Although there is a critical issue, limitation of the number of detectable analytes, in MALDI system, screening or development ofnovel matrix is believed to improve such drawback in coverage.
Herein, we report the development of MALDI-MS system towards the rapid analysis of metabolic dynamics and tissue metabolite
imaging. Furthermore, we also describe an attempt to develop novel matrix for expanding the coverage of detectable metabolites
in MALDI-MS.
Methods
For high-throughput analysis, E. coli was used as a model organism. Cell suspensions were directly mixed with matrix solution（ 5mg 9 -aminoacridine（ 9 -AA）in methanol）for rapid sample preparation. This suspension were spotted onto MALDI sample plate
and analyzed directly. For MSI experiment, rodent brain tissues were sectioned at 10 µm thickness with cryostat and then thawmounted onto an ITO-coated glass slide. A matrix solution was applied by spray-coating or sublimation method. In both types ofexperiments, data were acquired in negative ionization mode and signals between m/z = 50 to 1,000 were collected. Metabolites
were identified or estimated by comparing MS/MS spectra with standard compounds or databases online database. For matrix
development, more than 40 compounds including N- or C-substituted 9 -AA analogues, anthracene derivatives and quinoline
derivatives, were newly synthesized as matrix candidates. Using these synthetic matrices, 230 kinds of low-molecular-weightmetabolite standards were measured by MALDI-MS in negative ionization mode.
Results and Discussion
Firstly, we demonstrated the feasibility of a high-throughput MALDI-MS system for tracing intracellular metabolic dynamics ofbacteria. In a model system using E. coli, the time-dependent metabolite change was observed during environmental carbon
source perturbation following the rapid relief from glucose limitation. This technique enabled to trace time-dependent transitionsof the levels of phosphorylated metabolic intermediates and corresponding cofactors. Our proposed high-throughput analyticalplatform was expected to easily apply for a large-scale data acquisition of metabolite levels responsible for the central
metabolism. Metabolite correlation network analysis using these data is now under way. Furthermore, this highly sensitive
metabolite analysis technique was applied for in situ metabolite imaging. We could simultaneously visualize the distributions of abroad range of metabolites including nucleotides, cofactors, phosphorylated sugars, amino acids, lipids, and carboxylic acids innormal mouse brain tissue with single-cell sensitivity. Although we showed the findings using 9 -AA as a matrix, recently several
new synthetic matrices were found to be more efficient than 9 -AA, depending on the metabolites. Detailed results will be
discussed in the session.
（ 1 ）Miura D. et al. Anal. Chem.（2010）82, 498-504.（ 2 ）Yukihira D. et al. Anal. Chem.（2010）82, 4278-4282.（ 3 ）Miura D. et al. Anal.
Chem.（2010）82, 9789-9796.（ 4 ）Miura D. et al. J. Proteomicsin press

Oral Session
Monday, 17th SeptemberRoom D

15:00 － 17:00

Session 9: Imaging-II
Chair:Jiro Matsuo（Kyoto University, Japan）

S09-1640
16:40 － 17:00

Imaging of metabolites in plants and insects with high resolution APMALDI MSI

Dhaka R Bhandari, Andreas Roempp, Bernhard Spengler
Justus Liebig University Giessen, Giessen, Germany

Keywords:
Imaging mass spectrometry, metabolites, high mass resolution and spatial resolution.
Novel aspects:
High resolution in space and mass method for understanding metabolites distribution in plants and insects.
Abstract:
Mass spectrometry imaging（MSI）, introduced in the mid-90s, has shown encouraging progress recently in explaining themolecular basis of biological systems and processes. The technique has developed to become a promising tool for clinicalinvestigation and biomolecular research（ 1 - 3 ）. Herein, we present mass spectrometry images generated from plant and insect
tissues that reveal spatial distributions of metabolites with high resolution in mass and space（HR2）. MSI is able to visualize the
distribution of metabolites with high spatial resolution, aiding to explain mechanisms of complex biological processes.
A home-built atmospheric-pressure scanning microprobe matrix-assisted laser desorption/ionization（SMALDI）ion source for
imaging was used for ion generation（ 1 ）. To acquire uniform sections from the sample, embedding materials such ascarboxymethyl cellulose（CMC）and tragacanth gum were used for cryosectioning of insects. For root samples, on the other hand,
sections were prepared manually using a common shaving blade. Pneumatically assisted spraying was employed for matrix
deposition on the tissue, using a dedicated home-built apparatus. Matrices 2,5-dihydroxybenzoic acid（DHB）for positive ion modeand p-nitroaniline（pNA）for negative ion mode were used. The SMALDI source was operated with a nitrogen laser at 337 nm
wavelength with a 60 Hz repetition rate. The source was coupled to an orbital trapping mass analyzer（Exactive or QExactive mass
spectrometer, Thermo Fisher Scientific GmbH, Bremen）set to a mass resolving power of 50,000 to 140,000 at m/z = 200. The homebuilt software package ’Mirion’ was used to generate mass images from the raw files generated by the mass spectrometer. Ions
formed by 30 laser pulses were accumulated prior to detection, and matrix cluster peaks were used for internal calibration. All
measurements including mass spectra from 10 micrometer pixels were acquired with a mass accuracy 2.5 ppm（root meansquare）. Molecular images were generated with a bin width of m/z = 0.01.
Images from seeds（rape and wheat）, roots（rice and wheat）and insects（Paederus and Harmonia）were generated with 5 to 25
micrometer spatial resolution. For rapeseed at 10 micrometer pixel size, it was possible to image the ion at m/z = 496.24（cyclic
spermidine conjugate）that is only exhibited in the germinating region of the seed. For wheat seeds, several different metabolites
were imaged which were located in different regions of the seed. Whole insect imaging of Paederus was performed at 10
micrometer spatial resolution and the defensive agent pederin, known to be produced by Paederus, was observed as sodium
adduct at m/z = 526.29, as well as its metabolite pseudopederin with high mass accuracy. Similarly for Harmonia, MS images weregenerated using the signal of the resistance-related substance harmonine（m/z = 283.31）, as well as using the signals of its
metabolites, detected in different regions of the insect.
Literature：
1. Koestler et al.（2008）Rapid Commun Mass Spectrom. 22（20）：3275-3285.
2. Roempp et al.（2010）Angewandte Chemie International Edition. 49（22）：3834-3838.
3. Roempp et al.（2011）Anal Bioanal Chem. 401（ 1 ）：65-73.

Oral Session
Monday, 17th SeptemberRoom E

15:00 － 17:00

Session 10: Ion Mobility Spectroscopy Based on Instrument & Theoretical Development
Chair:

Toshiki Sugai（Toho University, Japan）

S10-1500
15:00 － 15:40[Keynote Lecture] Pushing the frontier of high-definition ion mobility
spectrometry using FAIMS

Alexandre A Shvartsburg, Gordon A Anderson, Richard D Smith
Pacific Northwest National Laboratory

Keywords:
IMS, FAIMS, isomer separation, PTMs
Novel aspects:
Novel waveform generators and gas compositions raise FAIMS resolving power by >10 times（to ~500 for peptides）, enabling new
approaches to proteomics（especially for characterization of post-translational modifications）and metabolomics.
Abstract:
Fast gas-phase separations using ion mobility spectrometry（IMS）prior to mass spectrometry（MS）are becoming ubiquitous, as
the recent introduction of IMS/MS platforms by multiple major vendors rapidly expands applications into new areas. As singlestage drift-tube IMS analyses developed since 1970-s turn routine, research and technology development are moving into novel
approaches beyond simple ion separation by low-field mobility.
The mobility of all ions in gases depends on the electric field intensity, and a major new IMS paradigm is differential or fieldasymmetric waveform IMS（FAIMS）based on the difference between mobilities at two intensities. That difference is often
substantially uncorrelated to the ion mass/charge ratio or absolute mobility at either intensity, rendering the FAIMS dimension
significantly orthogonal to MS or conventional IMS. While FAIMS/MS had been reported ~20 years ago and found promising forbiological analyses, its utility was severely restricted by low resolving power, R（typically ~10）.
We have strived to improve the R metric and resolution of FAIMS and explore the new application classes afforded by such gains.
Modeling and experiments have revealed that high resolution requires homogeneous electric fields established in planar electrodegaps. New planar-FAIMS devices employing helium/nitrogen buffers with up to 75% He or power supplies that produce waveforms
with amplitudes（dispersion voltage, DV）up to 5.4 kV have raised R for multiply-charged peptides to ~200, but electricalbreakdown in gas precluded further increases of He fraction and/or DV. The resolving power of separations in media, including
FAIMS, generally scales as the square root of separation time. Extending ion residence in the gap from the previously longest 0.2 s
up to 0.8 s had raised the resolving power by up to twofold（to >300 for peptides）. Hydrogen has much higher breakdown
threshold than He, and the greatest H2 fraction at maximum DV in our FAIMS unit is 90% vs. 50% for He. Hydrogen-rich mixtures
allow improving the resolution further, especially for larger singly-charged ions, while accelerating analyses. Finally, new highdefinition voltage generator with a more stable and precise output has narrowed the features at highest resolving power by ~ 1 /3,
pushing the maximum R to ~500.
Upon reviewing the latest advances in FAIMS technology, we shall discuss exemplary applications to proteomics andmetabolomics. As a filtering technique, FAIMS is best suited for targeted analyses, such as separation and identification of isomeric
peptides with differently localized post-translational modifications（PTMs）. High-resolution FAIMS can broadly separate thesevariants for many modifications, including phosphorylation, glycosylation, and even smallest PTMs like acetylation or methylation,and various attachment sites. The capability extends to larger“middle-down“（~ 3 - 4 kDa）peptides such as histone tails thatare richly modified and potentially exhibit millions of localization variants. Crucially, the variant separation is unrelated to the PTM
shift involved and even those with adjacent alternatively modified sites（which challenge LC and MS/MS methods most）are wellresolved. Separations for different charge states are also independent, and the variants“co-eluting“ for one are often resolved foranother. The peak capacity is thus multiplied by the number of observed states, which is increasingly consequential for larger
peptides that exhibit more states.
We shall also look at lipidomic applications, where FAIMS can assist lipid classification and effectively resolve regioisomers
differing in the fatty acid position or other details.

Oral Session
Monday, 17th SeptemberRoom E

15:00 － 17:00

Session 10: Ion Mobility Spectroscopy Based on Instrument & Theoretical Development
Chair:

Toshiki Sugai（Toho University, Japan）

S10-1540
15:40 － 16:00

Mobility of atomic and small molecular ions in cooled helium gas forinvestigation on interaction potentials and collision dynamics

Hajime Tanuma1, Ryosuke Isawa1, Jun-ichi Yamazoe1, Shiro Matoba2
1

Tokyo Metropolitan University, Tokyo, Japan, 2Japan Atomic Energy Agency, Takasaki, Japan

Keywords:
ion mobility, interaction potential, collision dynamics
Novel aspects:
Extensive analysis of atomic and molecular ion mobiliy using ab initio potentials and two-temperature theory has revealed thecollision dynamics at very low energy.
Abstract:
Typical measurements of ion mobility spectrometry（IMS）for chemical analysis have been performed in ambient air. Because theambient atmospheric pressure is high of 0.1 MPa, the reduced electric field strength E/N in the drift tube is smaller than 1 Td even
if the electric field is stronger than 100 V/cm. Therefore, the dependence of ion mobility on E/N is not important for IMS and thezero-field mobility has been used for the identification of the chemical compound. On the other hand, the traditional ion mobility
measurements in rarefied gases have been performed in various electric fields and gas pressures, and the ion mobility as afunction of the E/N has been investigated to discuss the interaction potentials and the elastic collision cross sections between theion and the gas molecule.
In Tokyo Metropolitan University, we have developed a drift tube mass spectrometer operated at liquid helium and nitrogentemperatures, and measured the mobility of various atomic and small molecular ions at low temperatures. For the closed-shell
systems, only one potential energy curve as a function of the internuclear distance is involved in elastic collisions. Therefore themeasured ion mobility of the alkali ions, namely Li +, Na+, K+, Rb+ and Cs+, in helium at 4.3 and 77 K can be reproduced by theclassical calculation based on the two-temperature theory with the ab initio interaction potentials between the ions and helium.
For the open-shell systems, the interaction potential curve on the ground electronic state could split into different molecularstates at the small internuclear distance. Using the averaged cross sections for two interaction curves, the mobility of the openshell ions in helium can be calculated approximately. We have measured the mobility for C+, N+, O+, He+, Ne+, Ar+, Kr+, and Xe+ions, and the calculation with the ab initio potentials shows fairly good agreement with the experimental results.
Not only atomic ions, diatomic and triatomic molecular ions have been used as the incident ions for the selected-ion-injection drift
tube experiments. The molecular ion mobility as a function of the E/N shows quite different behavior from that of the atomic ions.
For the atomic ions, the theoretical calculation of the mobility can be performed shortly. On the other hand, the mobility ofmolecular ions is extremely difficult to be calculated because of the multi-dimensional potential surfaces. To analyze the molecularmobility, the classical trajectory calculations have been carried out for N2 + and O2 + ions in helium, and the experimental results ofthe mobility can be reproduced quite well. In these calculations, it was found that the temporal rotational excitation plays a very
important role.
Recently, we have measured the mobility of OH+ and OD+ in helium and observed a minimum in the mobility as a function of the
E/N. The features of these results are quite different from those of O2 +, CO+, NO+, CO2 +, and NO2 +, which have no minimum. The
possible mechanism in collisions at few meV will be discussed with the ab initio potentials of molecular ions between heliumatom.
We have also make a start on the development of an apparatus for the IMS in ambient air. The progress report on this new
project will be given in our presentation.

Oral Session
Monday, 17th SeptemberRoom E

15:00 － 17:00

Session 10: Ion Mobility Spectroscopy Based on Instrument & Theoretical Development
Chair:

Toshiki Sugai（Toho University, Japan）

S10-1600
16:00 － 16:20

Novel analytical approaches enabled by ultra-fast chip-based FAIMS
separation coupled to mass spectrometry

Michael Ugarov1, Yuqin Dai1, Michael Flanagan1, Michelle Romm2, Vaughn Miller2
1

Agilent Technologies, Santa Clara, USA, 2Agilent Technologies, Wakefield, USA

Keywords:
FAIMS, ion mobility spectrometry, high speed separation, microscale fabrication
Novel aspects:
Novel approaches to applications enabled by high speed FAIMS separation
Abstract:
There has been a considerable increase in interest for orthogonal gas phase separations in combination with mass spectrometry
during the last several years. High-Field Asymmetric Waveform Ion Mobility Spectrometry（FAIMS）uses variation of ion drift
velocity in atmospheric pressure environments at very high electric fields. It is often seen as a candidate for enhancing theseparation provided by liquid chromatography（LC）, or even potentially replacing LC stage entirely for some applications.
Previously, one disadvantage of all differential mobility devices has been the scanning duty cycle relative to fast LC. It proved achallenge to achieve performance that allows simultaneous effective separation, high transmission and scan speed. For thispresentation we have characterized performance of a new generation of microscale FAIMS devices, which enable this ability andwhich promise to revolutionize high throughput multi-dimensional separations. Several mass spectrometry applications are
expected to benefit from high speed pre-separation. We describe preliminary experimental results related to some of thesepotential applications.
A newly designed chip-based FAIMS device from Owlstone, Ltd., UK has been incorporated into the Agilent TOF mass
spectrometry system. This generation of chip FAIMS devices has an analytical gap of 100μm and ion drift path of 700μm. The
device comprises multiple channels with parallel electrodes designed to achieve optimum ion collection and transmission. In
addition, this configuration offers enhanced selectivity while maintaining exceptional speed of separation. Entire CV scan can beaccomplished in under 100μs, while switching delay between different CV settings can be less than 5 μs. This speed is enabled
by using very high values of dispersion field inside miniature channels.
As a result, the new device is compatible with TOF MS and QQQ MS data acquisition rates, as well as time scales of ultra-fastchromatography and solid phase extraction（SPE）based analytical methods such as Agilent RapidFire.
Preliminary testing has demonstrated that the performance is in good agreement with theoretical expectations. We reportsignificant improvements in separation power and transmission, particularly for the high mobility ions compared to the previous
series of prototypes. In addition, the FAIMS device demonstrates very low ion losses in“off-line“ mode which supports highperformance of the mass spectrometer in its regular operation regime. High dispersion voltages and short ion residence times areshown to provide high stability and reproducibility of operation with respect to specific conditions of analytical sample, solvent or
chemical matrix.
Several potential applications have been demonstrated using the unique fast scanning and switching capabilities of the new
system. They include separation of drugs, metabolites and interfering compounds, isomer identification, as well as peptide
selection based on charge state distribution.
For example, the ability to separate components of a peptide mixture by charge state allows enhanced detection of higher chargeions with respect to singly charged ions as well as chemical noise. This proves beneficial for the identification confidence. Thisapplication requires adjustable resolution, excellent transmission and the possibility to switch dispersion and compensation fieldsettings quickly to fully realize this potential benefit.
In another example, we were able to demonstrate conditions for selective transmission of phenacetin vs. acetaminophen,morphine vs. morphine glucuronide as well as some other pairs of analytes. This capability is very attractive, for example, in highthroughput SPE/MS analytical methods such as RapidFire/MS which do not include liquid chromatography. Incorporating the
FAIMS device into the RapidFire QQQ MS system provides an orthogonal mode of separation allowing more accurate quantitationof analytes that are susceptible to interference from in-source fragmentation.

Oral Session
Monday, 17th SeptemberRoom E

15:00 － 17:00

Session 10: Ion Mobility Spectroscopy Based on Instrument & Theoretical Development
Chair:

Toshiki Sugai（Toho University, Japan）

S10-1620
16:20 － 16:40

Real-Time 2D separation with LC-DMS-TOF-MS: Increasing the Selectivityof High Resolution MS in Qualitative and Quantitative Analysis

J C Yves Le Blanc, Larry Campbell, Nic Bloomfield, Alexander V Loboda, Brad Schneider,
Igor V Chernushevich
AB SCIEX, Concord, Canada

Keywords:
Quadrupole-TOF MS； Differential mobility spectrometry； LC-MS； Selectivity
Novel aspects:
Improving high resolution MS detection of isobaric species based on atmospheric pressure differential mobility and liquid
modifiers
Abstract:
Introduction
Complex samples mixture of low molecular weight and polar compounds are particularly challenging from an LC-MS acquisition
perspective. In order to extract all relevant analyte information from such samples, higher peak capacity is frequently required,
and this leads to coupling of comprehensive two-dimensional（ 2 D）separation technique such as GCxGC and LCxLC. However, insuch coupling, the second dimension of separation requires to be very fast, which can be challenging to LC. Here we propose thatthe second dimension can be obtained with chemically-assisted differential mobility separation（DMS）to provide morecomprehensive detection of analyte from complex mixture. DMS provides a separation that is orthogonal to MS as well as LC, andthe addition of chemical modifier in the transport gas can increase significantly the separation power of the DMS system.
Methods
A DMS cell was mounted in the atmospheric region between the entrance aperture of a hybrid quadrupole-time-of-flight mass
spectrometer and TurboVR source. Nitrogen was used as the transporter gas, and constant gas flow in the DMS cell was achievedby the primary stage vacuum pumping of the MS system. Addition of 1.5% of either isopropanol（IPA）or acetonitrile（ACN）to thecurtain gas was used to alter the DMS separation. LC was performed using a Shimadzu UFLC system operated at 400uL/min with a2 x100mm（2.6u）Luna Kinetex C18 column. A mixture of 30 compounds ranging from m/z 70 to 700, with different polarity,was
spiked in different samples matrix such as urine and protein precipitated plasma.
Preliminary Data
The DMS cell was operated at a separation voltage（SV）up to 4000 and the compensation voltage（CoV）was stepped from -30
to +10V with increments of 0.5V（81 steps）. Mass spectra were collected at each step for 20ms, thus producing a total cycle timeof 1.62sec. Using a gradient of 1 min from 2 % to 98 % organic, several of the lower mass compounds co-eluted. For species oflower mass（m/z < 350）, a wide spread of CoV values were observed and many of the isobaric and co-eluting species wereeffectively separated as a result of using the DMS with either IPA or ACN as modifier in the gas transport. It is noticeable thatsome of the species that could not be separated by LC, such as fendiline（m/z=316.2060）and chlorprotixen（m/z=316.0912）were
separated by the DMS, which enabled individual MS/MS data collection with precursor ion selection window equivalent to<0.1amu, as opposed to generating composite MS/MS spectrum. When dealing with samples of higher complexity, such as diluted
urine samples, the DMS with IPA provided the highest peak capacity that complemented LC selectivity. This enables faster andmore comprehensive coverage of analyte ions detected from complex samples while maintaining rapid sample throughput（i.e.
rapid LC gradient）. Additional results pertaining to selectivity gains for qualitative and quantitative analysis of TOF-MS and TOFMSMS data will be presented.

Oral Session
Monday, 17th SeptemberRoom E

15:00 － 17:00

Session 10: Ion Mobility Spectroscopy Based on Instrument & Theoretical Development
Chair:

Toshiki Sugai（Toho University, Japan）

S10-1640
16:40 － 17:00

Isomer-selected photodissociation of carbon and silicon cluster ions withion mobility mass spectrometry using a double reflectron time-of-flightmass spectrometer

Kiichirou Koyasu, Ryoichi Moriyama, Tomohiro Ohtaki, Jun Hosoya, Fuminori Misaizu
Tohoku University, Sendai, Japan

Keywords:
Ion mobility spectrometry； Carbon cluster； Silicion cluster； Photodissociation； Double reflectron mass spectrometer
Novel aspects:
We have developed a combined setup of an ion drift cell with a double reflectron time-of-flight mass spectrometer, for the studyof highly-sensitive isomer-resolved photodissociation with the tandem reflectrons.
Abstract:
In general, the number of coexisting isomers in atomic and molecular clusters increases rapidly with their sizes. Among suchclusters, silicon cluster cations, Si n + , have stable prolate structures for n< 30 and spherical isomers for n> 24. Carbon cluster
cations, Cm +, also have different type isomers depending on their size； cyclic isomers for m< 40 and fullerene isomers for the evennumber size at m> 30. Therefore the isomers of Sin + and Cm + have two structural motifs； spherical and non-spherical structures.
These different structures may show marked difference in their reactivity. Therefore the isomer-resolved measurement is necessary
in order to examine the reactivities and reaction mechanism rigorously. Ion mobility spectrometry, IMS, is a powerful tool todirectly separate isomers. However, the ion intensity drops by several orders of magnitude after the isomer-separation with thismethod. It is also important to obtain spatially focused ion bunches for unambiguous and highly-sensitive photodissociation
measurements of size-selected ions.
In order to fulfill the isomer-resolved photodissociation experiments against above problems, we have developed a combined
setup of an ion drift cell with a double reflectron time-of-flight mass spectrometer, TOFMS. In this setup, dissociation efficiency isenhanced by the focusing of parent ions at the irradiation point after a first reflectron. In this study, we have examined UV laser
photodissociation reactions for each isomer of Sin +and Cm + after mass- and isomer-separations. Sin + and Cm + generated by laser
vaporization were isomer-separated by a drift cell, in which the ions were collided with He under an electrostatic field. The cell
was cooled to 170 K. The separated isomers were mass-separated in a double reflectron TOFMS setup. The ions were thendissociated by irradiation with a 4.66 eV laser at the ion focal plane in the first reflectron. The photofragment ions were massanalyzed by the second reflectron.
In the isomer-separated experiments for the parent Sin +ions with n = 24-27, we have observed two types of fragment ions, Si6  -  11 +fragment clusters and Si 10 -loss fragment ions, from both the prolate and spherical isomers. In particular, relative intensities of
Si10-loss fragments from the prolate isomers were found to be slightly higher than that from the sphericals. This result was partly
explained from the calculated geometric structures containing Si10 units［ 1 ］.
On the other hand, forCm +（m = 32, 34, 36, 38）, we have observed clearly different fragmentation pathways between the cyclic
and fullerene isomers； C14-loss was preferentially observed from the cyclic isomers, whereas sequential C2-losses were obtained
from the fullerenes. The C14-loss products were already reported in dissociation studies of Cm + with m< 29, where stable isomers
have multi-cyclic structures［ 2 ］. This predominant pathway was explained by the neutral C 14 stability based on the Hueckel
model. The C2-loss pathways of fullerenes are also consistent with the previous photodissociation studies on fullerenes［ 3 ］.
［ 1 ］S. Yoo and X. C. Zeng, J. Chem. Phys. 124, 054304（2006）.
［ 2 ］P. P. Radi, T. L. Bunn, P. R. Kemper, M. E. Molchan, and M. T. Bowers, J. Chem. Phys. 88, 2809（1988）.
［ 3 ］S. C. O’Brien, J. R. Heath, R. F. Curl, and R. E. Smalley, J. Chem. Phys. 88, 220（1988）.

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 1: Developments in Tandem Mass Spectrometry - Hybrid Instrumentation“Thewhole is greater than the sum of its parts”(Aristotle).

PMo-001

11:10 － 12:20

Boundary Activated Dissociation in a Low Pressure Linear Ion Trap in thepresence of Nonlinear DC Fields

mircea guna

AB SCIEX, Concord, ON, Canada

Keywords:
BAD, CAD, nonlinear fields
Novel aspects:
Nonlinear dc fields modify the stability boundary of the quadrupole, increasing the BAD efficiency to 94% and extending the mass
range of the BAD fragmentation spectrum beyond previously observed limits.
Abstract:
Boundary-Activated Dissociation（BAD）of multiple charge ions has been investigated in a low pressure linear ion trap in thepresence of nonlinear dc fields. Nonlinear dc fields allowed ions to be stored at working points beyond the by= 0 stability
boundary of the regular quadrupole. The ions reached large stable radial amplitude trajectories and gained high kinetic energies
from the main drive RF field and thus the BAD efficiency increased from less than 20% to upto 94%. The broadening of the stability
diagram at the by = 0 boundary also enabled the observation of fragment ions of higher mass-to-charge ratio than the precursor
ions thus overcoming one of the major drawbacks of BAD of multiple-charged ions. A comparison between high pressure beamtype collision activated dissociation（CAD）and low pressure BAD spectra showed that similar internal energies can be depositedusing these two techniques however less fragmentation channels were observed using low pressure BAD.

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 1: Developments in Tandem Mass Spectrometry - Hybrid Instrumentation“Thewhole is greater than the sum of its parts”(Aristotle).

PMo-002

13:30 － 14:40

ECD in an RF-Free Electromagnetostatic Cell on a Triple Quadrupole Mass
Spectrometer

Yu Chu Lin1, Samuel Bennett1, Valery G Voinov1,2, Douglas F Barofsky1
1

Oregon State University, Corvallis, Oregon, U.S.A., 2Pacific Institute of Bioorganic Chemistry, Vladivostok, Rusia

Keywords:
Electron Capture Dissociation（ECD）； Ion Activation/Dissociation； Electromagnetostatic dissociation cell
Novel aspects:
Enhanced ECD in a radio-frequency-free electro-magnetostatic cell
Abstract:
Introduction
Last years we demonstrated a Radio Frequency-Free（RFF）electro-magnetostatic（EMS）cell for producing electron capture
dissociation without the aid of a cooling gas or phase-specific electron injection into the cell as part of a triple quad and a hybrid
Q-TOF tandem mass spectrometer［ 1 - 3 ］. That work further demonstrated not only ECD, but also combined ECD and collisioninduced dissociation（CID）in an RFF EMS cell［ 4 ］. In this presentation, we describe an improved EMS cell design and present
results that demonstrate that this improved cell’s ECD efficiency is at the threshold of analytical usefulness.

Methods
A triple quadrupole（Q-q-Q）Finnigan TSQ 700 mass spectrometer was converted to a Q-ECD-Q instrument ：·An indirectly heated, ring-shaped, Tantalum disc（Heat Wave Labs, Inc., Watsonville, CA）,located concentric with the cell’s axis
at the ion-entrance, serves as the source of electrons.·
The cell comprises an electromagnet and anaxially polarized Sm 2Co17 ring-magnet（Chino Magnetism Corp. Ltd., Fairfield, NJ）
that has a 12.54 mm diameter, 1.0 mm thickness, and 2.0 mm bore. Several common peptides were introduced into the instrument
by ESI in order to produce parent ions for the ECD experiments.
Preliminary Data
Using an indirectly heated disc as a source of electrons instead of a directly heated, ring-shaped, wire filament solved threeimportant problems. First, the greatly increased emitting area of the disc over that of the wire filament emitted a much larger
number of electrons. Second, the problem of voltage drop through the emitter was eliminated； in the case of the ring-shaped,
wire filament, this voltage drop could be up to 7 V resulting in an excessively wide electron energy distribution. Third, theproblem of the magnetic field induced by the current in the ring-shape wire filament negatively effecting electron emission was
totally eliminated. Taken together, the gains resulting from these three improvements increased the ECD efficiency of EMS cell up
to at least 2 % , which is at the threshold of analytical utility. As example, ECD fragments recorded while injecting 5 µg/mL
solution of substance P in methanol at a flow rate of 2 µL/min are present.
Summary
In summary, we have achieved ECD in a linear, RF-free, hybrid electrostatic/magnetostatic cell at efficiency level of at least 2 %without the aid of a cooling gas. The cell’s design and compact construction allow it to be incorporated into virtually any type oftandem mass spectrometer, e.g., triple quadrupole, hybrid quadrupole ion trap, hybrid quadrupole time-of-flight, or even FT-ICR.
Acknowledgement
This research was supported by the National Science Foundation under Grant No. 0924027, the National Institutes of Health underGrant No. 1 R01RR026275-02, and the National Institute of Environmental Health Science under Environmental Health Sciences
Center Grant ES00210. The authors also thank Elsworth T. Hinke for his technical assistance.
References
1.Voinov VG, Deinzer ML, Barofsky DF. Rapid Commun.Mass Spectrom. 2008,22,3087.
2.Voinov VG, Deinzer ML, Barofsky DF. Anal.Chem. 2009； 81： 1238.
3.Voinov VG, Deinzer ML, Beckman JS, Barofsky DF. J.AM.Soc.Mass Spectrom. 2011,22,607.
4.Voinov VG, Beckman JS, Deinzer ML, Barofsky DF. Rapid Commun.Mass Spectrom.2009, 23,3028.

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 1: Developments in Tandem Mass Spectrometry - Hybrid Instrumentation“Thewhole is greater than the sum of its parts”(Aristotle).

PMo-003

11:10 － 12:20

A rapid and sensitive liquid chromatography tandem mass spectrometry
method for quantification of etoposide and etoposide chatechol in
cerebrospinal fluid

Hsiao-Wei Liao1,2, Yen-Shen Lu3, Ching-Hung Lin3, Ching-Hua Kuo1,2National Taiwan University, Taipei, Taiwan, 2School of Pharmacy, College of Medicine, National Taiwan University,
Taipei, Taiwan, 3Department of Oncology, National Taiwan University Hospital, Taipei, Taiwan
1

Keywords:
Etoposide, liquid chromatography tandem mass spectrometry, cerebrospinal fluid
Novel aspects:
This is the first time using liquid chromatography tandem mass spectrometry method for quantification of etoposide andetoposide catechol in cerebrospinal fluid.
Abstract:
Cancer with brain metastasis is one of the leading causes of death in many major cancer subtypes. Etoposide is one of thechemotherapy drugs that can partially penetrate blood brain barrier. In human body, etoposide undergoes O-demethylation andcan be metabolized into etoposide catechol which exhibits signicant cytotoxic activity. Understanding the etoposide andetoposide chatechol concentration in CSF for patients with brain tumor can help to evaluate the drug efficacy. A rapid andsensitive ultra-high-pressure liquid chromatography/electrospray ionization tandem mass spectrometry（UHPLC-MS/MS）method
was developed for quantification of etoposide and etoposide chatechol in CSF.
Chromatographic separation was performed on an Agilent Extend RP-18 column（1.8 um, 50 mm×2.1 mm）with a gradient mobile
phase consisted of 0.1 % acetic acid in water and methanol delivered at 0.3 ml/min. Detection was achieved by an Agilent 6460
UHPLC-MS/MS system with selected reaction monitoring（SRM）in positive ion mode with the transition m/z 589.6/229.1 and m/z
575.2/229.0 for etoposide and etoposide chatechol, respectively. The limit of detection（LOD）is 0.5 ng/mL and the limit ofquantification（LOQ）is 1 ng/mL for both etoposide and etoposide chatechol. The total analysis time is below 3 minutes. The
method was validated in terms of precision, accuracy, matrix effect and linearity. Finally, this method was successfully applied tothe analysis of CSF samples from cancer patients with brain and leptomeningeal metastasis treated with etoposide, and thepharmacokinetic behavior of etoposide in CSF was determined.

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 1: Developments in Tandem Mass Spectrometry - Hybrid Instrumentation“Thewhole is greater than the sum of its parts”(Aristotle).

PMo-004

13:30 － 14:40

Qualitative analysis of impurities in enalapril using liquid chromatographyion trap time of flight hybrid mass spectrometry

FENG JI, QI Y LI, JING DONG
Shimdzu Co., Beijing,China

Keywords:
Enalapril, Impurities, LCMS-IT-TOF
Novel aspects:
To our knowledge, this is the first complete characterization of the fragmentation of enalapril impurities, mainly are its byproducts, using ESI-IT-TOF tandem mass spectrometry with accurate mass measurements.
Abstract:
Enalapril Maleate is the maleate salt of enalapril, the ethyl ester of a long acting angiotensin converting enzyme inhibitor. Enalapril
is a pro-drug； it is bioactivated by hydrolysis of the ethyl ester to enalaprilat, which is the active angiotensin conveting enzyme
inhibitor. Enalapril has side effects： fainting； urinating more； chest pain, etc. However, by-products, unknown impurities and theirstructures are a big concern. Currently, high-performance liquid chromatography（HPLC）has been the essential method foranalysis of enalapril. Atmospheric pressure ionization sources, such as electrospray ionization（ESI）are routinely used in drug
impurities analysis for sensitivity.
A liquid chromatography/electrospray ion trap time-of-flight mass spectrometry（LCMS-IT-TOF）was used in this study. It has theability to provide multistage tandem spectra（MSn）with accurate masses（error< 5 ppm）in both MS and MSn modes. Tof analyzer
allows fast acquisition of full spectra with high sensitivity and elevated mass resolution（12000 FWHM）. With this technique, we
can acquire reliable structural information about the ionized molecule and the product ions, based on the exact mass and themultistage tandem mass spectrometric analyses（MS n ）. In addition, as the mass/charge ratio gets smaller, the number ofcandidates gets smaller, too. Since mass/charge ratios of the product ions in the highest order spectra are usually less than 200 Da
and the neutral losses are usually less than 100 Da, their formulae could be confirmed with significantly higher confidence.
Enalapril samples were separated on a Shimadzu Shim-pack XR-ODS column（2.0 mm I.D.×75 mm L., 2.2 μm）with isocratic elution
using 10 mM ammonium acetate and acetonitrile as mobile phase. High concentration of enalapril（300 mg/L）was injected toconcentrate impurities. Flow path switching valve was used in the instrument, cutting the enalapril main constituent to waste line
in order not to contaminate MS detector. Four impurities were separated and detected by ESI-IT/TOF. MS n spectra（n= 4 ）ofeach impurity were obtained in both positive and negative ion mode. The combination of accurate mass measurement in MS
spectra and sequential MS n experiments enabled fragmentation pathways to be elucidated in detail. Formulae, chemical structuresof impurities in an Enalapril sample were suggested with supporting results on the probable fragmentation pathways.
Protonated molecules of impurity 1 （m/z 349.1758）gave product ions at m/z 303.1703 with a neutral loss of m/z 46.0055, which was
CH2O2. Using formula predict software impurity 1 was C18H24N2O5. Similar MSn measurement was observed between impurity 1and enalapril. This could be explained by loss of two methylene group（perhaps carboxylic acid ethyl ester）from enalapril
structure.
Formula predict of impurity 2 m/z 381.2395 shows a formula as C20H34N2O5,contains 6 more H atom compared to enalapril. Whenm/z 381 was selected as the precursor ion to perform the MS2 product ion scan experiment, a product ion at m/z 335.1976, 307.2027,
291.2078, 273.1972, 263.2129 and 192.1394, all have corresponding response loss of 6 H in enalapril. Product ion at m/z 183.0775,170.0823
were both observed at impurity 2 and enalapril. m/z 183.0775,170.0823 is formed by the loss of benzene ring form enalapril. So thestructure of impurities 2 may be explained by deoxidation of benzene ring from enalapril.
Also we may get the formulae for impurity 3 and 4 are C20H26N2O4 and C19H26N2O4. To our knowledge, this is the first completecharacterization of the fragmentation of enalapril impurities, mainly are its by-products, using ESI-IT-TOF tandem mass
spectrometry with accurate mass measurements.

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 1: Developments in Tandem Mass Spectrometry - Hybrid Instrumentation“Thewhole is greater than the sum of its parts”(Aristotle).

PMo-005

11:10 － 12:20

Qualitative Analysis of Gossypol, Free Gossypol, and Gossypol Derivatives
in Cottonseeds By Electrospray Ionization Tandem Mass Spectrometry

Luying Zhou1, Lai Chen2, Jinting Yao1, Hongyuan Hao1, Hengtao Dong1, Qiang Li1
1

Shimadzu (China) Co., Ltd., Shanghai, China, 2Shanghai Ocean University

Keywords:
Gossypol Derivatives, LCMS-IT-TOF, MSn Spectra
Novel aspects:
The study of derivatives struture by MSn Spectras got from LCMS-IT-TOF
Abstract:
Free gossypol and in cottonseed is a toxic chemical, which can affect human reproductive function, skin and stomach burning,
nausea, vomiting, diarrhea, headache, coma, convulsions and even death due to respiratory, circulatory system failure. Therefore,
strict control of the free gossypol and its derivatives is required in deep-processing process of cottonseed. In this article, according
to the MS n Spectra of gossypol, the structures of known and unknown gossypol derivatives were figure out by LCMS-IT-TOF.
Quantity and quality of free gossypol derivatives were different when using different extracting way. The qualitative results ofgossypol derivatives get from LCMS-IT-TOF could tell the best way of deep-processing process of cottonseed.

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 1: Developments in Tandem Mass Spectrometry - Hybrid Instrumentation“Thewhole is greater than the sum of its parts”(Aristotle).

PMo-006

13:30 － 14:40

Evaluation of the higher sensitive LC/MS/MS incorporates novel
desolvation technologies to achieve low femto-gram LOQ

Shinjiro Fujita, Natsuyo Asano, Kazuo Mukaibatake
Shimadzu Corporation, Kyoto, Japan

Keywords:
LC/MS/MS, desolvation, heated gas, off axis, orthogonal
Novel aspects:
Low femto-gram LOQs were achieved by using highly sensitive LC/MS/MS with heated gas and multi orthogonal heated device
Abstract:
The triple quadrupole mass spectrometer is widely used in various application fields to quantify the trace amount of compounds
in real world samples because of its excellent sensitivity and selectivity. In order to achieve lower limit of quantitation, many
researchers have been developing new desolvation technologies at either ESI sprayer or MS inlet.
Here, we report the higher sensitive triple quadrupole mass spectrometer equipped with novel desolvation devices such asaddition of the heated gas blowing along with electrospray and the heated multi-orthogonal ion path mounted at the inlet of mass
spectrometer. The combination of the heated gas at ESI and heated multi-orthogonal ion path enhanced desolvation efficiency
which resulted in low femto-gram limit of quantitation.
Several commercially available drug samples（Reserpine, Verapamil, Alprazolam, Carbamazepine, Cilostazol, Lidocaine,
Fluticasone and Testosterone） were prepared for the sensitivity evaluation of higher sensitive triple quadrupole mass
spectrometer equipped with the modified ESI source and the heated multi orthogonal ion path. All the samples were analysed by
the Multiple Reaction Monitoring（MRM）. MRM parameters including MRM transitions and collision energy as well as compound
dependent ion transfer voltages were optimised through automatic MRM optimization functionality incorporated in LabSolution
software（Shimadzu Corporation, Kyoto, Japan）.The temperatures of heated gas and heated multi-orthogonal ion path were
optimised for each sample.
Using modified ESI source and the heated multi orthogonal ion path helped to deliver a limit of quantitation for all eight drugs
below 5 fg/uL. The temperature of heated gas and heated multi-orthogonal ion path were independently optimised for eachsample with the range of 200 to 500 degree C and 150 to 300 degree C respectively. The ion intensity of reserpine was increased 8
times with the heated gas and 4 times with heated multi-orthogonal ion path operated at 300 degree C. Besides larger volume ofIon was introduced into the mass spectrometer by the effective desolvation, the base line noise was maintained at reasonably
low level as the noise derived from neutral droplet or unwanted ions were strongly reduced in the multi orthogonal region heatedat high temperature, and resulted in excellent signal to noise ratio which achieved low femto-gram sensitivity.

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 1: Developments in Tandem Mass Spectrometry - Hybrid Instrumentation“Thewhole is greater than the sum of its parts”(Aristotle).

PMo-007

11:10 － 12:20

Determination and pharmacokinetics of di-(2-ethylhexyl) phthalate in rats
by ultra performance liquid chromatography with tandem mass
spectrometry

Wan-Ling Chang-Liao1, Lie-Chwen Lin1,2, Tung-Hu Tsai1,3National Yang-Ming University, Taipei, Taiwan, 2National Research Insitute of Chinese Medicine, 3Taipei City Hospital,
Taipei, Taiwan

1

Keywords:
bioavailability, Di-（ 2 -ethylhexyl）phthalate（DEHP）, pharmacokinetics, plasticizer, tandem mass spectrometry.
Novel aspects:
This is the first time that a LC-MS/MS has been developed to investigate oral bioavailability in rat.
Abstract:
Di-（ 2 -ethylhexyl）phthalate（DEHP）is generally used to increase the flexibility of plastics for industrial products which has been
contaminated in food and beverages as clouding agent in Taiwan in the early 2011. Previous studies indicated that DEHP may leadto hepatotoxic, cytotoxic, teratogenic and mutagenic effects on animals. In order to assess the exact extent of absorbed DEHP via
the oral route, the aim of this study is to develop a reliable and validated ultra performance liquid chromatography with tandem
mass spectrometry（UPLC-MS/MS）method for the evaluation of the oral bioavailability of DEHP in Sprague-Dawley rats. The
optimal chromatographic separation of DEHP and Butylbenzyl phthalate（BBP； as internal standard）were achieved on a C18
column（2.1 mm x 100 mm, 1.7 μm）. The mobile phase consisted of 5 mM ammonium acetate-methanol（11：89, v/v）with a flowrate of 0.25 mL/min. The monitoring ion transitions were m/z 391.4 → 149.0 for DEHP and m/z 313.3 → 149.0 for the internal standard.
The average of matrix effect and recovery of DEHP at low, medium and high concentrations was 113 ± 20% and 98 ± 5 %. The
calibration curve is linear within the given concentration range from 0.1-2.5 μg/mL and produced linear correlation coefficient isgreater than 0.995. In conclusion, the validated UPLC-MS/MS method is suitable for analyzing the rat plasma sample of DEHP and
the oral bioavailability of DEHP was about 7 % in rats.

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 1: Developments in Tandem Mass Spectrometry - Hybrid Instrumentation“Thewhole is greater than the sum of its parts”(Aristotle).

PMo-008

13:30 － 14:40

Pharmacokinetics of Di-isononyl Phthalate in Freely Moving Rats by UPLCMS/MS

Mei-Ling Hou1, Tung-Hu Tsai1,2Institute of Traditional Medicine, School of Medicine, National Yang-Ming University, Taipei, Taiwan, 2Department of
Education and Research, Taipei City Hospital, Taipei, Taiwan

1

Keywords:
DINP, phthalate, freely moving rat, pharmacokinetics, tandem mass spectrometry
Novel aspects:
This is the first time that a LC-MS/MS has been developed to investigate oral bioavailability of DINP in rat.
Abstract:
Di-isononyl phthalate（DINP）is a general-purpose plasticizer for polyvinyl chloride. However, the industrial chemical plasticizer
had contaminated food and beverages as clouding agent, which had been inspected by Taiwan Food and Drugs Administration.
The aim of study is to develop a sensitive and specific ultra-high performance liquid chromatography coupled with electrosprayionization tandem mass spectrometry（UPLC-MS/MS）method to investigate the pharmacokinetics of DINP in freely moving rats.
Multiple reaction monitoring（MRM）was used to monitor the transition of the protonated molecule m/z of 419［M+H］+ to the
product ion 149 for DINP. Plasma and feces homogenates were extracted by protein precipitation with 100 % acetonitrile（0.1%
formic acid）for sample preparation. The analyte was analyzed by UPLC-MS/MS with C18 column（100 mm x 2.1 mm, 1.7 µm）which
was equilibrated and eluted with an isocratic mixture of acetonitrile-ammonium acetate water solution（90：10, v/v）at a flow rateof 0.3 mL/min. The limit of quantification（LOQ）of DINP in rat plasma and feces homogenate was determined to be 0.25 µg/mL.
The intra- and inter-day accuracy（% Bias）and precision（% CV）were less than 15 %. Linear calibration curves were obtained for
DINP concentration ranges of 0.05-2.5 µg/mL in plasma and feces. The feces were homogenized mechanically using 50 %acetonitrile as the media. The pharmacokinetic curve demonstrated that the disposition of DINP in rat plasma was fitted well by
the two-compartmental model after DINP administration（10 mg/kg, i.v.）. The elimination half-life of DINP was 5.87 ± 3.41 and150.41 ± 58.38 min for intravenous（10 mg/kg）and oral（100 mg/kg）administration, respectively. The pharmacokinetic data indicatethat the oral bioavailability of DINP in freely moving rats was about 1.19 %. The total DINP excretion up to 48 h was 12.30 ± 4.46 %in feces.

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 1: Developments in Tandem Mass Spectrometry - Hybrid Instrumentation“Thewhole is greater than the sum of its parts”(Aristotle).

PMo-009

11:10 － 12:20

Pharmacokinetics of dibutyl phthalate by ultra performance liquid
chromatography with tandem mass spectrometry in rats

Li-Wen Chang1, Tung-Hu Tsai1,21National Yang-Ming University, Taipei, Taiwan, 2Taipei City Hospital, Taipei, Taiwan

Keywords:
dibutyl phthalate, pharmacokinetics, plasticizer, tandem mass spectrometry.
Novel aspects:
This is the first time that a LC-MS/MS has been developed to investigate pharmacokinetics of DBP in rat.
Abstract:
Dibutyl phthalate（DBP）is a commonly used plasticizer for flexible polyvinyl chloride with a broad range of applications. However
several plasticizers have been illegally used as clouding agent to form dispersion in aqueous matrix of the drinks. The aim of study
is to establish a rapid and accurate analytical method to investigate the pharmacokinetics of DBP in Sprague-Dawley rat by Ultra
Performance Liquid Chromatography coupled to tandem mass spectrometry（UPLC-MS/MS）. The UPLC-MS/MS system equippedwith a positive electrospray ionization（ESI）source in multiple reaction monitoring（MRM）mode was used to monitor m/z 279.28→ 148.93 and m/z 225.09 → 102.96 transitions for DBP and chrysin（internal standard）, respectively. The analytes were separatedusing a C18 reversed-phase column（2.1 mm x 100 mm, 1.7 μm）, the mobile phase for column separation was 5 mM amonium
acetate mthanol（27：73, v/v）with a flow rate of 0.25 ml/min. The method has been applied to measure DBP in rat plasma andfaece by protein precipitation procedure. The limit of quantification for DBP in rat plasma and faese was 50 ng/ml and 125 ng/ml,
respectively. The pharmacokinetic results demonstrated that the maximum concentration（C max ）and the time to reach the
maximum concentration（Tmax）in rat plasma were 2.88±0.17 μg/mL and 7.11±0.50 min, respectively after DBP administration（30mg/kg, i.v.）. About 0.9 % of the administered dose was recovered from the faece within 48 h. The results showed that DBP
degraded within 2 h, suggesting a rapid metabolism of DBP in the body.

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 1: Developments in Tandem Mass Spectrometry - Hybrid Instrumentation“Thewhole is greater than the sum of its parts”(Aristotle).

PMo-010

13:30 － 14:40

Separation of polar solutes by hydrophilic methacrylate-based monolithic
columns via capillary electrochromatography coupled with UV-Vis andmass spectrometry detection.

Yung-Han Shih, Ching-Yi Wu, Cheng-Lan Lin, Pei-Zhu Liang, Hsi-Ya Huang
Chung Yuan Christian University, ChungLi, Taiwan

Keywords:
hydrophilic organic polymer-based monolith； capillary electrochromatography； CEC-MS； phenolic acid； amino acid
Novel aspects:
This study demonstrated a novel poly（TAIC-EDMA-SMA）monoliths which are highly potential stationary phases for hydrophilicsolutes separation.
Abstract:
Abstract
In traditional methacrylate ester-based monoliths, it is very hard to separate polar compounds. In this study, we describe triallyl
isocyanurate as hydrophilic cross-linker to synthesize poly methacrylate ester monolithic columns for the separation of polar
solutes in capillary electrochromatography（CEC）. Different ratios of triallyl isocyanurate to ethylene dimethacrylate（EDMA）andalkyl methacrylates were optimized and characterized by SEM to evaluate the morphology, N2 adsorption/desorption isotherm todetermine the surface area and thermogravimetric analysis for thermal stability. Compared with traditional methacrylate esterbased monoliths, the triallyl isocyanurate-co-methacrylate monoliths exhibit hydrophilicity, better thermal stability and goodbaseline separation ability for all polar analytes at a mobile phase composed of 18~27.5 % acetonitrile. Moreover, these triallyl
isocyanurate-co-methacrylate columns were also applied in CEC-ESI/MS.

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 2: Advances in Methods and MS Instrumentation for Biomolecule Characterization

PMo-011

11:10 － 12:20

The COrrelation Ion-Neutral Time Of Flight Mass Spectrometry (COINTOF_MS)

Victor Buridon1, Cecile Teyssier1, Hassan Abdoul-Carime1, Bernadette Farizon1, Michel Farizon1,
Tilmann D Mark2
Universite de Lyon 1, Institut de Physique Nucleaire Lyon, Villeurbanne, France, 2Institut fur Ionenphysik undAngewandte Physik, Leopold Franzens Universitat, Technikerstrasse 25, A-6020 Innsbruck, Austria

1

Keywords:
Ion-Neutral correlation, event-by-event analysis technique
Novel aspects:
COIN-TOF MS as a new and versatile instrument for the investigation of collision processes and analytical chemistry
Abstract:
Mass-spectrometry is nowadays widely used not only for the analysis of（bio-）chemistry products［ 1 ］but also for theinvestigation of processes（e.g., ion, electron or photon-nano-system collisions）［ 2 ］. Collecting and correlating all the nascentfragments produced in a given dissociation pathway represent a tremendous challenge for an accurate characterization of theparent molecule or the description of the interaction mechanisms. The mass of charged products can be determined with highaccuracy by time-of-flight mass spectrometry［ 3 ］. In contrast, ascribing the neutral species requires very specific conditions［ 4 ］.
The coincidence measurements between sets of fragments may be obtained only by using multi-detectors technique［ 5 ］,
however the correlation between fragments within a given dissociation event are still not accessible. These become even morechallenging for complex nano-systems when branching ratios between several competitive channels for neutrals production are
opened.
We present the COIN-TOF Mass Spectrometer［ 6 ］. This new and versatile instrument is based on a single detector for thedetection of the ion and the neutral fragments produced within a single dissociation event and the“event-by-event“ analysis ofdata. Such analysis provides information on the ion and neutral products, their correlations for a given dissociative channel andalso the nature of the prior interaction that leads to the dissociation of the investigated molecular system. Thus, physicalparameters i.e., branching ratios for neutral fragments, kinetic energy releases., can be directly accessible. Illustration of the COINTOF_MS will be discussed through selected examples obtained from recent collision induced dissociation measurements［ 7 ］.
［ 1 ］J.B. Fenn et al., Science246 64（1989）； R.G. Cooks et al., Science311 1566（2006）； S.K. Kim, T. Ha, J ；-P. Schermann, Phys.
Chem. Chem. Phys., 121366（2010）［ 2 ］J.S Brodbelt, Mass Spectrom Rev., 16 91（1997）； R. Zenobi, R. Knochenmuss, Mass Spectrom. Rev.17 337（1998）； R.E.
Continetti, Ann. Rev. Phys. Chem., 52 165（2001）［ 3 ］N. Misraleh-Kohan, W.D Robertson, R.N. Compton, Mass Spectrom. Rev.27 207（2008）［ 4 ］D.P. deBruijn, J.Los, Rev.Sci.Instrum.53 1020（1982）； ）R.D. Thomas, Mass Spectrom. Rev.27 485（2008）；［ 5 ］P. Jukes, A. Buxey, A. B. Jones, and A. J. Stace, J. Chem. Phys., 109 5803（1998）；K.A. Hanold, A.K. Luong, T.G. Clements, R.E.
Continetti, Rev. Sci. Instrum.70 2268（1999）［ 6 ］M. Farizon, B. Farizon, H. Abdoul-Carime, G. Bruny, S. Eden, S. Feil, C. Montano, Depôt de brevet n °09 58991 du 15
Decembre2009, PCT/FR2010/052733 patent pending.
+［ 7 ］H. Abdoul-Carime et al., Direct observation of neutral water dimer from collision induced dissociation of H （H
2 O）
2 by
COINTOF mass spectrometry, submitted 2012

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 2: Advances in Methods and MS Instrumentation for Biomolecule Characterization

PMo-012

13:30 － 14:40

Automatic Disulfide Bond Assignment Using a1 Ion Screening by Mass
Spectrometry for Structural Characterization of Protein Pharmaceuticals

Sheng-Yu Huang2, Yu-Ting Hsieh1, Chun-Hao Chen1, Chao-Chi Chen2, Wang-Chou Sung3,
Min-Yuan Chou4, Sung-Fang Chen1

National Taiwan Normal University, Taipei, Taiwan, 2Mithra Biotechnology Inc., Taiwan, 3National Health ResearchInstitutes, Taiwan, 4Industrial Technology Research Institute, Taiwan

1

Keywords:
disulfide bonds, dimethyl labeling, pharmaceuticals
Novel aspects:
An automatic method for disulfide bond assignment using dimethyl labeling and computational screening of a 1 ions by MS
analysis with customized software, RADAR, is developed.
Abstract:
An automatic method for disulfide bond assignment using dimethyl labeling and computational screening of a1 ions by MS analysis
with customized software, RADAR, is developed. Utilizing the enhanced a1 ions generated from labeled peptides, the N-terminal
amino acids from disulfide-linked peptides can be determined. In this study, we applied this method for structural characterizationof recombinant monoclonal antibodies, an important group of therapeutic proteins. In addition to a1 ion screening and molecularweight match, new RADAR is capable of confirming the matched peptide pairs by further comparing the CID fragment ions. With
the N-terminal amino acid identities as a threshold, the identification of disulfide-linked peptide pairs can be achieved rapidly at ahigher confidence level. Unlike majorities of the current approaches, prior knowledge of disulfide linkages or a high- end mass
spectrometry is not required, and tedious labor work or deliberate interpretation can be avoided in this study. Our approachmakes it possible to analyze unknown disulfide bonds of protein pharmaceuticals as well as their degraded forms without further
protein separation. It can be used as a convenient quality examination tool during biopharmaceutical development andmanufacturing processes.

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 2: Advances in Methods and MS Instrumentation for Biomolecule Characterization

PMo-013

11:10 － 12:20

Determination of chloramphenicol in biological samples by gas
chromatography-mass spectrometry

Suh-Jen J Tsai, Hsiu-Hua Huang, Pei-Yin Hsieh, Che-Wei Chang
Providence University, Taichung, Taiwan

Keywords:
Chloramphenicol； Derivatization； GC-MS； Biological samples
Novel aspects:
An analytical method has been established for the determination of chloramphenicol（CAP）. The derivatization of non-volatile
CAP with either N,O-bis（trimethylsilyl）trifluoroacetamide（BSTFA）or N-（tert-buthyldimethylsilyl）- N-methyltrifluoroacetamide（MTBSTFA）reduced the polarity of CAP.
Abstract:
Determination of chloramphenicol in biological samples by gas chromatography-mass spectrometry
Suh-Jen Jane Tsai*, Hsiu-Hua Huang, Pei-Yin Hsieh, Che-Wei Chang
Department of Applied Chemistry, Providence University, Taichung , Taiwan 43301
E-mail： sjtsai@pu.edu.tw

Abstract
An analytical method has been established for the determination of chloramphenicol（CAP）. CAP is one of the most important
antibacterials. CAP has often been used in the animal and fish farms to prevent diseases, to fight parasites, and to promote
growth. GC-MS is less expansive then LC-MS, and consequently, is more popular in the routine laboratories. However, it is notapplicable to analysis the non-volatile CAP directly by GC-MS. The derivatization of non-volatile CAP with either N,O-bis（trimethylsilyl）trifluoroacetamide（BSTFA）orN-（tert-buthyldimethylsilyl）- N-methyltrifluoroacetamide（MTBSTFA）reduced thepolarity of CAP. The derivatives were effectively determined by GC-MS. TThe experimental conditions for GC-EI-MS were
optimized. These included the optimum injection temperature, injection volume for the derivatization condition, reaction solvent,and reaction time...etc. GC-EI-MS was done with extracted ion mode. The derivatization efficiency of silation and acetylation werecompared. The accuracy and precision of this proposed method for the determination of chloramphenicol in biological samples
including pig liver, pig kidney, and Bovine liver were also evaluated.

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 2: Advances in Methods and MS Instrumentation for Biomolecule Characterization

PMo-014

13:30 － 14:40

Combining quantum mechanical and MS analysis to understanding thesource of H+ during APPI of PAH dissolved in toluene

Arif Ahmed, Chell Ho Choi, Kim Sunghwan
Kyungpook National University, Daegu, Korea

Keywords:
quantum mechanics, appi, mass spectromery
Novel aspects:
Mechanism of APPI of studied by combining experimental MS data and quantum mechanical calculation
Abstract:
Atmospheric pressure photo ionization（APPI）is a powerful technique for analysis of polycyclic aromatic compounds（PAH）which
are very important in environmental and crud analysis. In a previous study published in our group, it has been shown that toluene
is the source H+ for APPI when only toluene was used as a solvent. In this study, APPI mechanism, especially, the source of H +during the generation of protonated PAH compounds were studied by quantum mechanical calculation and mass spectrometry
analysis.
Antharacene, acridine, HPLC grade toluene, water, MeOH, anisole and benzene were purchased from Sigma-Aldrich（MO, USA）
and used without further purification. Standard samples were dissolved in toluene, anisole and benzene solvent to final
concentration of 1 µM~10 µM. The solutions were analyzed by positive mode APPI LCQ Fleet ion trap mass spectrometer（Thermo
Scientific）. The General Atomic and Molecular Electronic Structure System（GAMESS）program was used for all of thecomputations. Density functional theory with B 3 LYP functionals was utilized in combination with an all-electron 6 -31G（d,p）basis
set. Minimum energy reaction paths were determined by first optimizing the geometries of the minima and transition states.
Anthracene was dissolved in toluene, anisole and benzene and analyzed by APPI MS. Only anthracene dissolved in toluene
produced protonated ions. This strongly suggests that the hydrogen in methyl group of toluene molecules but not the one in thearomatic ring is used to produce protonated ion. Additionally, APPI MS spectra of acridine dissolved in toluene, anisole andbenzene were obtained and compared. It was observed that all three combinations of anthracene-toluene, anthracene-benzene
and anthracene-anisole dominantly produced protonated ion. The data suggest that both hydrogen in methyl group and aromaticring can be used to produce protonated ions for acridine. To further study the findings, theoretical calculation is currently
undergoing. The general atomic and molecular electronic structure system（GAMESS）program is being used for computations.

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 2: Advances in Methods and MS Instrumentation for Biomolecule Characterization

PMo-015

11:10 － 12:20

Transformation of the Green Fluorescence Protein upon transfer fromsolution to the gas phase.

Vladimir Frankevich, Konstantin Barylyuk, Paviel Sagulenko, Renato Zenobi
Swiss Federal Institute of Technology, Zurich, Switzerland

Keywords:
Fluorescence, GFP, FT-ICR, spectroscopy
Novel aspects:
GFP conformation in the gas phase. Novel method to probe the properties of gas-phase molecular ions
Abstract:
The combination of laser-induced fluorescence（LIF）with mass spectrometry opens up new possibilities for structural studies of
biomolecular ions in the gas phase. What will happen to a protein when the solvent molecules are removed? Will it maintain anear-native conformation, unfold, or assume a new structure? The green fluorescent protein（GFP）is highly fluorescent in its
native conformation, while it does not fluoresce upon unfolding. Observing the fluorescence of GFP in the gas phase can therefore
serve a very direct measure of its conformation.
A Fourier-transform ion cyclotron resonance mass spectrometry（FTICR-MS）coupled with laser spectroscopy was chosen as anexperimental platform to probe the properties of isolated biomolecular ions. A unique home-built optical setup was constructedthat allows laser-induced fluorescence/dissociation experiments on trapped ions in the gas phase. Ions can be produced
externally by electrospray ionization（ESI）and trapped for high resolution mass analysis and spectroscopic studies in the FTICR
cell.
We performed a series of laser spectroscopy experiments in order to probe the conformation of gas-phase GFP ions directly
inside the ESI plume and when trapped in the high vacuum of the FTICR mass spectrometer. GFP fluorescence spectra in the ESI
source were found to be unchanged along the plume, but the signal intensity gradually decreased. In additional experiments, atrapped GFP ion population was subjected to CW laser irradiation（from 351 nm to 514 nm）of various powers（up to 1.5 W）and
exposure times（up to 2 min）. Interestingly, neither fluorescence nor dissociation was observed for trapped GFP ions. On thecontrary, synthetic cR 6 G labeled protein-dye conjugates prepared from ubiquitin and GFP showed fluorescence signal whenisolated in the FTICR cell under same conditions. The lack of fluorescence from GFP could be directly attributed to protein
unfolding in vacuum. On the other hand, the fluorescence could disappear due to the loss of structural water molecules. Water
molecules are important contributors to the stability and functioning of biomolecules. There is evidence that the structural waterparticipates in the GFP fluorescence process. Molecules of structural water build a hydrogen bond network that is necessary for
GFP fluorescence. Our high-resolution MS experiments show the complete absence of structural water molecules in GFP. This
does not affect the GFP conformation too much, but dramatically changes its fluorescence properties. Our preliminary datasuggest that this is the mechanism responsible for the lack of GFP fluorescence in vacuum.

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 2: Advances in Methods and MS Instrumentation for Biomolecule Characterization

PMo-016

13:30 － 14:40

Elucidation of CID and ETD based MS/MS Fragmentation for Analysis of
Doubly Derivatized Steroids

Yu-Min Juang, Tzu-Fang She, Chien-Chen Lai

Institute of Molecular Biology,National Chung Hsing University, Taichung, Taiwan

Keywords:
Steroids, ETD, Microwave-accelerated derivatization
Novel aspects:
ETD conjunction with CID supplied more structure information of doubly derivatized steroids, and expected that a novel andsensitive metohd could be applied for steroid analysis.
Abstract:
The specific measurements of target steroids play an important role in the clinical diagnosing of a number of common endocrine
disorders and testing drug abuse in athletes. More recently, liquid chromatography/electrospray ionization tandem mass
spectrometry（LC/ESI-MS/MS）with collision-induced dissociation（CID）has advantage that detection of complex sample andtime-reduced of pretreatment, and it is a rapid and convenient technique for examination of disease and drugs. Nevertheless,steroid assay is difficult to develop specific methods for the detection of an individual steroid in the presence of many structurally
similar compounds. To further develop a specific method with sensitivity and precision, we conducted a complementary MS/MS
analysis of doubly Girard’s reagent P derivatized steroids by using combined CID and electron transfer dissociation（ETD）. Thisapproach proved that derivatization with Girard’s reagent P by microwave irradiation led to higher ESI sensitivity of steroids andreduces the reaction time less than 10 min. As the result of derivatized steroids underwent two fragmentation methods, CID
provided much product ions which are low m/z and the fragment mechanism was random and some was perplexity. However,ETD provided high m/z ions and sequential fragment mechanism which was easy to recognize. These findings indicate that theanalysis of doubly derivatized steroids by ETD in conjunction with CID can provide more structural information of B- and C-ring,
and with side chain on it. Furthermore, establishment of both CID and ETD fragment ions database that from nine doubly
derivatized steroids expected to improve steroid identification.

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 2: Advances in Methods and MS Instrumentation for Biomolecule Characterization

PMo-018

13:30 － 14:40

Identification of protein ligands by FT-ICR-MS/MS

Adam A Dowle, Abbas Maqbool, Gavin H Thomas, Jerry Thomas
University of York

Keywords:
FT-MS/MS SORI CID Bruker solariX ligand
Novel aspects:
Protein ligands were identified by FT-ICR-MS/MS using CID and SORI with a Bruker solariX, which allowed revision of proposed
metabolic pathways.
Abstract:
The presence of non-covalently bound ligands in proteins can have a variety of significant biological effects； examples are
numerous including biological synthesis, cell signalling, and transport. Accurate identification of the ligands involved is of vital
importance in these processes. However, conventional mass spectrometry and even X-ray crystallography cannot always provide
definitive identification, especially where sample is limited in amount. The high mass accuracy of FT-ICR-MS, in tandem with its
ability to couple with a variety of ion dissociation options, offers good scope for the identification of these biological molecules.
We have demonstrated that the Bruker solariX equipped with a 9.4-tesla magnet can be used to accurately identify non-covalent
protein ligands.
Ligand identification by FT-ICR-MS was performed under denaturing conditions to maximise signal quality and mass accuracy.
Acquisition parameters were adjusted to aid release of the ligand and optimise sensitivity in the low m/z region where the ligands
are found. Important acquisition parameter changes included increasing the source accumulation time and reducing the TOF time.
Typically, identification of elemental composition was possible from the intact m/z alone, with indications of structure arising fromfragmenting the ligand using different dissociation methods. The solariX affords a variety of dissociation options； so far we have
performed fragmentation of ligands using CID in the quadrupole and SORI in the Infinity™ cell. These approaches have yielded
differing and complementary product ion spectra that aid the assignment of ligand structure. In the case of L-alanyl-γ-D-glutamylmesodiaminopimelate the y1-ion pedominated in the SORI spectrum while the y2-ion was only detected following CID. Example ofligands identified include： L-alanyl-γ-D-glutamyl-mesodiaminopimelate； glutathione； sialyl amide and S-adenosyl,
S-carboxymethylhomocysteine. Identifications were consistent with results of structural binding assays or X-ray crystallography
data.

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 2: Advances in Methods and MS Instrumentation for Biomolecule Characterization

PMo-019

11:10 － 12:20

Absolute Internal Mass Calibration with Carbon Soot Ions for High
Resolution MALDI 15T FT-ICR Mass Spectrometry

Choi Myoung Choul, Kyu Hwan Park, Hyun Sik Kim, Se Gyu Lee
Korea Basic Science Institute, Ochang, Korea

Keywords:
Mass Calibration, Carbon Soot, FTICR MS, 15T
Novel aspects:
With the high resolution carbon cluster peaks, the mass accuracy of internal calibration was improved in 15 T MALDI FT-ICR MS.
Abstract:
Introduction
Since Fourier Transform Ion Cyclotron Resonance Mass Spectrometer（FT-ICR MS）was introduced by Comisarow and Marshall in
1974, many research groups have gone into mass measurement accuracy（MMA）enhancement using the advantages of highresolving power of FT-ICR MS in wide mass range with external or internal calibration. MMA is crucial for characterization andidentification of small molecules such as natural products, drug substances, metabolites as well as peptides. Due to simultaneous
detection of calibrants and analytes, internal calibration shows higher MMA by compensating time-dependent tolerances. It has
been reported to improve MMA using matrix assisted laser desorption ionization（MALDI）FT-ICR MS.
MethodAll mass spectra were obtained using positive ion mode on a MALDI FT-ICR-MS with a 15 T superconducting magnet（ApexQe
Bruker Daltonics, Bremen, Germany）. Desorption/ionization was carried out with 355 nm Nd：YAG laser of 6 ns pulse width.
Carbon soot（CS ； combustion from liquid butane）was prepared for a source of carbon clusters and used for a calibrant as well
as for a matrix of positive ion MALDI FT-ICR MS. The enhancement of MMA by internal calibration with a number of standard
peaks from CS was demonstrated via analysis of a peptide mixture, which was directly spotted onto CS layer.
Results
Carbon clusters（soot）with 24 u mass spacing were generated in the range of m/z 720-2500 and successfully adopted for internal
calibration of a peptide mixture which is usually used as internal standards. With the high resolution carbon cluster peaks, themass accuracy of internal calibration was improved in 15 T MALDI FT-ICR MS. The average mass error of analytes was ~29 ppb andthe RMS error of ~31 ppb.

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 2: Advances in Methods and MS Instrumentation for Biomolecule Characterization

PMo-020

13:30 － 14:40

Identification of epitopes recognized by monoclonal antibodies raised
against the meningococcal factor H binding protein

Agnese Faleri1,2, Sebastien Brier2, Laura Santini2, Silvana Savino2, Marzia Monica Giuliani2,
Mikkel Nissum2, Mariagrazia Pizza2, Vega Masignani2, Nathalie Norais2
1

University of Siena, Siena, Italy, 2Novartis Vaccines and Diagnostics, Siena, Italy

Keywords:
Epitope mapping, H/D exchange
Novel aspects:
Mapping of epitopes recognized by different monoclonal antibodies raised against the fHbp of Neisseria meningitidis using
Hydrogen/Deuterium exchange coupled with Mass Spectrometry.
Abstract:
The factor H binding protein（fHbp）is included in the multicomponent 4 CMenB developed to prevent disease caused by
Neisseria meningitidis serogroup B. fHbp is a 27-kDa membrane-anchored lipoprotein that was discovered by reverse vaccinology.
It binds to the complement factor H regulator and down-regulates the alternative pathway thus allowing survival of the bacteria inhuman plasma. fHbp has also the capability to induce bactericidal antibodies in mice and humans, and it was proposed that
antibody binding to fHbp could increase the immune response by preventing the bacteria to inhibit the complement alternative
cascade.
The identification of protective epitopes is an important step to understand the mechanism of an effective immune response andto improve the rational design of more protective antigens. In this study, we investigated the interactions between fHbp anddifferent monoclonal antibodies using hydrogen-deuterium exchange associated with mass spectrometry（H/DX-MS）. The H/DXMS approach relies on the differential solvent accessibility of the free and bound antigen to a deuterated buffer. Thus, the antigen
epitopes in interaction with the antibodies are protected from the solvent and incorporate less deuterium compared to the free
antigen. We report here the mapping of the epitopes recognized by different monoclonal antibodies and present a structuralcomparison of the different antigenic regions identified.

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 2: Advances in Methods and MS Instrumentation for Biomolecule Characterization

PMo-021

11:10 － 12:20

Variable Temperature Ion Mobility Mass Spectrometry of MultimericProtein Complexes

Kamila J Pacholarz, Perdita E Barran

University of Edinburgh, Edinburgh, United Kingdom

Keywords:
ion mobility, variable temperature, protein complexes, thermal stability
Novel aspects:
Variable temperature IM-MS studies investigate the influence of buffer gas temperature on the mutlimeric protein structures, andhighlight the difference between the gas phase and solution thermal stability.
Abstract:
Despite an increasing success of ion mobility mass spectrometry（IM-MS）to study intact biomolecules, there are still some
controversies regarding structure preservation in the gas phase and data calibration obtained from the commercially available
travelling wave ion mobility spectrometry（TWIMS）instrument Synapt HDMS. The issue with any mobility analyzer used toinvestigate the conformations of biomolecules is that the measurement itself may alter the conformation of the ion. Application ofhigh injection energies and long ion storage times can cause structure collapse or reorganisation via unfolding. Recent studies
show, that the ion effective temperature within TWIMS for small proteins can exceed 500 K which will cause an ion to re-order tomore elongated conformations under typical settings recommended by the manufacturer. We set out to investigate the influenceof buffer gas temperature on the mutlimeric protein structures and gain more insights into the thermal stability those complexes.
Ion mobility and mass spectrometry data of mutlimeric complexes was acquired on a home built linear temperature variable drift
tube DT IM-MS instrument. Ion species selected for this study were trimeric Protein X（52 kDa）, and two commercially available
tetrameric protein complexes： avidin（64 kDa）and concanvalin A（102.6 kDa）. All three systems yield stable ion currents andhave simple preparation requirements. Prior to IM-MS analysis, the sample buffer was exchanged to 100 mM ammonium acetateusing chromatography spin columns. The data were obtained at different buffer gas（helium）temperatures ranging from 200 to 500
K to examine the thermal stability of those protein complexes, their collapse, unfolding and complex dissociation.
All three protein complexes under investigation present narrow charge state envelopes（protein X trimer n =13-16, avidin tetramern = 15-18 and concanvalin A tetramer n = 21-24）. Smaller complexes such as trimeric protein X and avidin）present a shift towards
higher charge states upon exposure to higher temperatures such as 400 K. Ion mobility data analysis show a decrease in collisioncross sections（CCSs）of those complexes in comparison to CCSs determined at 300 K, suggesting a structural collapse. Moreover,in case of Protein X the stochastic increase in CCSs usually observed with increasing charge state resulting from protein unfolding
due to coulombic repulsions, is no longer observable； the CCSs remain more-less constant across charge states, again supportingthe complex collapse. When exposed to temperatures above 500 K, the protein complexes dissociate into monomeric（Protein X,
avidin, concanvalin A）or dimeric（concanvalin A）species. Capturing the ions prior to the dissociation（at 470 480 K）reveals adrastic increase in the CCSs associated with protein unfolding. Based on the IM-MS data, we speculate that investigated protein
complexes exposed to high temperatures collapse first prior to unfolding. Interestingly, cooling down the drift cell to 200 K allows
preservation of tetrameric concanvalin A, which otherwise exhibits a significant amount of dimeric and monomeric species even at
300 K. Presented variable temperature IM-MS studies pinpoint the importance of the analysis temperature for protein structural
studies, and highlight the difference between the gas phase and solution thermal stability.

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 2: Advances in Methods and MS Instrumentation for Biomolecule Characterization

PMo-022

13:30 － 14:40

Full H/D Exchange for Improving Small Molecule and Peptide
Identification with Electrospray Ionization

Sebastian Tittebrandt1, Bernhard Spengler2, Wolf D Lehmann11German Cancer Research Institute, Heidelberg, Germany, 2Institute for Inorganic and Analytical Chemistry, University
Giessen, Germany

Keywords:
Full H/D exchange, gas-phase H/D exchange, electrospray
Novel aspects:
Complete H/D exchange of small analytes and peptides and their analysis by ESI-MS and -MS/MS without significant loss of label.
Abstract:
N-, O-, and S-bound hydrogens undergo fast H/D exchange in solution. Determination of the number of exchangeable hydrogens
in an analyte strongly substantiates the confidence of mass spectrometric identification. So far complete H/D exchange of
peptides with subsequent analysis by MS is reported only sporadically［ 1 ］. In this study we show the systematic investigation ofgas-phase and solution H/D exchange for a variety of compound classes with the aim of full H/D exchange. As a result of this fullexchange, MS and MSMS spectra of peptides are better suited for de novo sequencing via CBS［ 2 ］and for database-linked
peptide identification. The identification of unknown analytes by MS is generally put on a more reliable basis using full H/D
exchange as additional analytical tool.
By in-solution H/D exchange, virtually complete exchange can be achieved for N-, O-, and S-bound hydrogen atoms in intact
molecules（metabolites, amino acids, lipids and peptides）. In an electrospray ion source, H/D exchange may occur by collisions
with gas-phase components containing exchangeable hydrogens［ 3 ］. While this phenomenon is of disadvantage for exact
measurements of the number of exchangeable hydrogens, it offers the possibility for gas phase introduction of deuterium within
an ESI source. For small molecules, we observed that using a D 2 O-saturated ESI source, hydrogens present in -COOH, -OH, -SH,
-NH2, non-amide -NH, as well as in phosphate and phosphate esters groups efficiently undergo H/D exchange. Amide hydrogen in
peptides is only poorly exchanged under these conditions, as confirmed by gas phase in- and out-exchange of deuterium.
However, full H/D exchange could be established using highly enriched D 2 O（99.9 % atom% D）as solvent plus a D 2 O-saturated
ion source atmosphere. In this way, degrees of deuteration of up to 99.8 atom% at all exchangeable positions could be achieved.
Tandem mass spectra of fully deuterated peptides show that b- and y- ions carry the number of deuterium atoms calculated on
the basis of their structure. Thus, full H/D exchange is avaluable tool for improving de novo and database-linked peptide
sequencing.

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 2: Advances in Methods and MS Instrumentation for Biomolecule Characterization

PMo-023

11:10 － 12:20

SALDI-MS of oligopeptide using gold nanorods on ITO plates.

Masanori Fujii1, Naotoshi Nakashima1,2,3, Yasuro Niidome1,2
1

Kyushu University, Fukuoka, Japan, 2WPI I2CNER, 3JST-CREST

Keywords:
Gold nanorods, SALDI-MS
Novel aspects:
We demonstrated a high sensitivity SALDI-MS measurement to the angiotensin on an ITO plate using photoabsorption of the gold
nanorods.
Abstract:
Surface Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry（SALDI-MS）is frequently used for analysis of
biomolecules. In modern SALDI-MS measurements, nanostructured surfaces or nanoparticles absorb photons and assist indesorption and ionization of sample molecules. In this work, we detected of oligopeptide（angiotensin I）in SALDI-MS
measurements using gold nanorods fixed on an indium-thin oxide（ITO）plate. The gold nanorods on the ITO plate are expected
to capture and concentrate the target molecules.
A solution of the prepared gold nanorods with aspect ratios of about 5 （51 ± 7 nm and 9.7 ± 1.1 nm in longitudinal andtransverse directions, respectively）was centrifuged twice at 15000 g for 10 min and then decanted to remove excess CTAB
molecules from the solution. After this, gold nanorods were coated with poly（sodium styrenesulfonate）（PSS）to obtain polyanionic surfaces. The PSS layers contributed to decrease the concentration of CTAB in the solution and stabilize the colloidal
dispersion of the gold nanorods in the absence of CTAB. An ITO plate modified with 3 -aminopropyltriethoxysilane（APTES）was
immersed in an aqueous solution of the PSS-coated gold nanorods to deposit the anionic nanorods on the cationic surfaces. The
nanorod-ITO plates that were dropped and dried angiotensin I（100 mL）were used for SALDI-MS measurements using a MALDI-MS
instrument（Autoflex III, Bruker Daltonics）. The light source was a nitrogen laser oscillated at 337.1 nm. An aqueous solution of
trifluoroacetic acid（0.1wt.%, 100 mL）was dropped and dried onto the plate before measurements.
The extinction spectra of the nanorod-ITO plate are shown the two SP bands near 520 and 900 nm. This indicates that the gold
nanorods were deposited on the ITO plate. The mass spectra of the nanorod-ITO plate with angiotensin（0.5 mM 10 fM）areshown a peak of protonated angiotensin（m/z = 1297）. Even when adilute angiotensin solution was dropped on a nanorod-ITO
plate（10 fM）, a peak of protonated angiotensin was found at m/z = 1297 with a good signal/noise ratio（S/N = 11）. In theabsence of gold nanorod, no signal was detected even when 10 mM angiotensin I was dropped on an ITO plate. In the absence ofangiotensin I, the nanorod-ITO plates with and without trifluoroacetic acid showed several peaks, but there were no peaks at m/z= 1297. These control experiments indicate that the peaks at m/z = 1289 and 1291 are background signals from the PSS or APTES
on the nanorod-ITO plate, but the signal at m/z = 1297 of angiotensin I comes from the SALDI processes induced by thephotoabsorption of gold nanorods.
The ITO plate, which was modified with gold nanorods, was found to have effects in collecting angiotensin I molecules close to
the gold nanorods, and the SALDI processes that were induced by the photoabsorption of the gold nanorods efficiently
contributed the desorption and ionization of the angiotensin I.1,2)References
1 ）Y. Niidome, Y. Nakamura, K. Honda, Y. Akiyama, K. Nishioka, H. Kawasaki and N. Nakashima, Chem. Commun., 1754（2009）.
2 ）Y. Nakamura, Y. Tsuru, M. Fujii, Y. Taga, A. Kiya, N. Nakashima and Y. Niidome, Nanoscale, 3, 3793-3798（2011）.

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 2: Advances in Methods and MS Instrumentation for Biomolecule Characterization

PMo-024

13:30 － 14:40

Collision/Reaction cell for ICP-MS - a new Concept for an improved
removal of low masses

Lothar Rottmann, Jung Gerhard, Tomoko Vincent
Thermo Fisher Scientfic, Bremen, Germany

Keywords:
ICP-MS, collision cell, new cell design with stepped or curved rods, interference removal
Novel aspects:
The sensitivity of a collision cell for ICP-MS is significantly improved by using a rod structure with variable cross-section.
Abstract:
One of the fundamental challenges in ICP-Q-MS, polyatomic interferences can be suppressed with the use of a collision/reaction
cell. Although a universal gas mixture can be suitable for most routine applications, some analytes in more challenging matrices
will either benefit from an alternative gas or more aggressive cell conditions to preferentially suppress or remove the polyatomic
interferences. However, in the reaction mode it is also preferable to remove the reacting gas ions and other low mass ions along
the cell.
A new design of a collision cell comprising flatapole rods with a curved or stepped shape was developed. This allows a hightransmission caused by a larger r0 at the entrance of the cell and a low mass cut-off at the lower r0 e.g. in the middle of the cell.
The underlying principle is the variation of q along the collision cell. This allows achieving for the same RF amplitude highsensitivity and robustness as well as sufficiently high low-mass cut-off to ensure elimination of reaction products.

Experiments were carried out that clearly showed the following performance improvement：· High transmission of the system even for low masses in kinetic energy discrimination（KED）mode in a He pressurized cell. At theiCAP Q > 400 cps for 7Li could be detected in a 1 ppb standard solution in this mode.
· Low-mass cut-off in order to remove unwanted masses of lower mass. The background from high intensity peaks of e.g. 40Ar iscompletely removed at higher masses and thus allows higher signal-to-noise at these masses.
Excellent interference removal properties. In KED mode a value of > 20 for the ratio of the intensity of 1 ppb 59 Co and theinterference of 35Cl16O could be achieved easily for a 1.5 % HCl matrix.

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 2: Advances in Methods and MS Instrumentation for Biomolecule Characterization

PMo-025

11:10 － 12:20

Applications of online- bioaffinity- mass spectrometry to structure andaffinity determination of neurodegenerative proteins from brain material

Stefan Slamnoiu1, Camelia Vlad1,2, Adrian Moise1, Mihaela Stumbaum1,2, Thomas Gronewold2,
Markus Perpeet2, Michael Przybylski1

Laboratory of Analytical Chemistry and Biopolymer Structure Analysis, Department of Chemistry, University ofKonstanz, Germany, 2SAW Instruments GmbH, Bonn, Germany

1

Keywords:
affinty mass spectrometry, SAW-ESI-MS, biosensor, neurodegenerative proteins, alpha synuclein
Novel aspects:
The new affinity-MS method based on the online coupling of SAW-biosensor to ESI-MS provides information of biopolymer
interactions at molecular level, enabling affinity detection and quantification, and ligand structure identification.
Abstract:
Introduction:
In the analysis of affinity interactions biosensors are a key analytical tool, being able to detect and quantify bioaffinity bindings.
Nonetheless, biosensors do not provide the chemical structure identification of ligands, for which mass spectrometry is a powerfultool［ 1 ］. Here we show that the online combination biosensor- mass spectrometry provides simultaneous chemical structure
identification and quantification of bioaffinity interactions.
Methods:
In this study we report affinity interaction and structural studies of antigen-antibody and peptide-protein complexes, as well as of
biological samples： mouse brain homogenate containing
α-Synuclein and human CSF containing Aß peptides. The SAWbiosensor was directly coupled to（i）ESI ion trap, and（ii）ESI FT-ICR-MS. The analysed complexes included Aß（ 1 -16）peptide /anti- Aß（ 1 -16）antibody； Aß（12-40）/ anti- Aß（17-28）antibody； Tau protein（isoform 2 N/ 4 R）/ anti-Tauspecific antibody（clone
TAU 5 ）； melitin / calmodulin, and α-Synuclein / anti- α-Synuclein antibodies. An anti- α-Synuclein antibody was used for theaffinity characterisation of mouse brain homogenate, while anti- Aß antibodies were employed in the analysis of human CSF.
Antibodies / proteins were immobilised via a thiol linker on the surface of a gold chip inside the biosensor, and their interactions
with the specific peptide or protein were monitored with the SAW biosensor. Using the online interface developed in ourlaboratory, the bound peptide or protein was eluted at acidic conditions from the gold chip and analysed by ESI mass
spectrometry, which provided the structural characterisation of the ligands［ 1 ］.
Results:
The online combination of SAW biosensor and mass spectrometry allowed the structural identification and affinity determinationof affinity-bound Tau, Aß and α-Synuclein. The mass spectrometric characterisation provided details on post translational
structure modifications of proteins and peptides present in biological samples, while the analysis of the binding kinetics delivered
the K D values for the antibody-antigen interactions. The key achievement of the method consists in the ability to structurally
characterize specific biopolymer molecules（Aß, α-Synuclein）, occurring at low concentrations in biological material. Using thisnew hybrid tool, screening for biomarkers of neurodegenerative diseases will become more efficient and accurate, leading toeasily quantifiable and interpretable results.
（Supported by grant KF2026662 from the Bundesministerium für Wirtschaft und Technologie, Berlin Germany）［ 1 ］Dragusanu, M. et al. J. Am. Soc.Mass Spectrom. 21, 2010, 1643-1648.

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 2: Advances in Methods and MS Instrumentation for Biomolecule Characterization

PMo-027

11:10 － 12:20

Determination of Opiates in Dried Blood Spots using Novel Flow-throughTechnology coupled to LC/MS/MS

Kenneth Lewis2, Dennis NAGTALON1
1

Agilent Technologies, 2OpAns

Keywords:
Dried Blood Spot, LC/MS, Opiates, Blood
Novel aspects:
Automated flow-through dried blood spot LC/MS analysis for opiates in blood
Abstract:
Dried blood spot technology（DBS）coupled to highly sensitive LC/MS/MS systems promises significant advantages in bioanalytics
both in the toxicology and pharmaceutical industry. However, two major disadvantages that limit the acceptance of DBS
technology are：（ 1 ）the laborious, time-consuming, error prone sample pre-treatment required, and（ 2 ）poor assay sensitivity
due to reduced sample volume. An automated card extraction system virtually eliminates off-line sample preparation with direct
online sample extraction of DBS cards without the need for hole punching while triple quadrupole mass spectrometry technology
provides unparalleled sensitivity and superior quantitation for the low sample volumes typically encountered with DBS analyses.
This presentation will describe our application of this integrated analysis system to the quantification of opiates in blood in the
ng/mL concentration range.

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 2: Advances in Methods and MS Instrumentation for Biomolecule Characterization

PMo-029

11:10 － 12:20

Optimization of serum N-linked glycan profiling using MALDI-TOF mass
spectrometry

Yongha In1,2, Heysun Maeng1,2, Sun Young Ahn1,2, Chang Won Park1,2, Yangsun Kim1,2
1

HUDSON SURFACE TECHNOLOGY, FORT LEE, NJ, USA, 2APPLIED SURFACE TECHNOLOGY INC., SUWON, KOREA

Keywords:
glycan, glycomics, homogeneous, crystallization, reproducibility
Novel aspects:
Uniform and homogeneous crystallization of matrix material by automatic matrix dispensing system is of critical importance toconsistent and quantitative analysis of glycans with MALDI-TOF MS
Abstract:
Most of currently available cancer biomarkers are glycoproteins such as CA125（MUC16）, alpha-fetoprotein（AFP）, and CA 15- 3（MUC 1 ）, and many tumor antigens are derived from glycans which are supposedly shed by glycoproteins. In addition, quite afew individual proteins have been reported to change their glycosylation patterns in cancer. On the basis of these findings, thestudy of global pattern of protein glycosylation, so called ’glycomics’, is now considered to be highly promising source of newcancer biomarkers. The method of choice for the precise profiling of glycans is mass spectrometry（MS）, which provides a wide
range of analytical power to successfully distinguish molecules with tiny differences in the molecular mass. A couple of successful
applications of glycan analysis by MS to the identification of potential biomarkers have been already published. Here, we report
the optimization of high-throughput profiling of N-linked glycans by MALDI-TOF MS with high reproducibility. N-linked glycans
were released from glycoproteins by peptide N glycosidase F（PNGase F）, and analyzed by MALDI-TOF MS. We focused on theminimization of variations in MS peak intensities that could be generated in each steps of MALDI-TOF MS. For the stableconsistency of the analysis, uniform and homogeneous crystallization of the matrix material on the sample spot was found to be
critical. Therefore, we developed automatic matrix dispensing system so as to enhance the homogeneous matrix preparation, andtested its performance regarding reduced variations in the MS data. Through the correlation of MALDI parameters, optimized
MALDI analysis conditions were determined, leading to stable reproducibility. The coefficient of variations（CV）in normalized
intensities of glycans decreased below 5 %, and the variations in absolute intensity were in a range of 0.69-9.25% for 32 samples,indicating significant improvement in minimizing variations of MS data. These results suggest that uniform and homogeneous
crystallization of matrix material by automatic matrix dispensing system is of critical importance to consistent and quantitative
analysis of glycans with MALDI-TOF MS.

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 2: Advances in Methods and MS Instrumentation for Biomolecule Characterization

PMo-030

13:30 － 14:40

Analysis of Panax ginseng extracts by comprehensive Two-Dimensional
Ultra High Performance Liquid Chromatography coupled with IT-TOF

Tatsunari Yoshida1, Shin-ichi Yaguchi2, Tsutomu Nishine1, Hirohisa Mikami1
1

Shimdzu Co., GADC, Japan, 2Shimadzu Co., MSBU, Japan

Keywords:
comprehensive 2 D-LC, LC × LC, two-dimensional-UHPLC, LCMS-IT-TOF, Panax ginseng extracts
Novel aspects:
In this study, we employed comprehensive two-dimensional UHPLC coupled with LCMS-IT-TOF for the analysis of Panax ginseng
extracts, a popular Chinese herbal medicine.
Abstract:
The great sample complexity often overgrows the separation power of mono-dimensional（ 1 D-）HPLC techniques； in such cases
implementation of multidimensional chromatographic systems is mandatory. In this respect, comprehensive two-dimensional liquid
chromatography（ 2 D-LC）is a powerful tool for the analysis of complex samples including pharmaceutical, biological, and natural
products. The favourable features of 2 D-LC were already demonstrated more than thirty years ago； since then, a lot of efforts
have been put in the development of multidimensional techniques of ever higher peak capacity. Recently, UHPLC（Ultra High
Performance Liquid Chromatography）has been successfully employed to remarkably decrease analysis time in the second
dimension of comprehensive LC system, and it is becoming popular to speed up 2 D-UHPLC separations.
In this study, we employed two-dimensional UHPLC for the analysis of Panax ginseng components, a popular Chinese herbal
medicine. A combination of RPLC（low pH）x RPLC（high pH）was selected and the chromatographic behavior of five major
ginsenosides（ginsenoside Rg1, Re, Rb1, Rc, and Rd）and other minor compounds was monitored. All experiments have been run
on a 2 D-UHPLC system（Shimadzu Prominence）, and interfaced through ESI to an hybrid mass spectrometer（Shimadzu LCMS-ITTOF）, which possesses both MSn ability of an ion trap and the excellent resolution and mass accuracy of time-of-flight. The
LCMS-IT-TOF and Composition Formula Predictor Software allowed the structural assignment of ginsenosides in Panax ginseng
with similar formula below 5 ppm for analysis.

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 2: Advances in Methods and MS Instrumentation for Biomolecule Characterization

PMo-031

11:10 － 12:20

Influence of Sample Preparation Techniques on the Sensitive Detection of
Peptides by MALDI-MS

YUSAKU HIOKI, Hiroki Kuyama, Chikako Hamana, Kohei Takeyama, Koichi Tanaka

Koichi Tanaka Laboratory of Advanced Science and Technology, Shimadzu Corporation, Kyoto, Japan

Keywords:
MALDI preparation technique, highly sensitive analysis
Novel aspects:
The optimization of matrix concentration and solvent system significantly improved the quality of MALDI mass spectra of peptides.
Abstract:
Introduction：
The quality of MALDI mass spectra of peptides is known to be significantly influenced by sample/matrix preparation procedures.
So far, many different kinds of sample/matrix preparation methods have been developed. The most widely used among them is adried-droplet method with the use of a saturated matrix solution on a stainless steel sample plate. Systematical understanding,however, of preparation techniques in terms of highly sensitive detection of low-abundance peptides without using special plateor anything complicated have been lacking in detail. To this end, we started a systematic study on optimizing conditions for matrix
concentration and solvent system for both sample and matrix. Results demonstrated a detection limit of below 10 amol peptides
using alpha-cyano- 4 -hydroxycinnamic acid（CHCA）as a matrix with sufficient signal intensities.
Methods：
A 0.5 uL volume of peptide solutions with an equal volume（0.5 uL）of matrix solutions were mixed on a stainless steel MALDI
plate（Shimadzu/Kratos, UK）and allowed to dry at ambient atmosphere. Sample and matrix solutions were prepared using 0.1%（v/v）aqueous TFA for acidification. MALDI-TOF MS（AXIMA Performance™； Shimadzu/Kratos, UK）measurement using CHCA asa matrix was performed in positive reflectron mode. MALDI-QIT-TOF MS （AXIMA Resonance™； Shimadzu/Kratos, UK）
measurement using 2,5-dihydroxybenzoic acid（DHB）as a matrix was performed in positive mode. Peptides and protein digest
standards were purchased from Waters（USA）.
Results：
To optimize the concentration of CHCA in matrix solutions, we examined several points of the concentrations ranging from 0.01 to
10 mg/mL. The CHCA concentration dependence of MALDI-MS response was examined by using the signal-to-noise ratio. Results
demonstrated the extremely high S/N ratio of peptide peaks in MALDI mass spectra at a CHCA concentration of around 0.1 mg/
mL. The enhanced MALDI-MS response（about 10-100 times）compared with the use of a saturated（10 mg/mL）matrix solution
was observed. For example, limits for the detection of human ACTH 18-39 was 10 amol under optimal condition using the drieddroplet technique in MALDI-TOF MS measurement（positive reflectron mode）. Furthermore, the effect of varying solvent systems
for sample and matrix preparation was examined in terms of matrix solubility, reducing adsorption of sample to plastic tubes, andsmall deposition on target plate. The condition by optimizing matrix concentration and solvent system obtained in this study using
ACTH 18-39 was verified with four other peptides and achieved significant improvement of sensitivity by two to three orders ofmagnitude, comparing with the conventional conditions. The optimization of concentration and solvent composition of both CHCA
and DHB matrices significantly improved the quality of MALDI mass spectra of peptides, and details will be discussed in thissession.

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 2: Advances in Methods and MS Instrumentation for Biomolecule Characterization

PMo-032

13:30 － 14:40

Development of modified micro-array MALDI plates with carboxylated
dopamine for the analysis of small molecules.

Duhee Park1,2, Hyunjung Seo1,2, Chang Won Park1,2, Yangsun Kim1,2
1

Applied surface technology Inc., Suwon, Korea, 2Hudson Surface Technology, Inc., Fort Lee, NJ, USA

Keywords:
dopamine, matrix, carboxylation, coating, DHB
Novel aspects:
Micro-array carboxylated dopamine plates can efficiently assist the ionization of samples for the detection of small molecules
without matrix
Abstract:
Dopamine in mussel is known as adhesive material, which is abundant in the proteins found at the interface between adhesive
pads and opposing surfaces, and helps mussels bind to organic or inorganic surfaces. This adhesive characteristics and molecular
structure of dopamine is similar to 2,5-dihydroxybenzoic acid（DHB）with substitution of a carboxyl group to an amine group. Since
DHB is one of the preferred matrix material for MALDI-TOF mass spectrometry（MS）, we attempted to make dopamine-coateddisposable target plates for the analysis of small molecules by MALDI-TOF MS, and tested the plates for possible use as a sample
platform for MALDI-TOF MS without additional matrix material, anticipating that dopamine on the plate can play a role of matrix
material. Micro-patterning with chemical etching and photolithography were used to make an array of 50-500 μm hydrophilic spots
with 100-300 μm gap on hydrophobic plates. It has 5000 more spots in comparison with those of standard microscope slide.
Patterned stainless steel surfaces have been coated with 3,4-dihydroxy-L-phenylalanine（DOPA）by immersing the stainless steel
plates in DOPA solution with gentle shaking. The adhesion of dopamine is confined only within the hydrophilic spot area. The
thickness of coated film increased with coating time, and the color of hydrophilic spot area turned dark brown when it wasmonitored by a microscope. A homogeneous and fine surface structure was shaped eventually. The ionization of some small
molecules on the dopamine-coated plates for MALDI process of MS was increased when observed in MS spectra of the small
molecules, indicating that coated dopamine layer on the sample spot could replace MALDI matrix material. Further modification（carboxylation）of dopamine-coated surface with succinic anhydride has been performed and applied to the detection of small
molecules by Laser Desorption Ionization MS. The molecular ion peaks were obtained from carboxylated dopamine plates andcompared with those from different plates. The ionization yields from micro-array carboxylated dopamine-coated plates werecomparable with DHB matrix spots. The clear identification of small molecules by MALDI-TOF MS was possible due to theabsence of interference from matrix peaks. In conclusion, micro-array carboxylated dopamine plates can efficiently assist theionization of samples for the detection of small molecules without matrix, and can provide a preferred sample platform for highthroughput MALDI-TOF MS analysis of small molecules.

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 2: Advances in Methods and MS Instrumentation for Biomolecule Characterization

PMo-033

11:10 － 12:20

Bonded Zwitterionic Stationary Phases and Mass Spectrometric Detection
for Bioanalysis

Patrik Appelblad, Wen Jiang, Jonsson Tobias, Petrus Hemstrom
Merck SeQuant, Umea, Sweden

Keywords:
LC/MS, HILIC, Liquid chromatography, Detection sensitivity
Novel aspects:
Hydrophilic interaction chromatography（HILIC）has excellent compatibility and provides enhanced detection sensitivity whencombined with mass spectrometric（MS）detection.
Abstract:
Introduction
Hydrophilic interaction chromatography（HILIC）is a useful tool for separation of polar compounds in biofluids.HILIC has excellent
compatibility and provides enhanced detection sensitivity when combined with mass spectrometric（MS）detection. HILIC uses ahigh percentage of organic solvent in the mobile phase, hence easy to volatilize in the detector interface.
Biofluids is generally of aqueous character and contain many polar compounds, and their metabolites. The liquid part of blood,
devoid of cells and platelets, is termed either plasma or serum depending on how the sample has been prepared. By applying
protein precipitation protocols prior to analysis, matrix（proteins, salts, minerals and lipids）can effectively be removed and allow
sensitive methods to be developed. In practice, this means adding a volume portion of plasma, serum, or urine in a minimum offour volume portions of organic solvent to stimulate protein precipitation. Besides effective matrix removal, the procedure makes
the biological sample perfectly aligned to a HILIC separation, and large sample volumes can be injected without a risk ofcompromising the separation efficiency. This poster will illustrate how different bonded zwitterionic HILIC phases have beenutilized for bioanalytical purposes.

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 2: Advances in Methods and MS Instrumentation for Biomolecule Characterization

PMo-034

13:30 － 14:40

Quantitative Analysis of Free and Total Thyroid Hormones in Serum Withand Without Online Sample Cleanup using LC/MS/MS

Rory Doyle, Kevin McCann
AGILENT TECHNOLOGIES

Keywords:
thyroid, hormones, LC/MS, serum, online sample cleanup
Novel aspects:
Analysis of thyroid hormones in serum by LC/MS/MS with/out online sample cleanup
Abstract:
Liquid chromatography triple quadrupole mass spectrometry（LC/MS/MS）has become an essential tool for small molecule
quantitation due to its high sensitivity and specificity, excellent reproducibility and the ability to perform simultaneous analysis ofmultiple analytes. Thyroid hormones can be challenging compounds to analyze due to the low levels relevant to clinical research.
In order to address this challenge, sensitive liquid chromatography-tandem mass spectrometry methods for the simultaneous
analysis of Thyroxine（T 4 ）,3,3’,5-Triiodothyronine（T 3 ）and 3,3’,5’-Triiodothyronine（rT 3 ）in serum samples were developed.
Both positive and negative ionization techniques were investigated, but optimal results were achieved using positive electrosprayionization（ESI）. Quantitative analysis was performed using multiple reaction monitoring（MRM）transition pairs for each analyte
and internal standard. Final concentrations were calculated by comparing the response of the analyte to a known concentrationof internal standard and plotting the result on a calibration curve developed using stripped human serum spiked with standards.
Standard liquid chromatography（LC）was used initially.The LC system was also configured for sample enrichment, comprised of
two pumps, a 2 position/ 6 port valve, an enrichment column and a reverse-phase C18 analytical column to assess the added
value of online sample cleanup. Methods were compared using measurements made in serum Standard Reference material（SRM9971）. The LC-MS/MS parameters were optimized and calibrated over the range of 1 pg/ml to 500 pg/ml for free and 1 ng/
ml to 500 ng/ml for total Thyroid hormone concentrations. The LOQ was the lowest calibrator. The calibration curves show
excellent linearity and reproducibility across the entire range of analysis.

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 2: Advances in Methods and MS Instrumentation for Biomolecule Characterization

PMo-035

11:10 － 12:20

Selective introduction of 4-amidinobenzyl moiety into peptide N-terminus
for straightforward de novo sequencing

Toru Yoshikawa, Masahiro Miyashita, Hisashi Miyagawa
Kyoto University, Kyoto, Japan

Keywords:
de novo sequencing, charge derivatization, fragmentation, selective modification, peptide
Novel aspects:
This study offers much easier and more straightforward sequence determination of peptides from complex mixtures usingchemical modification approach
Abstract:
MS/MS has become a major technique for protein and peptide sequencing, owing to its high sensitivity, rapid analysis, andapplicability to complex peptide mixtures. In particular, de novo peptide sequencing（that is, elucidation of amino acid sequence
without the prior knowledge of the sequence information）is powerful when the target peptides are from unexplored organisms
lacking genomic/proteomic information, or those undergone post-translational modifications. However, due to incomplete
fragmentation of the peptide backbone, de novo peptide sequencing remains a challenging task. In this context, we havedemonstrated that N-terminal derivatization with a 4 -amidinobenzoic acid greatly improves peptide fragmentation under lowenergy CID conditions, such as in a triple quadrupole or an ion trap mass spectrometer. 4 -Amidinobenzoyl moiety can be
introduced into N-terminal amino groups by using a succinimide active ester method, which is commonly used for derivatization ofprimary amines. However, introduction of 4 -amidinobenzoyl moiety usually occurs not only at N-terminal a-amino groups, but also
unfavorably at side chains of lysine and tyrosine residues by this method. This often results in the production of a complex mixture
which makes the analysis even more difficult. Recently, a new method for selective modification at N-terminal a-amino groups of
peptides using an oxidative amide synthesis method via metalloporphyrin-catalyzed aromatic alkyne oxidation was reported. In
this study, we propose a new method to selectively introduce 4 -amidinobenzoyl moiety to the peptide N-terminus by utilizing thisreaction.
As an alkyne carrying a 4 -amidinobenzoyl moiety, N-（ 4 -ethynylphenyl）- 4 -amidinobenzamide was designed, and synthesized bymixing 4 -amidinobenzoic acid and 4 -ethynylaniline in the presence of BOP reagent in dry pyridine. The product was purified by
HPLC. Metalloporphyrins［Mn（2,6-Cl 2 TPP）Cl］were prepared by refluxing porphyrins（2,6-Cl 2 TPP）and MnCl 2 in DMF. Peptide
derivatization with N-（ 4 -ethynylphenyl）- 4 -amidinobenzamide was accomplished as follows： to a solution of lysine-containing
peptides in 50 % aqueous acetonitrile, N-（ 4 -ethynylphenyl）- 4 -amidinobenzamide（500 eq）, Mn（2,6-Cl2 TPP）Cl（ 6 eq）in a
NaHCO 3 aqueous solution containing H 2 O 2 were added. The mixture was kept at room temperature overnight, and partiallypurified with a MonoSpin C18 spin column（GL Sciences, Inc）to remove the unreacted reagents before LC/MS n analysis. The LC/
MSn analysis was performed using Shimadzu LCMS-IT-TOF hybrid mass spectrometer. All mass spectra were obtained in a positive
mode.
LC/MS analysis of the reaction solution showed that only a single moiety of N-（ 4 -ethynylphenyl）- 4 -amidinobenzamide wasintroduced into each of the tested lysine-containing peptides in a high yield without significant production of by-products. Production spectra of derivatized peptides confirmed that the N-terminal a-amino group was specifically modified and that the lysine andtyrosine side chains remained intact. The improving effect of amidino benzoyl moiety on the peptide fragmentation was similar tothat observed in the case of derivatization with 4 -amidinobenzoic acid using the succinimide active ester method. In conclusion,
we have established a method that can selectively derivatize the N-terminal a-amino group for de novo peptide sequencing. Thismethod offers much easier and more straightforward sequence determination of peptides from complex mixtures.

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 2: Advances in Methods and MS Instrumentation for Biomolecule Characterization

PMo-036

13:30 － 14:40

Extractive electrospray ionization mass spectrometry (EESI-MS) for
sensitive detection of iodine-129

Zhongchen Wu1, Yafei Zhou2, Bin Jia2, Huanwen Chen2

1
Shandong Universitiy at Weihai,Weihai city,China/Shandong, 2East China Institute of Technology,Nanchang city,China/
Jiangxi

Keywords:
EESI-MS, iodine-129
Novel aspects:
an EESI-MS based method has been developed for sensitive detection of ultratrace iodine-129 in various liquid samples.
Abstract:
Detection of radioactive species plays an important role in public safety. Due to the high afnity to the human body and very long
physical half-life（15.7 million years）, Iodine-129 is considered one of the most hazardous substances and must be monitored withhigh accuracy and precision.
EESI is an ambient ionization technique targeting to direct ionize samples for mass spectrometric detection with minimal（orwithout）sample pretreatments. Ultratrace uranyl species1 and its isotopic ratio（235U/238U）2 have been accurately detected using
multiple-stage EESI mass spectrometry in natural water samples.
In this work, an EESI-MS based method has been developed for sensitive detection of ultratrace iodine-129 in various liquidsamples. The base peak at m/z 129（ 129I-）was adopted for quantification. The calibration curve showed good linearity within awide range of 6.5-208 ppt（R=0.996）. The limited detection（LOD）is 1.38 ppt, which was measured at three times of the standard
deviation of the blank determination, and with the corresponding relative standard deviation（RSD）values of 4.4 %-6.8%.
Since most iodine species are easy to be enriched and absorbed in liquids in the form of ions, our method offers a rapid, sensitiveand specific approach for the detection of iodine-129 ions, providing potential applications many cases including nuclear leakage
or nuclear explosions.
Acknowledgement(s)Financial support from Jiangxi Key Laboratory for Mass Spectrometry and Instrumentation（JXMS201106）is highly appreciated.
References
1. Mingbiao Luo, Bin Hu, Xie Zhang, Daofeng Peng, Huanwen Chen, LIli Zhang and Yanfu Huan. Extractive electrospray ionization
mass spectrometry for sensitive detection of Uranyl Species in Natural water samples. Anal. Chem., 2010, 82, 282-289
2. Chunxiao Liu, Bin Hu, Jianbo Shi, jianqiang Li, Xinglei Zhang and Huanwen Chen. Determination of uranium isotopic ratio（235U/238U）using extractive electrospray ionization tandem mass spectrometry. J. Anal. At. Spectrom., 2011,26, 2045-2051

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 2: Advances in Methods and MS Instrumentation for Biomolecule Characterization

PMo-037

11:10 － 12:20

De novo peptide sequencing facilitated by N-terminal charge
derivatization with high proton affinity

Yoichiro Nihashi, Hiroyuki Awane, Masahiro Miyashita, Hisashi Miyagawa
Kyoto University, Kyoto, Japan

Keywords:
de novo sequencing, peptide, derivatization, stable isotope-labeling, low-energy CID
Novel aspects:
The approach utilizing derivatization reagents with high proton affinity as shown in this study offers a novel de novo peptide
sequencing method under low-energy CID conditions.
Abstract:
Recent advances in various tandem mass spectrometric techniques have enabled the facile determination of the amino acidsequences of peptides. In addition, the development of comprehensive databases of protein primary sequences predicted fromthe genomic sequences of various organisms has boosted the efficiency with which proteins can be identified by mass
spectrometry. However, since database coverage is limited to the components of major organisms in most cases. Amino acidsequencing without the assistance of a database（de novo sequencing）is still essential in the identification of peptides andproteins from unexplored organisms.
De novo sequencing can be carried out by interpreting mass differences between a series of consecutive ions of N- or C-terminal
fragments generated by collision-induced dissociation（CID）. However, fragmentation of peptides often gives complicated andincomplete product ion spectra, making de novo sequencing difficult. Newly developed mass spectrometric techniques for betterfragmentation, such as ECD or ETD, and for highly accurate mass measurements, such as FT-ICR mass spectrometry or Orbitrap,
have greatly improved the efficiency of de novo sequencing； however, there still remain difficulties in obtaining mass spectra thatallow easy identification of key fragment ions for sequencing. The difficulties come mainly from incomplete fragmentation of thepeptide backbone. To overcome these problems, chemical modifications at the N-terminus of peptides have been attempted formore regular and informative fragmentation. The introduction of a positively charged group, such as quaternary or tertiaryammonium and quaternary phosphonium groups, at the N-terminus is known to enhance the relative intensity of N-terminal
fragment ions（b-ions）in product ion spectra, significantly facilitating the formation of whole series of b-ions. Similarly, it has beenreported that the introduction of a negatively charged group at the N-terminus enhances the generation of C-terminal fragmentions（y-ions）with suppression of the occurrence of b-ions.
In this study, we evaluated the improving effect of N-terminal charge derivatization on peptide fragmentation under low-energy
CID conditions using compounds with high-proton affinity. Derivatization reagents containing guanidino or amidino moiety wereintroduced into the N-terminus of peptides, and their effects on fragmentation were compared using an ion trap mass
spectrometer. Among four reagents differing in proton affinity, derivatization with 4 -amidinobenzoic acid, which has moderately
high proton affinity, brought about the most effective fragmentation in all peptides used in this study. This result clearly indicatedthat there was an optimal affinity for efficient fragmentation of peptides.
However, even with the aid of derivatization, identification of b-ions was still not very easy in some cases due to the overlap of
b-ions with other series of ions, This was especially the case when multiply charged ions were selected as a precursor. In thisstudy, to specifically identify b-ions from other fragment ions, stable isotope-labeling of derivatization reagents was further
investigated. 15N was introduced into 4 -amidinobenzoic acid, and its effect on specific identification of b-ions was evaluated. As
expected, b-ions were effectively identified by comparing the spectra between peptides derivatized with 15N-labeled or nonlabeled 4 -amidinobenzoic acid, which allowed straightforward interpretations of spectra.
In conclusion, N-terminal derivatization with 4 -amidinobenzoic acid undoubtedly improves the peptide fragmentation pattern,which may offer a novel de novo peptide sequencing method under low-energy CID conditions.

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 2: Advances in Methods and MS Instrumentation for Biomolecule Characterization

PMo-038

13:30 － 14:40

Efficient and rapid multienzymatic limited digestion (MELD) method forcomplete protein characterization and bottom-up de novo sequencing

Gabriel Mazzucchelli1, Tyler A Zimmerman1, Marie-Alice Meuwis1, Nicolas Smargiasso1,
Michel Degueldre1, Dominique Baiwir1, Laurent Leclerq2, Edwin De Pauw1
1

University of Liege, Liege, Belgium, 2Johnson & Johnson Pharmaceutical R&D, Beerse, BELGIUM

Keywords:
de novo sequencing, maximal sequence coverage, multienzymatic digestion, post translational modification, proteincharacterization
Novel aspects:
By allowing missed proteolytic cleavages, multienzymatic limited digestion method provides better sequence coverage, accurate
PTM characterization and allows de novo sequencing of long sequence tag using our“sequence assembly“ software
Abstract:
This method relies on a combination of multienzymatic and limited proteolytic digestions on a purified protein, for completesequence and posttranslational modification（PTM）characterization. It generates numerous different peptides with misscleavages, greatly increasing the probability of analyzing the entire protein sequence.
This 2 hours digestion protocol was validated on several commercial proteins and applied to the identification and localization of
Beta-Lactoglobulin posttranslational modifications induced by a reactive metabolite of acetaminophen（NAPQI）. This experimentshows 100 % sequence coverage involving information on peptide identity and modifications localization. Strong identification
confidence was obtained due to multiple overlapping peptides matches with the given sequence tag and to a high number ofoverlapping peptides assignments. A program to assemble amino acid sequences of entire proteins from such data wasdeveloped.
HSA, Myoglobin, Lysozyme, Invertase and α-Amylase（ 5 μg）after reduction-alkylation, were independently digested by proteases
mixtures, 2 h, in standard buffer and 2 enzymes mixtures were tested, consisting of Trypsin, GluC and Chymotrypsin at weight
enzyme/weight total protein ratios of respectively： 1 /500, 1 /375, 1 /250 for Mix 1 and 1 /3000, 1 /2250, 1 /1500 for Mix 2. Eachdigest（ 1μg）was analyzed by nano-LC ion trap MS（Ultimate 3000, Dionex, Amazon, Bruker）by nano-UPLC Q-TOF（nanoAcquity Synapt G2, Waters）with identical 60min. gradient. Data were treated with Data Analysis 4.0, Protein Scape 3.0（Bruker）
and PLGS 2.5（Waters）. β-Lactoglobulin modifications（NAPQI addition）were induced by activated acetaminophen using anelectrochemical flow-through cell（model 5125, ESA Biosciences）.
Complete multi-enzymatic digestion presents the advantage of compatibility with most existing protein sequences, due to highnumber of accessible specific cleavage sites（ 6 using trypsin, Glu-C and chymotrypsin）, but the limitation of such a protocol isthat the very small proteolytic peptides generated are not compatible for LC-MS analysis. By controlling and limiting digestion
reaction time, miss-cleavages are generated and after database searches, the number of unique peptides matching the protein
sequence highly increases. The analysis of the five proteins gave the following results： HSA, Myoglobin and Lysozyme wereidentified with 100% sequence coverage with respectively 421,108, and 49 unique peptides； Invertase： 75.9% sequence coverage 158 unique peptides,α-Amylase： 78.5 % sequence coverage - 60 unique peptides. The missing sequence coverages in the last two
proteins mainly correspond to sequences known to contain post-translational modifications（PTM）sites. For example, when
invertase was N-deglycosylated the sequence coverage reached 92.9 % showing clearly which peptides were modified. A
comparison analysis of NAPQI modified and unmodified β-Lactoglobulin clearly indicated which cysteine residues were modified
by NAPQI. By integrating the extracted ion chromatogram of the unmodified peptides found in common for both conditions,quantification could be achieved and the occupation frequency of this specific modification was evaluated. With our method, atleast an average of 10 different peptides covering each sequence stretch of the protein could be obtained. This greatly increased
the robustness and the significance of the identifications and the localization of the modifications. The protocol as presented here
and tested on several pure proteins digested in solution certainly improves the general“bottom-up“ strategy applied for highly
confident protein identification and would allow better protein characterization, even for those having PTMs, such as
N-glycosylations. Moreover this simple and rapid method could also find helpful applications to assemble amino acid sequence ofnovel proteins using our“sequence assembly“ software.

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 2: Advances in Methods and MS Instrumentation for Biomolecule Characterization

PMo-039

11:10 － 12:20

ANALYSIS OF 8 KINDS OF ESTROGENS IN ENVIRONMENTAL WATER BYULTRA HIGH PERFORMANCE LIQUID CHROMATOGRAPH HYBRID TRIPLEQUADRUPOLE MASS SPECTROMRTER

hongyuan Hao, Jinting Yao, Luying Zhou, Hengtao Deng, Qiang Li
Shimadzu (China )Co., LTD. shanghai, china

Keywords:
UHPLC-MS/MS； Estrogen； environmental water；
Novel aspects:
This poster is development of an Ultra fast analysis method for determingestrogens in envirmental water.
Abstract:
A UHPLC-MS/MS method was developed for the determination of Estrogens in environmental water. 8 kinds of Estrogens（ Estriol,
17-alpha-Estradiol, 17a-Ethinylestradion, Estrone, 17-beta-Estradiol, Diethylstilbesterol ,hexestrol, dienestrol）were seperated by
UHPLC system with a gradient elution program, and detected by Shiamdzu Triple Quadrupole MS LCMS-8030. The linear range wasfrom 0.5 to 500 μg/L with correlation coefficients（r）more than 0.999. Retention times and peak areas results were highly
reproducibility. The limit of quantification（LOQ）were less than 2 ng/L for all of Estrogens. This method is rapid, efficient andhighly sensitive for quantitative analysis of 8 Estrogens in environmental water.

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 2: Advances in Methods and MS Instrumentation for Biomolecule Characterization

PMo-040

13:30 － 14:40

Identification of metabolic pathways related to xylose fermentation
performance in recombinant yeast by metabolome analysis

Hitoshi MITSUNAGA1, Danang Waluyo1, Haruyo Hatanaka2, Takeshi Bamba1, Eiichiro Fukusaki1
1Osaka University, Osaka, Japan, 2Suntory Business Expert Limited.

Keywords:
xylose, ethanol, performance, PLS, methionine
Novel aspects:
Based on metabolome analysis, PLS might semi-rationally be able to determine the pathway related with xylose fermentation
performance.
Abstract:
Xylose is the second most abundant sugar after glucose in the hydrolyzate of lignocellulose. Thus, construction of recombinant
yeast that efficiently converts xylose into ethanol became an important task for successful ethanol production from lignocelluloses.
However, due to the slow consumption of xylose and relatively high yields of by-products such as xylitol and glycerol, the ethanol
yield is very low, which suggests that some metabolic regulations might（have）been induced during xloseassimilation in therecombinant yeast. However, there is no effective strategy of molecular breeding to rationally improve the artificial xylose
metabolism, because metabolism in the recombinant yeast has not yet been elucidated. This study aims to develop a semirational metabolome analysis-based method for identification of metabolic pathways related to xylose fermentation performancein recombinant yeast.
Seven independent xylose assimilating yeast strains were subjected to the ethanol fermentation experiment in artificial synthetic
media containing xylose as a sole carbon source in order to quantitatively evaluate the performance of fermentation. Time courseof several important compounds, including xylose, ethanol, glycerol and xylitol were monitored by means of HPLC. Seventy-fiveand forty-two compounds（117compounds in total）from yeast cells were determined by GC-MS and CE-MS, respectively.
Orthogonal projection to latent structure was conducted to reveal the relationship between the 117 metabolites and ten indicesof fermentation performance. Analysis of important metabolites for prediction model construction revealed that several aminoacids biosynthesis are important for xylose fermentation performance of the recombinant yeast. Several genes which might
correlate with these important pathways were suggested to be the target for genetic modification for improvement of xylose
fermentation performance. Based on the OPLS analyses, some genetic modification were introduced to the xylose assimilating
recombinant yeast. The result of the fermentation test of this recombinant yeast revealed that the modulation of methionine
biosynthesis-related genes would contribute to reduction of xylitol production without significant loss of function for both xylose
consumption and ethanol production.
To prove the universality of the contribution of methionine for reduction of xylitol production in the recombinant yeast, wild-typestrain was subjected to the fermentation experiment with a methionine-rich medium. The result showed that xylitol production
was decreased without significant changes in xylose consumption and ethanol production. This suggested that methionine
biosynthesis might be generally related to xylitol production in recombinant yeasts.

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 2: Advances in Methods and MS Instrumentation for Biomolecule Characterization

PMo-041

11:10 － 12:20

Study using synthetic organic acids in Thermo Finnigan LTQ Orbitrap
APCI. Application to the acidic fraction, from Brazilian marine oil.

Luzia Koike1, Eugenio V Santos Neto2, Rosane A Fontes2, Rosane N Castro3

Campinas State University - Campinas- Brazil- southeast, 2Petrobras R&D Center,Rio de Janeiro, RJ., 3Federal Universityof Rio de Janeiro, RJ.

1

Keywords:
Biodegradation, Brazilian oils, Aciod Components
Novel aspects:- Direct injection in High resolution UPLC-LTQ-Orbitrap APCI（-）,geochemistry parameters from acid fractions compare whithydrocarbons and obtain biodegradations parameters
Abstract:
Study using synthetic organic acids as standards in Thermo Finnigan LTQ Orbitrap APCI. Application to the acidic fraction, from
Brazilian marine oil.
Autores： Luzia Koike 1； Rosane Alves Fontes 2； Eugenio Vaz dos Santos Neto 2； Rosane Nora Castro 3；Flávia Airoldi
Aguayo 1,Nayara S.Inoue1, Luiz G.Bonato1 and Luiz Fernando Lima1
1 Campinas State University, Organic geochemistry Group, 2Petrobras R&D Center,Rio de Janeiro, RJ.and 3 Federal University of
Rio de Janeiro, RJ.
Study of biodegradation of the evaporitic marine oils from northeastern Brazil Potiguar Basin named ME-a to Me-d, the acid
fractions, were analyzed using Thermo-Finnigan-LTQ-Orbitrap in APCI（-）mode. Standards acids were synthesize： 5 a（H）
homopregnanic 1 and 5 b（H）cholanic 2with similar basic structures of the steranes（K. Peters and M. Moldowan, 1993 andRaphael Ikan,1993）, 8,13-tetrahydrocopalic 3 and 15-isocopalic acid 4 with similar basic structures to studied terpanos（K. Peters e  .The compounds were analyzed using isopropanol and toluene（ 1 ： 1 ）in the APCI source negative mode to
M. Moldowan,1993）
study the profiles and fragmentation patterns. Although the main fragments in the all standard spectra show the loss H2O or CO（William J. Griffths 2003）from the molecular ion, we also see important fragments that are at 217.16 and 191,08 and 249.25 Da, gives
respectively, as in steranes and terpanes. In previous work（L.Koike et al., 1992； Sidney G. De Lima et al. 2010）the acid fractions
originating from Brasilian oils were studied using biodegradation and we derivatized through four steps of the chemical reactions.
After this process the yield of the acidic fractions were near 20 % . In the present work the acid fractions were extracted andcleaned up using SPE-NH2 , increasing the recuperation to 80-90 %. After the extraction and clean up, the samples were injected
directly in the LTQ Orbitrap UPLC by APCI in the negative mode. Spectra were acquired at high resolution.
Comparing biomarker parameters from the hydrocarbon fraction with those of the acid fraction reveals that biodegradation
increases from ME-a to ME-d. All the related parameters are closely similar in maturity from the ME-a to Me-d and the ratio
between steranes/terpanes are characteristic of marine oils. Carboxylic acids can help to refine the classification of degrees ofbiodegradation of oils, and can be applied to other studies such as secondary cracking of oil, which is a crucial step ofquantification. After obtaining sample profiles we used the software COMPOSERR to summarize the data and related parameters
for the acid fraction. Because the software summarizes using the insaturation índex DBE（Double Bond Equivalent）with thissoftware it was possible to see the serial linear acid（DBE 1.5）biodegradations increasing from ME-a to ME-d. In the O2（DBE 2.5）
we saw linear substituted in monocyclic acids. From the intracelular layer of alicyclic bacyllus, showing increases from Me-a to
ME-d. The O 2 distribution results show that in components from acid compounds with molar masses until C 39 carbons. Thisphenomenon increases in the pentacyclic and tetracyclic compounds from ME-a to Me-d. Using a lacustrine fresh water oil sample
together with the marine ares ME-a to ME-d in the chart y axis the ratio hopanes/steranos（S/H）versus hopanes/steranes using
GC-MS data in the x axis, we can see that the in the lacustrine oil are ordered up in the range 20 to 25（S/H）and in marine
samples this S/H range remain in range 0 -5. This work is the first in the literature and represents a major advance in the study ofbiomarkers acids.
L. Koike, L.M.C. Rebouças, F. de A.M. Reis, A.J. Marsaioli, H.H. Richbow and W. Michaelis,1992, Naphtenic acids from crude oils ofCampos Basin, , Org. Geochem. 18, 851

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 2: Advances in Methods and MS Instrumentation for Biomolecule Characterization

PMo-042

13:30 － 14:40

Standard Samples to Establish Benchmark Metrics and Enable
Optimization of Quantitative Proteomics Methods

Sean L Seymour2, Xu Wang1, Brian Williamson1, Ignat Shilov2, John F MacNamera1, Christie L Hunter2,
Fadi A Abdi1
1

AB SCIEX Framingham MA, 2AB SCIEX Foster City CA

Keywords:
iTRAQ, validation, benchmark, standard samples, quantitative proteomics
Novel aspects:
Standard samples designed to enable quantitative workflow benchmarking
Abstract:
We have been working to generate a family of carefully designed standard samples comprised of proteins spiked with known
variation into complex mixtures to enable the measurement of performance metrics of quantitative proteomics experiments.
Our goal is to generate benchmarks of key measures like precision and accuracy as a function of dominant variables such as
analyte concentration and extremity of fold change and then to use these benchmarks as a foundation to compare and improvemethods. The first quantitative strategy that is being evaluated using the standard sample set is iTRAQ reagent quantification.
First, a control experiment was undertaken to measure the identification rate impact on a constant quantity of just the E.coli
lysate component (1) unlabeled, (2) 4plex-labeled, and (3) 8plex-labeled, employing extensive replicates. Measuring with a
TripleTOF 5600TM system, the average peptide-level identification loss relative to the unlabeled sample was 11% and 24% for
4plex and 8plex, respectively (only 15% loss from 4plex to 8plex). In the current round of the study, the quantification accuracyof the known ratios will be evaluated and false discovery rate of detection of differential proteins will be measured by a targetdecoy approach. Once benchmarks have been successfully established, we will use these to improve our informatics
methodologies used to analyze isobaric quantitative data, compare performance in multiple laboratories, and compare to otherquantitative proteomics workflows using the same samples and metrics.

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 2: Advances in Methods and MS Instrumentation for Biomolecule Characterization

PMo-043

11:10 － 12:20

Improvement in Mass Measurement Accuracy and Resolving Power Usinga New Compensated Fourier Transform Ion Cyclotron Resonance Cell

Nobuyuki Shimura1, Magnus Palmblad2, Jens Fuchser3, Yuri E M Van Der Burgt2, M Goekhan Baykut3,
André M Deelder2
Bruker Daltonics K.K., Yokohama, Japan, 2Leiden University Medical Center, Leiden, Netherlands, 3Bruker Daltonik
GmbH, Bremen, Germany

1

Keywords:
Resolving Power Increase, FT-ICR-MS, New Compensated Cell
Novel aspects:
A new FTICR cell is tested with a number of realistic use cases
Abstract:
Introduction
A recently described Fourier transform ion cyclotron resonance（FTICR）analyzer cell（Nikolaev et al., JASMS 2011）with dynamic
harmonization of the electric fields in the Penning trap, together with improvements in vacuum system, electronics and instrument
control and data acquisition software, enables ultrahigh resolving power（>1,000,000）in m/z 200-2000 in a 12 tesla FTICR system.
We will show the improvements in a number of realistic use cases by comparing the new system with the previous generation
instrumentation at the same magnetic field. Examples include the resolution and accurate mass determination of peptides,alignment and hybridization with ion trap data in bottom-up proteomics, urine metabolomics and combinatorial chemistry.
Methods
Urine samples were prepared using standard desalting protocols on C18 solid phase extraction tips and spiked with known
concentrations of different analytes. Zebrafish embryos were homogenized, proteins extracted and digested using trypsin.
Standard peptides were synthesized from the C- to the N-terminus by Fmoc-based solid phase peptide synthesis with Tentagel S
AC as a resin. Peptide mixtures were separated by nanoscale liquid chromatography using a 15-cm 75 um i.d. PepMap C18（Dionex）column on a Dionex Ultimate 3000 system with a flow rate of 300 nL/min. All FTICR mass spectra were acquired on 12 T
solariX systems, with two different cell designs（Bruker Daltonics）. Tandem mass spectra were acquired on an amaZon ETD
speed ion trap（also Bruker）.
Preliminary Data
A resolving power that could easily be achieved in the new system with the compensated cell was significantly better than thebest that was ever reached in the old system. For instance, three standard peptides of elemental composition H100C73N14O26S,
H100C75N18O17S3 and H100C62N20O29S at m/z 811（doubly protonated）that could only with difficulty be resolved on the old systemwith a resolving power of 1,500,000, could easily be resolved to the baseline in the new system with a resolving power in excess of
4,000,000. Two synthetic peptides, with sequences MIM and HTH and elemental compositions H 31 C 16 N 3 O 4 S 2 and H 23 C 16 N 7 O 5
respectively, could be clearly resolved. The mass difference between these two peptides is only 0.000469 Da the well-known H 2S8vs
N4O split, which is the smallest common mass difference between any two peptides. These two peptides are also those closest
to 400 Da of all possible peptide pairs with this mass difference, and therefore useful for evaluation of instrument resolution at m/z
400.
Also for realistic samples such as urine metabolites and complex protein digests such as the zebrafish whole embryo, thepractically achievable resolving power on a time scale compatible with liquid chromatography（i.e. a spectral acquisition rate of
approx. 1 Hz）the new system outperformed the old, especially when internally calibrating each FTICR spectrum using peptides
identified in a separate ion trap analysis of the same sample, after chromatographic alignment of the FTICR and ion trap datasets.

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 3: Structures and Dynamics of Atomic and Molecular Clusters

PMo-044

13:30 － 14:40

Gas phase ion-molecule reactions of C2H5+ and C2H3+ with C2H2 using
low-temperature drift tube mass spectrometer

Kenichi Iwamoto, Haruto IKUTAOsaka Prefecture University, Osaka, Japan

Keywords:
ion-molecule reaction, low-temperature Drift tube
Novel aspects:
the low temperature rate constants of hydrocarbon ions and acetylene were measured
Abstract:
The results of a study of the ion-molecule reactions of C2H5 + and C2H3 + with acetylene will be reported. The association reaction
forming C 4 H 7 + is important to understanding the ion chemistry in Titan’s atmosphere. A few experimental data on association
reactions exist for temperatures below the temperature of liquid nitrogen. The thermal and low-temperature（125K）rate constants
will be reported. The experiments were carried out using the low-temperature drift tube mass spectrometer.

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 3: Structures and Dynamics of Atomic and Molecular Clusters

PMo-045

11:10 － 12:20

Assembly Modulated Metallosupramolecular Polymer Formation
Observed by CSI-MS and Other Analytical Methods

Masatoshi Kawahata1, Kadzuya Hirata2, Hiroshi Danjo2, Kentaro Yamaguchi1
1

Tokushima Bunri University, Kagawa, Japan, 2Konan University, Kobe, Japan

Keywords:
CSI-MS, Metallosupramolecular Polymer, cyclophane, Boron
Novel aspects:
Assembly modulated metallosupramolecular polymer formation of D 3 -symmetric tris（spiroborate）cyclophanes（Twin Bowls）
and［Fe（tpy） 2 ］ 2 + complex by adjusting countercharges, K+ or Ba 2 +, was definitely observed by CSI-MS and other analyticalmethods.
Abstract:
Much attention has been paid to the development of supramolecular polymers for the creation of functionalized nanomaterials,
and the development of metallosupramolecular polymers is recognized as one of the most important approaches for the creationof functionalized nanomaterials because these polymers show potential for charge or electron transfer and other optoelectronic
characteristics. We have reported the design and preparation of a new class of back to back twin bowls（Twin Bowls）of anionicD3-symmetric tris（spiroborate）cyclophanes that exhibit molecular recognition behavior via π - and electrostatic interactions at
both sides of a symmetry plane. Twin Bowls were readily prepared from 2,2’,3,3’-tetrahydroxy-1,1’-binaphthyls（THBs）and boricacid. Equimolar amounts of THBs and boric acid were mixed in DMF at 150 °C to afford Twin Bowls3-·（Me2NH2 +）3 in 85% yield.
Dimethylammonium would be generated by hydrolysis of DMF. In this reaction, almost no signals of other oligomeric structures
were detected by ESI-MS analysis. This molecular coupling behavior makes it possible to glue various cationic guest molecules toeach other via dual host-guest interaction. Particularly in the case of cationic metal complexes, iterative clathration takes place toform metallosupramolecular polymers. Twin Bowls recognize cationic［Ir（tpy）2］3+（tpy = 2,2’： 6 ’,2’’-terpyridine）complex guests
by electrostatic and π interactions to form metallosupramolecular polymer structures in the crystal and solution state. The
metallosupramolecular polymer structure of Twin Bowls and［Ir（tpy）2］3+ was unequivocally determined by single-crystal X-ray
diffraction analysis and iterative encapsulation of［Ir（tpy）2 ］3+ by Twin Bowls took place to form one-dimensional array of theguest molecules. In addition, two［Ir（tpy）2］3+ are in van der Waals contact each other through the cavity of Twin Bowls, showingthat the spherical cationic guests were effectively glued by Twin Bowls. And from the mearsurements of cold-spray ionization（CSI）-MS, more than 13 molecule-aggregates were observed. We hypothesized that this supramolecular polymerization wascaused from the balance of charge between hosts and guests, and changing this balance would affect the degree ofpolymerization, i.e. we could regulate the metallosupramolecular polymerization（elongation and dissociation）by adding a metal
cation as a control factor. By using CSI-MS, we examined the change of metallosupramolecular polymerization degree of Twin
Bowls and ［Fe（tpy）2 ］2+ complex when another metal cation （Ba 2+ or K + ） was added as a control factor. The
Metallosupramolecular polymer of Twin Bowls and［Fe（tpy）2］2+ complex showed the ion peak corresponding to Twin Bowls /［Fe（tpy）2］2+ complex = 1 / 1. The addition of potassium trifluoromethanesulfonate gave the degree of polymerization increased andthe ion peak corresponding to elongated metallosupramolecular polymer of Twin Bowls /［Fe（tpy）2］2+ complex = 2 / 3 wasobserved. Whereas the addition of barium trifluoromethanesulfonate gave the dissociation of the mettallosupramolecular polymer
and only the Ba 2+ ion was observed. These indicate that the polymerization with Twin Bowls and［Fe（tpy）2 ］2+ complex wasenhanced by the addition of a potassium cation, whereas the presence of a barium cation disturbed the association of themonomers.

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 3: Structures and Dynamics of Atomic and Molecular Clusters

PMo-046

13:30 － 14:40

Clusters of Water, Acetonitrile and Azeotropic Mixtures in Gas and Liquid
Phases Studied by Liquid-ionization Tandem Mass Spectrometry

Masahiko Tsuchiya1, Haruhiko Fukaya2, Yoshiaki Kurita3
1

Yokohama National University, Yokohama, Japan, 2Tokyo University of Pharmacy, 3Housei University

Keywords:
Clusters of water, Water, Azeotropic mixture, Ethanol, Acetonitrile
Novel aspects:
Liquid-ionization mass spectrometry was applied to clarify clusters existing in gas and liquid phases. Azeotropic mixtures showed
the presence of stable clusters which evaporate as they are in the liquid.
Abstract:
Studies of clusters are important for understanding the properties of liquids and the mechanisms of organic chemical reactions ina solution.
A tandem mass spectrometer（Extrel CMS, TMAX 2000）equipped with an improved Liquid-ionization（LPI）1,2) ion source was used
to measure mass spectra of hydrogen-bonded clusters in gas and liquid phases. The voltages applied to various electrode forfocusing sample ions affect the peak intensities. Therefore these voltages were adjusted to be appropriate. LPI mass spectral
patterns observed by the first mass spectrometer（Q 1 ）are affected only by the voltage applied to the needle tip（where sample
ions are formed）and the voltage difference between skimmer- 1 and skimmer-2. The influences of these voltages and mass
spectral patterns clearly indicate that neither adiabatic expansion nor condensation occur in the LPI mass spectrometer. Namely,
LPI mass spectra observed by Q 1 indicate information about the composition distributions of clusters（neutral）in gas and liquid
phases, that is, observed clusters are already in the liquid.
Mass spectra of water in the air have been measured and shown cluster ions of water, expressd as（H 2 O）n H+, with n up toaround 50. The numbers of water molecules（n）are related to the humidity in the air.3) Mass spectra of pure ethanol（99.5 %）showed neat ethanol cluster ions（C2H5OH）mH+ with m up to 12 and hydrate ions（C2H5OH）m（H2O）nH+ with m larger than 8 andn = 1 and 2. Hydrate ions, such as 8 -1, 9 - 1 and 10-1, were always observed, regardless of the quantity of background water. Even
when the quantity of water increased slightly, no hydrate cluster ions with m smaller than 7 were observed. Molar ratio（water/
ethanol）of the 9 - 1 cluster is 0.111 and agreed with the molar ratio（0.107）of azeotropic mixture of ethanol-water. The latter datasuggest the existence of stable clusters like the 9 -1, which evaporate as they are in the liquid, and that is the reason why theazeotropic mixture of ethanol-water exist. Results indicate that ethanol clusters with m = 2 to 7 and hydrates like the 9 - 1 exist
in the liquid phase.
Mass spectra of acetonitrile were affected by the presence of a small amount of water and showed the intense peaks
corresponding to 2 - 0 and 2 - 1 of（CH3CN）m（H2O）nH+. Mass spectra of azeotropic mixture of acetonitrile-water showed intense
peaks corresponding to the cluster ions of 2 -0, 2 - 1 and 3 -1, which are often observed in HPLC-MS when using acetonitrile andwater as the reversed phase eluate. Results indicate that these clusters are stable enough to evaporate as they are in the liquid
phase.
When using high flow rate（>10 micro-L/h）of the sample solution, mass spectra observed by the second mass spectrometer（Q 3 ）showed larger cluster ions than those observed by Q1. The primary ions must be the ions produced in the ion source andreact with the sample vapor flowing into the vacuum. Ion-molecule reaction must occur in the collision chamber（Q 2 ）, wheremultiple collisions should occur to produce larger cluster ions. Mass spectra observed by Q 3 indicate the results of ion-cluster
reactions.
References
1 ）M.Tsuchiya, Mass Spectrom. Rev. 17（1998）51
2 ）M.Tsuchiya, Y.Shida, K.Kobayashi, O.Taniguchi, S.Okouchi, Int. J. Mass Spectrom., 235（2004）229.
3 ）M.Tsuchiya, T.Tashiro, A.Shigihara, J. Mass Spectrom. Soc. Jpn. 52（2004）1.

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 3: Structures and Dynamics of Atomic and Molecular Clusters

PMo-047

11:10 － 12:20

ICP-MS and IR techniques used in metal detection and control of somenew synthetized materials with bioactive potential

NICOLETA C VEDEANU1, Ramona Stanescu2, Dana Alina Magdas3, Cezara Voica3

1’’IULIU HATIEGANU’’ UNIVERSITY OF MEDICINE AND PHARMACY, CLUJ-NAPOCA, ROMANIA, 2Babes-Bolyai University,
Faculty of Physics, RO-400084, Cluj-Napoca, Romania, 3National Institute for Research and Development for Isotopic
and Molecular Technologies, RO-400293, Cluj-Napoca, Romania

Keywords:
ICP-MS, IR, bioactive material
Novel aspects:
This work proposes the investigation of new synthetyzed materials with bioactive potential for bone rapair and reconstruction
using ICP-MS and IR as a necessary method for toxic metals determination
Abstract:
ICP-MS is a flexible technique that offers many advantages over more traditional techniques for elemental analysis and it has
become a widely used tool, for both routine analyses and for research in a variety of areas. Besides the classical tests of soil,
water, food, currently a wide range of biological samples, both solid and liquid can be analyzed by ICP-MS： blood, urine, plasma,serum, fluid interstitial, internal organs, hair, teeth or bones. In many cases it is required to determine not only the total amount of
a particular item, but also its chemical form, because it may have a significant impact on the bioavailability, mobility and toxicity ofthe element .
The research in the field of reconstructive medicine is in a continuous search of synthetic biomaterials, but also of new solutions
for prosthetic or regeneration of bones. Synthesis and marketing of improved biomaterials is always required as this automaticallyleads to an improve in the life quality. In this respect the present work uses two complementar techniques（ICP-MS and IR）in themetal ions detection and control of a new synthetized potassium-phosphate material doped with vanadium ions. Potassium,
phosphorus and vanadium are bio-elements required for materials used in bone repair but in suitable amounts. Thus, potassium
minimizes the loss of calcium from the body and thus reduces the risk of osteoporosis, providing a healthy skeletal system andvanadium is an active agent in helping to the body metabolism, focusing on the growth of bones and teeth strengthening.
By using ICP-MS technique, 9 samples of V 2O 5-P 2O 5-K 2Owith various quantities of potassium, phosphorus and vanadium were
investigated and suitable/expected amounts of metals were detected： for the sample with the lowest content of vanadium（ 3mol%）308225,184 mg/kg P, 60548,927 mg/kg K and 3292,332 mg/kg V were detected and for the sample with the highest content of
vanadium（ 5 mol %）135363,798 mg/kg P, 28925,277 mg/kg K and 294601,682 mg/kg V were detected. We applied Total Quantanalysis method with external standard of 10 μg/l Mg, Cu,Rh, Cd, in, ba, Ce, Pb, U with a level of certainty of ± 25 %. Such anelemental technique is strongly required in the study of materials with bioactive potential for the detection of impurities and toxic
elements； such materials should be from a biological point of view non-toxic and without filtered products.
IR spectra of the studied 9 samples show for low concentration of vanadium ions the specific bands belonging to the phosphategroups, but at high concentration of vanadim（x > 10 mol%）the bands belonging to the vibration of V2O5 groups dominates the
spectra. In the same time the bands belonging to the phosphate groups are strongly reduced except the specific bands of theshort chain phosphate units. This suggest that in the studied materials vanadium acts as network modifier for low concentration（x < 10 mol%）but may act also as a network former at high concentration of V2O5 .
1. J. Sabine Becker, H. Sela, J. Dobrowolska, M. Zoriy, J. S. Becker, International Journal of Mass Spectrometry 270（2008） 1 - 7
2. R.Viswanatha, M. V. S. Reddy, C. N. Reddy, R.P. S.Chakradhar, Journal of Molecular Structure 889 (2008)197-203

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 3: Structures and Dynamics of Atomic and Molecular Clusters

PMo-048

13:30 － 14:40

Size-dependent structures of zinc oxide cluster ions studied by ion
mobility mass spectrometry

Kunihiko Komatsu, Kiichirou Koyasu, Fuminori Misaizu
Tohoku University, Sendai, Japan

Keywords:
zinc oxide, clusters, ion mobility spectrometry, annealing, isomerization
Novel aspects:
Three types of structures of zinc oxide cluster ions have been obtained by ion mobility mass spectrometry coupled withtheoretical calculations.
Abstract:
Zinc oxide ZnO is a compound of central interest in the field of functional nanomaterials, such as blue light emitting diode,electrode, and photocatalyst. This compound is also used for cosmetics because of its low toxicity. Clusters of zinc oxide,（ZnO）ncan be regarded as local structure models of nanomaterials or condensed phases, and thus the clusters have been studied bothexperimentally and theoretically［1,2］. Ion mobility spectrometry is one of the powerful methods to obtain geometricalinformation of ions. This method utilizes the fact that the interaction of ions with buffer gas depends upon their geometricalstructures in an ion drift cell, in which He buffer gas is filled and electrostatic field is applied. The injected ions suffer thousands ofcollisions in the cell, and the time spent in the cell depends on their collision cross sections with He. Thus the isomer ions are
separated depending on their collision cross sections with the buffer gas； compact isomers pass through the cell faster than thebulky isomers.
In this study, structures of zinc oxide cluster ions（ZnO）n +（n = 2 -15）have been investigated by ion mobility mass spectrometry
coupled with quantum chemical calculations. The cluster ions were generated with laser vaporization of Zn rod followed byreactions with O2 contained in He supersonic jet. The ions were injected by pulsed electric field into an ion drift cell with kinetic
energies of 100-400 eV. The cell was filled with He as buffer gas with a pressure of 0.8 Torr, and an electrostatic field of 7.5 V/cm
was applied in order to guide the ions downstream. The cell temperature was cooled down to 170 K by liquid N 2 circulation.
Separated isomer ions were then accelerated by pulsed electric fields and analyzed in a reflectron time-of-flight（TOF）mass
spectrometer. We thus obtained TOF mass spectra sequentially by scanning the time difference, called“arrival time“, between
the injection pulse and the acceleration pulse in the TOF mass spectrometer after the cell. As a result, different isomer ions ofvarious size of clusters were separately detected corresponding to the differences of the arrival times in the two dimensional plotsof TOF vs. arrival time. The former corresponds to mass and the latter is proportional to the cross sections. Arrival time
distributions can be obtained as slices of the two dimensional plots on arrival time axis.
Three series of arrival time distribution were observed for（ZnO）n + depending on the cluster size and the ion injection energy into
the drift cell. The fastest series is observed for n = 6, 8 -15, which corresponds to cage or tube type structures, from thecomparison with density functional theory calculations and estimation of ion mobility from an averaged collision cross section
calculation. The second corresponds to ring structures, which is predominantly observed for n< 8. The last series is the zigzag or
linear structures, which were only weakly observed at the lowest injection energy of 100 eV in the present measurement. Most ofthese structures are consistent with the previous theoretical predictions［ 2 ］, except for the cage structure at n = 6. Isomerization
to stable cages or tubes from other structures was observed as an annealing effect with increasing ion injection energy.
［ 1 ］A. Dmytruk, I. Dmitruk, I. Blonskyy, R. Belosludov, Y. Kawazoe, A. Kasuya, Microelectronics J. 40, 218（2009）.
［ 2 ］B. L. Wang, S. Nagase, J. J. Zhao, G. H. Wang, J. Phys. Chem. C111, 4956（2007）.

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 3: Structures and Dynamics of Atomic and Molecular Clusters

PMo-049

11:10 － 12:20

Photodissociation dynamics of mass-selected cluster ions examined byfragment-ion imaging

Hiroshi Hoshino, Yoshihiro Yamakita, Yoshitomo Suzuki, Masataka Saito, Kiichirou Koyasu,
Fuminori Misaizu
Tohoku University, Sendai, Japan

Keywords:
Photodissociation； Imaging； Cluster； Reaction dynamics； Reflectron
Novel aspects:
Imaging detection of mass-analyzed photofragment ions from mass-selected cluster ions has been fulfilled using a newly
developed experimental setup.
Abstract:
Imaging detection has extensively been applied to neutral species so far for the investigations of photodissociation dynamics. We
recently developed a new experimental setup for image detection of mass-analyzed photofragment ions from mass-selectedcluster ions, using a conventional angular reflectron time-of-flight mass spectrometer coupled with an imaging detector［ 1 ］. In
this apparatus, the ion image of fragments can be obtained after mass-selection of parent ions. In the present study, we havefulfilled the ion image detection of fragments induced by photolysis of mass-selected Mg+ -Ar and Mg + -ICH 3 complex ions, andcompared the obtained dependences of recoil angular distributions on laser polarization directions and the obtained kinetic
energy release of the photodissociation reaction with previously obtained results.
+ 2P）fragment after photolysis of a mass selected Mg+-Ar complex. Mg+Arn wereAt first we have observed an ion image of Mg（
produced by laser vaporization of Mg rod and supersonic expansion of Ar gas. The mass selected Mg + -Ar ion was
photodissociated by irradiation with 4.66-eV laser（the 4 th harmonic of a pulsed Nd：YAG laser）in the first drift region of thereflecrton. The Mg+ fragment ions were reflected with a reflectron and their ion images were detected by a microchannel plate（
MCP ）with phosphor screen. Amplified electrons were converted to photons at the phosphor screen and finally detected with
the CCD camera. From the analysis of the image we obtained the results that the translational energy Eex and the anisotropy
parameter β was 604±100 cm -  1 and 0.98, respectively. From obtained image the Mg + ion was dissociated immediately after
excitation with a direction parallel to the laser polarization direction, although the selectivity was lower than the pure paralleltransition（ β = 2 ）. We successfully indicated that very low fragment-ion velocity on the order of several hundreds of ms-  1 isdetectable by this apparatus. From the observed translational energies we also estimated the binding energy of the Mg+ -Ar
ground state as D0= 1222±124 cm-1. This value is consistent with that reported previously, 1237 ± 40 cm-1［ 2 ］.
We have also investigated an ion image of MgI+ fragment after photolysis of a mass selected Mg+-ICH3 complex. As was alreadyreported by our group［ 3 ］, Mg+ICH3 has a bent geometry in its ground state. The positive charge is almost localized on Mg, andthus the ultraviolet absorption bands are originated from 2P←2S transition of Mg+. Present laser excitation at 4.66 eV corresponds
to 3 pz← 3 s transition, in which the transition dipole moment μ is parallel to the Mg-I bond axis. This excitation localized on Mg+
is followed by predissociation from a C-I bond dissociative state producing MgI + fragment ion. Based on these considerations,obtained ring image was explained by the dissociation process after excitation much faster than the rotational period of thecomplex. Maximum value of the kinetic energy release distribution, 1.24±0.13 eV, which was determined from the observed ringradius was also consistent with the previous result, 1.37 eV［ 3 ］.
［ 1 ］Y. Yamakita, H. Hoshino, Y. Suzuki, M. Saito, K. Koyasu, and F. Misaizu, in preparation.
［ 2 ］S. Massick and W. H. Breckenridge, Chem. Phys. Lett. 257, 465（1996）.
［ 3 ］A. Furuya, F. Misaizu, and K. Ohno, J. Chem. Phys.125, 094310（2006）.

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 4: Imaging I

PMo-050

13:30 － 14:40

Chemical digestion for imaging mass spectrometry

junpei naito, shuya satoh, hiroyuki hashimoto
Canon Inc., Tokyo, Japan

Keywords:
digestion, NTCB, imaging, TOF-SIMS, MALDI
Novel aspects:
Chemical digestion using NTCB for imaging mass spectrometry.
Abstract:
In recent years, imaging mass spectrometry has received considerable attention, owing to its capability to monitor many
molecules at the same time. Mass spectral peaks corresponding to particular molecules can be used as biomarkers in diseasediagnosis（ 1 - 3 ）. In most cases, matrix-assisted laser desorption/ionization（MALDI）is used to softly ionize the molecules, but
this method gives poor spatial resolution. We have focused on time-of-flight secondary ion mass spectrometry（TOF-SIMS）, whichenables two-dimensional mass spectrometry imaging of components with high spatial resolution（ 4 - 6 ）.
Recently we reported a new method for enhancing peptide signals in TOF-SIMS（ 7 ）. The modification of peptide with 2 -nitro5 -thiocyanobenzoic acid（NTCB）achieves high sensitivity for TOF-SIMS detection. Peptides containing only one cysteine at the
N-terminus were used as model samples in order to evaluate the ionization efficiency of the NTCB-modified peptides, because
peptides with internal cysteine residues give multiple digestion fragments. For the quantitative evaluation of ionization efficiency,
an inkjet printing technique was also used to obtain reproducible samples. The TOF-SIMS signal intensity of the parent ion wasincreased 20-fold through NTCB modification of a peptide with the amino acid sequence CKVASLRETYGDMAD. These results
demonstrate that the NTCB method has great potential for detecting the peptides by TOF-SIMS because it has the additionalfeature of chemical digestion of polypeptides containing cysteine. But little has been reported on the chemical digestion using the
NTCB method in imaging mass spectrometry.
The purpose of this study was to improve the NTCB method for measurement of two-dimensional distribution of peptides
because this method has a problem of migration of the NTCB-modified peptides. The NTCB method consists of three steps：（step1 ）cleavage of S-S bond,（step 2 ）modification of SH to SCN, and（step 3 ）hydrolysis of modified polypeptides with base. Amongthese steps, step 3 has a great influence on the migration since this step involves water treatment of the modified peptides. In
the present study we show an advanced NTCB method, that is, this method uses both an ammonia vapor as base and adeliquescent salt in order to keep a trace amount of water on a sample.
Chemical digestion of insulin was demonstrated. We prepared an insulin solution using step 1 and step2. NaCl as a deliquescentsalt was then added to the solution, and the solution was deposited onto a Si-wafer by ink jet printing. The Si-wafer was exposed
to ammonia vapor at 37 &#8451； in moist condition. After the treatment, insulin digest peptides were detected by TOF-SIMS.
Comparison of optical and TOF-SIMS images of the printed dots showed that the migration caused by step 3 treatment is
negligible. We are planning to apply this advanced NTCB method to imaging mass spectrometry of tissue sections.
（ 1 ）S. Rauser, C. Marquardt, B. Balluff, S. Deininger, C. Albers, E. Belau, R. Hartmer, D. Suckau, K. Specht, M. Ebert,
M. Schmitt, M. Aubele, H. Hoefler, A. Walch, J. Proteome Res. 9 （2010）18541863.
（ 2 ）D. Taverna, A. Pollins, G. Sindona, R. Caprioli, L. Nanney, Wound Repair Regen. 19（2011）A56.
（ 3 ）M. Setou, Y. Saito, T. Hayasaka, K. Onoue, Y. Takizawa, Drug Metab. Rev. 43（2011）18.
（ 4 ）H. Nygren, P. Malmberg, Proteomics 10（2010）16941698.
（ 5 ）S. Parry, N. Winograd, Anal. Chem. 77（2005）79507957.
（ 6 ）F. Benabdellah, A. Seyer, L. Quinton, D. Touboul, A. Brunelle, O. Laprevote, Anal. Bioanal. Chem. 396（2010）
151162.
（ 7 ）J. Naito, S. Satoh, Y. Otsuka, H. Hashimoto, Int. J. Mass Spectrom.（2012）, doi：10.1016/j.ijms.2012.03.003.

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 4: Imaging I

PMo-051

11:10 － 12:20

Enhanced peptide molecular imaging by depositing ice in Time-of-Flight
Secondary Ion Mass Spectrometry

Naofumi Aoki, Manabu Komatsu, Masafumi Kyogaku, Hiroyuki Hashimoto
Canon Inc., Tokyo, Japan

Keywords:
TOF-SIMS, imaging, ice
Novel aspects:
This report brings out a quantitative relationship between ionization enhancement and the amounts of the adherent ice on thesample.
Abstract:
INTRODUCTION； This report presents a method of enhancing ionization of a sample while retaining the original distribution of
the sample constituents in time-of-flight secondary ion mass spectrometry（TOF-SIMS）imaging. In recent years, mass spectrometry
imaging has received considerable attention, owing to its capability to monitor many molecules at the same time. Mass spectral
peaks corresponding to particular molecules can be used as biomarkers in disease diagnosis. In most cases, matrix-assisted laser
desorption/ionization（MALDI）is used to softly ionize the molecules, but this method gives poor spatial resolution. On the other
hand, time-of-flight secondary ion mass spectrometry enables two-dimensional mass spectrometry imaging of components with
sub-micrometer resolution. However, one of the major issues in TOF-SIMS is that the ionization probability of components with m/z > 500 is extremely low.
In this research, we report on ionization enhancement in TOF-SIMS imaging by a reproducible method of ice deposition onto thesurface of biomaterials. One of the three purposes of this report is to bring out a quantitative relationship between ionization
enhancement and the amounts of the adherent ice on the sample. The other purposes are to show that the ice deposition
method is suitable for mass imaging analysis because of retaining the original distribution of the sample constituents, and toinvestigate how ice behaves as ionization promotor.
METHODS； Peptide samples were prepared on a silicon substrate by using a inkjet printing apparatus to assess signal intensities
among samples quantitatively.The area of each spot was about 8 × 10-3 mm2（diameter 0.1 mm）, and the peptide amount ofthat was 67 fmol.
Ice deposition onto the sample was carried out in a preparation chamber of TOF-SIMS. After introducing the peptide sample inthe vacuum chamber, H2O gas was discharged into the chamber through a variable leak valve. At this time, by previously coolingthe sample to a temperature equal to or lower than a freezing point or a sublimation point of H2O, H2O gas that adsorbed on thesample surface turned to ice. The amount of ice adsorbed on the sample surface was estimated by two methods. One method is
to use a crystal oscillator sensor, and the other one is to calculate the incident flux of H2O molecules impacting onto the sample.
By using these methods, the intended amount of ice can be deposited on the sample surface. Following ice deposition onto theinkjet printing dots, the protonated parent ion was measured using TOF-SIMS.
RESULTS and DISCUSSION； In order to investigate the detailed change of the detection intensity of the sample with respect tothe amount of the adherent ice, we prepared several dot pattern samples which were covered with various amounts of ice from0 to 25 ng/mm2. It was found that the protonated parent ion intensity varies with the amount of ice, and approximately 1 ng/
mm2 of ice gives the highest intensity, which is roughly 10 times the intensity of the original. Comparison of optical and TOF-SIMS
images of the printed dots indicates that the constituents diffusion caused by ice deposition is negligible. It means that thismethod of ice deposition can keep the original distribution of the sample constituents, and is suitable for mass imaging analysis.
Experiments using deuterated ice showed that the adherent ice behaves as part of proton source.
From these results, it can be concluded that controlled ice deposition onto the biomaterials surface is effective for ionization
enhancement in TOF-SIMS imaging.

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 4: Imaging I

PMo-052

13:30 － 14:40

Direct and indirect imaging of secondary metabolites and enzymatic
reactions in plant material by Desorption Electrospray Ionization

Bin Li3, Nanna Bjarnholt2, Camilla Knudsen2, Janina Thunig3, Steen H Hansen3, Christian Janfelt1,3

1
University of Copenhagen, Copenhagen, Denmark, 2Dept. of Plant Biology and Biotechnology, University of
Copenhagen, Copenhagen, Denmark, 3Dept. of Pharmacy, Center for Advanced Drug Analysis, University of
Copenhagen, Copenhagen, Denmark

Keywords:
Imaging-MS, Plant-analysis, DESI-MS, cyanogenic-glucosides
Novel aspects:
Imaging of enzymatic reactions in plant material, and direct and indirect DESI imaging of plant material.
Abstract:
Introduction
We have developed an imprinting method, termed Indirect DESI imaging, where instead of imaging plant material directly, an
imprint is made on a porous Teflon surface which is then imaged by DESI, providing a very robust and simple imaging method.
We have now shown that although plant cells are disrupted during the imprinting process, initiating possible enzymatic reactions,the method still works for compounds which are degraded in these reactions. Indeed, the method can be used to image such
enzymatic reactions in selected areas of the plant material. Furthermore, by using a new DESI solvent composition we have also
been able to perform direct imaging of leaves which cannot be imaged using standard DESI solvents, due to the presence of a
cuticular wax layer on the plant surface.
Methods
Plant material was pressed with porous Teflon and a piece of tissue paper in order to absorb the surplus plant juice. A rubber
sheet was added to distribute the pressure and the sandwich was pressed in a vice for a few seconds. The Teflon imprint wasdried in a vacuum desiccator and imaged on a custom-built DESIimaging ion source mounted on a Thermo LTQ-XL linear ion trap
mass spectrometer.
Results from the imaging experiments were validated by quantitative LC-MS experiments performed on different small sections of
the plant material.
Preliminary Data
Indirect DESI Imaging was performed on leaves and petals of Hypericum perforatum, showing the distribution of hypericin andhyperforin. As previously reported, hypericin was found to reside in the black glands of the leaves and petals, while hyperforin
was found in translucent glands of the leaves and in spots and broad bands on the petals, respectively.
The method has also been applied for imaging of a variety of cyanogenic glucoside（CG）containing plants such as barley leaves,
Lotus japonicus leaves, sorghum etiolated seedlings and cassava tubers.
Many of these plants contain CGs together with active β-glucosidases capable of degrading CGs upon damage to the tissue.
Although the imprinting method inherently causes damage to the tissue it was found to be highly applicable for imaging of the CG
without observing significant degradation due to the imprinting process, since the imprinting time can be kept as low as a few
seconds and the enzymatic processes are“frozen“ when the imprint is dried in a vacuum desiccator. The fact that enzymatic
reactions do take place upon damage to the tissue was shown in an experiment where a leaf of Lotus japonicus was pressed witha metal ring and imprinted after 20 minutes. Indirect DESI images showed compounds distributed homogenously throughout theleaf（i.e. unaffected by enzymatic reactions）as well as compounds of decreased abundance in the pressed area（substrates ofthe enzymatic reaction）and newly formed compounds in the pressed areas（products of the enzymatic reaction）.
Direct DESI imaging was performed on leaves of Lotus japonicus and cassava tubers, showing elevated levels of CGs in the baseof the leaf and near the peel of the tubers, as confirmed by LC-MS. Direct imaging of the leaves was only possible using a special
spray solvent mixture. The cassava tubers were cut in 40 µm thick slices on a cryostat and mounted on glass slides, upon which
direct DESI imaging using normal spray solvent mixture was quite straight-forward.

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 4: Imaging I

PMo-053

11:10 － 12:20

Sub-Micrometer Imaging of Lipids and Trace Elements in Various Cells
with ToF-SIMS and Laser-SNMS

Heinrich F Arlinghaus1, Felix Draude1, Sebastian Galla1, Andreas Pelster1, Martin Koersgen1,
Jutta Tentschert2, Harald Jungnickel2, Andrea Haase2, Andreas Luch2, Tanja Schwerdtle3,
Johannes Muething4

1
Intitute of Physics, University of Muenster, Muenster, Germany, 2German Federal Institute of Risk Assessment,
Department of Product Safety, Berlin, Germany, 3Institute of Food Chemistry, University of Muenster, Muenster,Germany, 4Intiute of Hygiene, University of Muenster, Muenster, Germany

Keywords:
ToF-SIMS, Laser-SNMS, Cells, Lipids, Elements
Novel aspects:
ToF-SIMS and Laser-SNMS was used to optimize cell preparation techniques and to detect membrane molecules such as lipids, Ag
nanoparticles, and arsenic species in different cells with sub-micrometer spatial resolution.
Abstract:
In recent years, molecular imaging with submicron lateral resolution has become of more and more interest for characterizing
specialized compounds in biological samples. As an example, nanoparticles（NPs）gain great commercial interest in the medical
field due to their high mobility in human tissue. This enables enrichment in specific organs of the human body, which makes themsuitable in applications like drug delivery. On the other hand, high mobility of the nanoparticles might result in adverse healtheffects. Despite broad applications close to the human body, so far, there is only little knowledge of possible toxicity. In thiscontext, the distribution of nanoparticles within cells is of particular interest. Moreover, not only the distribution of nanoparticles
but also elemental and molecular cellular distributions such as metabolites and lipids are interesting for medical research.
In this study, we used time-of-flight secondary ion mass spectrometry（ToF-SIMS）and laser-secondary neutral mass spectrometry（Laser-SNMS）to investigate different cells both unexposed and exposed in vitro to silver nanoparticles（AgNPs）and arsenic
species. To optimize the analysis, a special silicon wafer sandwich preparation technique was employed； this entails freezefracturing and washing of cell cultures that were grown on silicon wafers. The data showed that during freeze-fracturing, the cell
membrane is often stripped from the cell, enabling direct analysis of the interior of the cells on one sandwich wafer and theremaining lipid membrane as a mirror image on the opposite wafer. During analysis, the signal from the nutrient materials wasobserved to diminish the contrast of the molecular signals in the images. By optimizing the sample preparation and washingprocedures, both the contrast and the imaging resolution could be significantly increased due to higher molecular yields andlower background. With these optimization procedures it was possible to detect lipid ions in a higher mass range, especially fromthose membranes that were stripped from the cells.
Under these optimized conditions, several studies were performed to detect the distributions of trace elements in cells. One study
dealt with AgNPs. These particles show antibacterial effects and are therefore applied e.g. in the medical field for wounddisinfection or as a coating material for surgical instruments and prostheses. In conjunction with the high mobility of nanoparticles,
the antibacterial effects may pose health dangers. In this context the uptake of AgNPs of human macrophages was measured withnanometer-scale resolution. 2 D and 3 D Laser-SNMS images clearly showed that AgNPs are incorporated by macrophages and inpart agglomerate to silver aggregates with a diameter of ~ 3 - 7 µm. In a similar approach, the distribution of arsenic in cells wasmeasured to obtain more information on the reasons why inorganic arsenic proves carcinogenic in humans. A comparison with
ToF-SIMS data showed that especially the high elemental sensitivity of Laser-SNMS makes it possible to detect theses traceelements in cells.

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 4: Imaging I

PMo-054

13:30 － 14:40

The potential use of reactive DESI to assess oxidative stress in the cell
cultureAnna Bodzon-Kulakowska, Piotr Suder, Tomasz Cichon, Jerzy Silberring
AGH University of Science and Technology, Krakow, Poland

Keywords:
DESI, oxidative stress, cell culture
Novel aspects:
Examination of oxidative stress in the cell culture using DESI -MS technique
Abstract:
Oxidative stress is a phenomenon involved in many pathological processes such as neurodegenerative diseases（for example
Alzheimer’s or Parkinson’s diseases）or other types of neurodegeneration. It is associated with decreased amounts of antioxidants,
and with elevated production of molecules known as reactive oxygen species（ROS）. This may lead to oxidative damage ofproteins, lipids, and DNA and, in consequence to apoptosis of the cell.
DESI（desorption by electrospray）is an example of MS imaging approach. This technique is capable of localization andidentification of a wide range of substances in biological tissues. DESI utilizes electrospray source to produce charged droplets oforganic solvent, which are focused on the analyzed material. After hitting the surface they produce a second generation of
charged droplets containing surface molecules. Addition of an appropriate reagent to the carrier solvent results in fast chemical
reaction of this substance with molecules of interest localized on the surface. The MS spectra are acquired concurrently, whichmeans that the product of this reaction may be analyzed at once, without additional sample preparation.
Malondialdehyde（MDA）is considered as a marker for lipid peroxidation in living organisms and cultured cells, since it is one of
the main products of lipids peroxidation. This compound may be detected with the aid of reactive DESI, where
dinitrophenylhydrazine（DNPH）is dissolved in the spray solvent. DNHP is generally used for derivatization of carbonyl compounds,
what improves sensitivity of their detection. This substance reacts with MDA to form dihydrazone MDA diDNPH, which might
easily be detected by the MS.
For the experiments we applied astrocyte cell culture, since there is a growing number of evidence for their potential role in many
processes in the brain, including pathological ones involved in neurodegenerative diseases. Two compounds, widely used inliterature that induce pharmacological stress： hydrogen peroxide（H 2 O 2 ）and tert-butyl hydroperoxide were utilized to evoke
oxidative stress in the cell cultures. The cell pellet, supernatant from the cells homogenized by sonification, and intact cells on theglass surface were analyzed.
To our best knowledge this is the first study to examine oxidative stress in the cell culture using DESI-MS technique. A possibilityof rapid measurement of the oxidative stress may provide an opportunity to investigate pathology of CNS, such as drug addiction
using cell culture as a model.

The research was supported by the Foundation for Polish Science - POMOST Programme co-financed by the European Union
within European Regional Development Fund（POMOST/2011- 3 / 1 ）and the grant from Ministry of Science and Higher Education
no. 3744/B/H03/2011/40 and 3048/B/H03/2009/37.

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 4: Imaging I

PMo-055

11:10 － 12:20

Higher sensitivity sub-micron spatial resolution TOF-SIMS bio-molecularimaging using interleaved Gas Cluster Ion Beam (GCIB) sputtering

Itsuko Ishizaki1, John S Hammond2, Takuya Miyayama1, Gregory L Fisher2
1

ULVAC-PHI, Inc., Kanagawa, Japan, 2Physical Electronics USA, Chanhassen, MN, USA

Keywords:
Imaging Mss Spectrometry, GCIB, TOF-SIMS
Novel aspects:
The combination of LMIG TOF-SIMS imaging of tissue samples interleaved with GCIB removal of the LMIG-induced damaged layers
provides higher sensitivity and optimum spatial resolution imaging for high mass molecules.
Abstract:
Time-of-Flight Secondary Ion Mass Spectrometry（TOF-SIMS）has recently evolved as a molecular surface analysis technique with
sub-micron spatial resolution. Based on these unique capabilities, applications of TOF-SIMS are rapidly expanding to lipid andmetabolite research in medical science, pharmaceuticals, and molecular biology. One limitation in these imaging studies has been
the low signal level of molecular ions from higher mass materials, e.g. lipids with molecular weights above 700 Da, when imaging
with sub-micron spatial resolution. This is due to the creation of a limited number of secondary ions per square micron from theoutermost layer of the sample while the Liquid Metal Ion Gun（LMIG）primary ion beam induces molecular damage in the nearsurface region. To enhance the quality of molecular ion imaging, there have been several efforts to increase the efficiency, or thesecondary ion yield, for high mass ions. These studies include the use of higher energy projectiles［ 1 ］, cluster ion beam
projectiles［ 2 ］, and the deposition of inorganic or organic secondary ion yield enhancing materials on the sample surfaces［ 3 ］.
In this paper, we discuss higher sensitivity TOF-SIMS molecular imaging with high spatial resolution using a combination of
Bi3 ++primary ions from a LMIG interleaved with Ar gas cluster ion beam（GCIB）sputtering. The GCIB beam is used to remove avery thin damaged surface layer after the LMIG acquisition of the maximum secondary ion signal intensity from the outermost
surface［ 4 ］. This interleaved process can significantly increase the signal intensity while maintaining optimum spatial resolution.
Results will be presented for imaging several lipids from tissue samples using LMIG data from only the outer surface of the sample
as well as data acquired with the interleaved LMIG / GCIB protocol. The comparisons of these results will be discussed relative tothe ultimate spatial resolution and the molecular ion sensitivity achievable for TOF-SIMS imaging of lipids in tissue samples.
References:［ 1 ］Y. Nakata, et al., J. Mass Spectr.,44（2009）128.
［ 2 ］K. Mochiji, et al., Rapid Comm. Mass Spectr.,23（2009）648.
［ 3 ］T. M. Brewer, et al., Rapid Comm. Mass Spectr.,24（2010）593.
［ 4 ］T. Miyayama, et al., Surf. Interface Anal.,42（2010）1453.

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 4: Imaging I

PMo-056

13:30 － 14:40

Drug and metabolites study in whole body animal by High Definition
MALDI imaging

Thanai Paxton1, Motoji Oshikata1, Emmanuelle Claude2, Lars Bendahl3, Ian Edwards2, Paul Murray2,
Johannes P Vissers2

1
Nihon Waters, Tokyo, Japan, 2Waters Corporation, Manchester, UNITED KINGDOM, 3H. Lundbeck A/S, Dk-2500 Valby,
DENMARK

Keywords:
Imaging, DMPK, MALDI, Ion Mobility, Pharmaceutical Applications
Novel aspects:
Parallel drug and metabolite localization in whole body section tissue samples in the presence of endogenous species separated
by IMS.
Abstract:
Introduction
Mass spectrometry Imaging（MSI）is increasingly used in pharmacokinetic studies during preclinical studies. It has been recognized
as a complementary technique to Whole-Body Autoradiography（WBA）, which is traditionally used for approval of a drug by the
Food and Drug Administration（FDA）agency. The two main advantages of MSI are cost savings compared to radio-labeling of the
drug and the absolute confirmation that the drug and its possibly produced metabolites are indeed visualized.
Here, we present the results from a study where two MS based imaging approaches were used to illustrate the spatial
distributions of a drug and its metabolites, along with untargeted analysis of endogenous molecules in a whole body section.
Methods
Olanzapine（OLZ）was administrated to rats at 8 mg/kg. Animals were euthanized at 2 or 6 hours post-dose. 30-µm-thick wholebody sagittal tissue sections were mounted on invisible mending tape. A single sagittal section of a whole rat spanned three
MALDI plates. CHCA matrix was applied evenly to the sample in several coats using a SunCollect nebulising spray device.
Data were acquired using a MALDI SYNAPT G 2 mass spectrometer in both MS and MS/MS mode with tri-wave ion guide optics
to separate ions according to their ionic mobility in the gas phase. The obtained data sets were subsequently processed using
High Definition Imaging（HDI）MALDI software for detailed image analysis.
Preliminary Data
OLZ is a drug of which its pharmacokinetics 1 have been intensively studied in different animal systems. However, spatial
distribution analysis solely by MSI lacks certain, specific information. The two main metabolites of OLZ（m/z 313.15）that havebeen identified in rat are N-desmethyl OLZ（m/z 299.13）and 2 -hydroxymethyl OLZ（m/z 329.14）. Typically, drug distribution
analysis by MSI is conducted by using a targeted analysis method for the drug of interest, aiming to achieve better sensitivity andspecificity, where the molecular ion is selected by a quadrupole, fragmented in a collision cell and finally the product ions m/z
separated before detection. In this study, traditional targeted and untargeted MSI analysis experiments have been explored andcontrasted as detailed below.
A first experiment was carried out on the 2 h post-dose tissue section in a multiplex targeted MS/MS, approach where the drug
and the two known metabolites were MALDI imaged from a single tissue section.
A second experiment was performed on the 6 h post-dose tissue section in an untargeted MS approach. Also in this instance,
both the drug and the two main metabolites were imaged. However, here a vast amount of information is also generated by theionisation of the endogenous species present in the whole body tissue section.
The final part of the study involved the performance evaluation of isobaric species separation in the gas phase by means of ion
mobility separation, which is integrated in the MALDI SYNAPT G 2 HDMS instrument, for untargeted MALDI-MS imaging
experiments of OLZ and its two main metabolites.

1

Kelem Kassahun et al., Drug Metabolism and Disposition, 1977, Vol. 25, No. 5, pp573-583

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 4: Imaging I

PMo-057

11:10 － 12:20

DESI-MS imaging of differences between physiological and morphinetreated rat brains.

Piotr Suder1, Anna Bodzon-Kulakowska1, Anna Drabik1, Joanna Ner1, Ewelina Paszynska1,
Jolanta H Kotlinska2
1

AGH University of Science and Technology, Krakow, Poland, 2Medical University of Lublin, Lublin, Poland

Keywords:
DESI, morphine addiction, metabolomics, brain
Novel aspects:
Comparison of brains low MW chemistry between control and morphine exposed brains. Application of DESI-MS technique incomplex, comparable analysis.
Abstract:
Drug addiction is one of the growing and still unsolved problems among societies of countries all over the world. Drug abusers
are involved in trade of illegal substances and are susceptible to dangerous, infectious diseases. They are also significant source ofcriminal behaviors. The society bears significant costs of treatment of drug abusers but, despite intense investigations, there is still
no effective therapy proposed for the addicts. The modern medicine offers psychotherapy supplemented by pharmacology withantagonists or weak agonists of the selected brain receptors. This strategy is definitely not sufficient, and such therapies usuallyshow low effectiveness. To evaluate and finally develop an effective, open and cheap treatment for drug addicts, the crucial task
is to understand molecular mechanisms involved in dependence, tolerance, and withdrawal. Based on our previous experiences inproteomic approach, we decided to use the rat morphine dependence model for the comparison of localization and distributionof low molecular mass molecules in the control, and morphine-exposed brains. Such approach allows for observation andcomparison of global changes in low MW molecules among major brain structures between physiological and pathological states.
Investigations were performed using DESI ion source（Prosolia, Indianapolis, USA）connected to the Amazon ETD mass
spectrometer（Bruker Daltonics, Bremen, Germany）. DESI-MS seems to be one of the most promising, mass spectrometry based,imaging techniques operating at ambient pressure, available since a few years. We examined density and localization maps of theselected, low molecular mass substances in the range of 100-1200 m/z. Mass spectra were taken in the positive and negativemodes. For the experimental purposes the rats were given 10mg/kg of morphine hydrochloride once daily（sc.）in days： 1, 3, 5
and 7, what stays in agreement with major morphine dependence models described widely in literature. Additionally, dependence
status was verified by precipitation of withdrawal signs by naloxone. Maps were constructed from the frontal sections（ca. 40
micrometers/slice）of the rat’s control brains. We compared them with the maps received from brains of rats after morphine
injections. Typically, at least 10 density maps from each brain were taken for experiments. Slices were acquired from various areas
in an interaural axis from +10 to + 2 mm range（corresponding to Bregma +1.5 to - 7 mm）what allowed for detailed visualizationof major structures of the reward system, as well as other structures, clearly visible on the slices, such as hippocampus, striatum,and cortex.
The obtained results bring a new insight into time-dependent activation of brain structures after morphine treatment, and show
differences in the low MW range between compared states, what might be useful for further research on the brain areas involvedin addiction. Presented findings may also be valuable from therapeutics point of view, showing early mechanisms during
development of addiction.
This work was supported by the grants from Polish National Science Center（grants number 3744/B/H03/2011/40 and 3048/B/
H03/2009/37）and Foundation for Polish Science - POMOST Program co-financed by the European Union within European Regional
Development Fund（POMOST/2011- 3 / 1 ）.

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 4: Imaging I

PMo-058

13:30 － 14:40

High-resolution TOF-SIMS Imaging reveals Multi-layered Barrier Structureof the Stratum Corneum of Skin

Akiharu Kubo1, Itsuko Ishizaki2, Akiko Kubo1, Hiroshi Kawasaki1, Yoshiharu Ohashi2, Masayuki Amagai1
1Keio University, Tokyo, Japan, 2ULVAC-PHI, Chigasaki, Japan

Keywords:
TOF-SIMS, skin barrier, stratum corneum, filaggrin, atopic dermatitis
Novel aspects:
Using TOF-SIMS imaging, we found that skin stratum corneum has at least three distinct layers that have different barrier functions
likely corresponding to metabolic process of filaggrin.
Abstract:
Stratum corneum（SC）is the fundamental structure of skin surface to protect the body of mammals from desiccation and foreign
insults. SC is a water repellent barrier constituted by corneocytes and inter-corneocyte lipids. Congenital SC barrier insufficiencies,
i.e. filaggrin deficiency, are hypothesized to predispose to atopic diseases such as atopic dermatitis, atopic asthma, allergic rhinitis,
and food allergy via augmentation of percutaneous allergen infiltration and subsequent sensitization to various allergens. Limitationof analyzing methods, however, hampers detailed analyses of SC barrier structure and barrier function. Here we introduce time-offlight secondary ion mass spectrometry（TOF-SIMS）imaging combining with immunofluorescence imaging to co-visualize SC
structure and infiltration of external molecules using mouse skin as a model. Based on the distribution of Na, K and arginine, we
temporary divide the SC into three distinct layers： an upper Na high K high arginine low layer（upper-SC）, a middle Na high K low
argininehigh layer（mid-SC）, and a thin bottom layer of Nahigh Klow argininelow（bottom-SC）. We found that the upper-SC works as asponge-like layer where several external ions easily infiltrated in. The mid-SC contains higher amounts of free arginine, one of the
major degradative products of filaggrin, and prohibited the infiltration of topically applied potassium and hexavalent chromium. In
contrast, trivalent chromium infiltrated into the mid-SC but not into the bottom-SC. Interestingly, filaggrin-null SC revealed frequent
infiltration of topically applied trivalent chromium into living cell layer, strongly suggesting that focal barrier breakage took place atthe bottom-SC in filaggrin-deficient conditions. Our results first demonstrated that SC has at least three distinct layers that havedifferent barrier functions likely corresponding to metabolic process of filaggrin. We anticipate our TOF-SIMS imaging technique tobe a starting point for the analysis of SC barrier deficiency in atopic diseases and the evaluation of medicines for skin barrier
recovery.

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 4: Imaging I

PMo-059

11:10 － 12:20

Quantitative MALDI Imaging of a Peptide Pharmaceutical for Drug
Distribution Studies

Nozomi Takai, Yukari Tanaka, Ayahisa Watanabe
Shionogi & Co., Ltd., Osaka, Japan

Keywords:
biopharmaceutical, peptide, MALDI, imaging MS
Novel aspects:
This study suggests that MALDI-IMS enables the analysis of the distribution of peptide pharmaceuticals. In addition, thequantitative analysis was achieved by MALDI-IMS.
Abstract:
Introduction

Biopharmaceuticals, such as peptide and antibody drugs, are expected to contribute to innovative drug therapy, and an increasing
numbers of pharmaceutical industries are challenging the development of biopharmaceuticals. In drug discovery and development,
studies about the distribution of a drug after its administration provide useful information on the mechanisms for the expression ofefficacy or toxicity. Therefore, quantitative and efficient methods for the analysis of the distribution of biopharmaceuticals will be
required.
Imaging MS（IMS）has been developed to visualize the distribution of drugs or biological molecules on tissue sections without
radiolabeling, and recently, several reports have demonstrated the quantitative performance by MALDI-IMS. Thus, MALDI-IMS is
expected to enable the quantitative analysis of the distribution of biopharmaceuticals.
In this study, MALDI-IMS was performed on tissue sections of mice administered with octreotide, a synthetic octapeptide analogue
of somatostatin.
Methods

Male 8 -week-old C57BL/ 6 J mice were injected via the lateral tail vein with octreotide（ 3 mg/10 mL/kg）. At 15 min after dose
administration, the liver and kidneys of each mouse were dissected. A half of the liver and the left kidney were frozen to perform
MALDI-IMS, while another half of the liver and the right kidney were homogenized with distilled water to determine theconcentration of octreotide in each tissue by LC-MS/MS. To perform MALDI-IMS, 10-µm-thick sections were obtained from thefrozen tissues, coated with 2,5-dihydroxybenzoic acid（DHB）as a MALDI matrix compound using an automatic vaporizer（ImagePrep, Bruker Daltonics）, and then analyzed using a linear ion trap mass spectrometer equipped with a MALDI source（MALDI LTQ XL, Thermo Fisher Scientific）.
Preliminary Data

Prior to MALDI-IMS, a standard solution of octreotide mixed with a MALDI matrix compound on the stainless plate was analyzed.
In MS analysis, the［M + H］+ ion of octreotide（m/z 1019）was detected with higher intensity in the use of DHB than in the use of
α-cyano- 4 -hydroxycinnamic acid. The doubly charged ion of octreotide（m/z 510）was not observed, unlike in ESI-based analysis.
In MS/MS analysis of protonated octreotide, product ions at m/z 914 and 1001 had relatively high intensities. Next, a standard
solution of octreotide spotted onto a liver section of a non-dosed mouse was analyzed by MALDI-IMS. A product ion at m/z 914
gave a better signal-to-noise ratio on the liver section than［M + H］+ ion（m/z 1019）or another product ion（m/z 1001）. Thus,
the m/z 1019→914 transition was used to obtain the octreotide-specific ion images in the following studies.
On the tissue sections of the mice injected with octreotide, the distribution of octreotide was clearly visualized by MALDI-IMS.
Furthermore, IMS-based signal intensity of octreotide correlated with the concentration of octreotide in the tissue samples.

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 4: Imaging I

PMo-060

13:30 － 14:40

Investigation of Zeolite Matrix for Analysis of Pharmacokinetics Using
Imaging Mass Spectrometry with Laser Desorption/Ionization

Naoki Moriguchi1, Hisanao Hazama1, Tatsuya Fujino2, Kunio Awazu1
1

Osaka University, Osaka, Japan, 2Tokyo Metropolitan University, Tokyo, Japan

Keywords:
Imaging Mass Spectrometry, Laser Desorption/Ionization, Pharmacokinetics, Zeolite Matrix
Novel aspects:
By using zeolite matrix, the ion signal intensity of a drug from a mouse brain section was 1.5 times higher than that using theconventional organic matrix.
Abstract:
Introduction: Acceleration of the processes of new drug development is desired in pharmaceutical industry. Recently, imaging
mass spectrometry（IMS）has increasingly got attention as a novel analysis method of pharmacokinetics, which investigatesabsorption, distribution, metabolism and excretion of an administered drug. Compared to the conventional method,
autoradiography, IMS has advantages in measuring time, cost, and ability of distinguishing the drug from its metabolites. However,IMS is inferior to autoradiography in the point of detection sensitivity of the drug1). Therefore, it is important to choose the optimal
ionization assisting reagent and its solvent, laser irradiation condition, and so on. The aim of this study is to investigate zeolite
matrix for analysis of pharmacokinetics using IMS in order to enhance detection sensitivity. Zeolite matrix is a mixture of anorganic matrix and zeolite, which is crystalline aluminosilicate with nanosized cages. It has been reported that zeolite matrix can
improve the signal intensities of protonated ion of peptides2). In the present study, ion signal intensity from a brain tissue section
obtained from a mouse administered a drug was measured using the zeolite matirix, and compared with that measured using theconventional organic matrix.
Materials and Methods: A mouse（C57BL/ 6 J）was administered buspirone by tail vein injection at a dose of 20 mg/kg. Buspirone
is an anxiolytic psychoactive drug. 30 minutes after the injection, the brain was removed and frozen. α-cyano- 4 -hydroxycinnamicacid（CHCA）and the zeolite（NaY5.6）were mixed in equal amount, and the zeolite matrix was dissolved in 70/30 acetonitrile（ACN）/water at a concentration of 8 mg/mL. The frozen brain tissue was sliced into 2 μm thickness with a cryostat microtome
and attached onto a metal sample plate. 1 μL zeolite matrix solution was spotted onto the brain section with a pipette and dried.
For comparison, 1 μL CHCA solution in 50/50 ACN/water at a concentration of 10 mg/mL was spotted onto the mouse brain
section and dried. A time-of-flight mass spectrometer（Voyager-DE PRO, Applied Biosystems）was used, and the third harmonic ofa Nd：YAG laser（GAIA II 30-T, Rayture Systems Co., Ltd.）was irradiated to the sample. 20 points randomly selected in the dried
matrix spot, and 100 laser pulses were irradiated to each point. Then, measured 20 mass spectra were averaged. Laser pulse
energy was set to 10, 15, 20 and 25μJ. Under the same conditions, five mass spectra were measured for zeolite matrix and CHCA,
respectively.
Results and Discussion: Using both ionization assisting reagents, the signal intensity of protonated ion of buspirone with the laser
pulse energy of 15μJ was the highest. When laser pulse energy was higher than 20μJ, the signal intensity was decreased with theincrease in the laser pulse energy. The signal intensity of protonated ion of buspirone at the laser pulse energy of 15μJ using thezeolite matrix was 1.5 times higher than that measured using CHCA at the same laser pulse energy. It seems that the water innanosize cages of the zeolite contributed to the protonation of buspirone. Therefore, it is supposed that the detection sensitivityof buspirone could be enhanced by using the zeolite matrix. In the future work, improvement of the deposition method, thesample preparation method, and so on will be investigated. In addition, the distribution of buspirone in the mouse brain tissue
section will be measured with IMS using the zeolite matrix.
References
1 ）E. G. Solon et al., The AAPS Journal, 12, 11-26（2010）
2 ）Y. Komori et al., J. Phys. Chem., 114, 1593-1600（2010）

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 4: Imaging I

PMo-061

11:10 － 12:20

Imaging the Penetration Behaviour of Topically Applied Drugs into Skin by
Desorption Electrospray Ionization Mass Spectrometry

Janina Thunig1, Rasmus Mortensen2, Louise Bastholm-Jensen2, Steen H Hansen1, Christian Janfelt1

Department of Pharmaceutics and Analytical Chemistry, University of Copenhagen, Copenhagen, Denmark, 2New
Products, LEO Pharma A/S, Ballerup, Denmark

1

Keywords:
DESI-MS, Imaging-MS, Skin, Creams-and-ointments, In-vitro-studies
Novel aspects:
DESI-MS imaging of the penetration of topically applied substances； DESI-MS imaging of skin sections
Abstract:
INTRODUCTION
Studying the penetration behaviour of substances into skin is of great importance for toxicologists and for the development of
new pharmaceutical products that are delivered into and through the skin. The stratum corneum, the outermost layer of the skin,acts as a protective boundary for exogenous substances, preventing them from entering the body. Several substances andformulations can permeate the skin barrier and this effect is used for topical delivery of substances that either need to bedelivered locally, or are rapidly metabolized upon oral delivery. In principle there are three different ways of penetration throughthe skin for exogenous substances： transcellular diffusion through the corneocytes, intercellular diffusion through the lipid sheets
between the corneocytes, and penetration of the compounds via perspiratory glands or hair follicles. The importance of the latter
pathway has long been underestimated due to a lack of appropriate experimental models, and has sparked scientists’ attention
only within the last ten years. As an example, for caffeine it was found that 50 % of the assimilated substance penetrated via thehair follicles［ 1 ］. The follicular pathway is normally studied by manually closing the single hair follicles with wax, a laborious
procedure, and measuring the drug level in blood or a receptor solution. Here we present an alternative qualitative approach forthe study of the skin penetration behaviour of topically applied substances, using pig ear skin as an in vitro model and desorption
electrospray ionization（DESI）mass spectrometry imaging for the detection of drug substances and lipids.
METHODAll hair is removed from the pig ear and the formulation is applied to the skin for different periods between 0 and 24 hours inorder to follow the penetration of the compounds into the skin over time. After the application period, surplus ointment isremoved from the skin and the treated area is cut from the rest of the pig ear. For the first time point（ 0 hours）the formulation isapplied and wiped off immediately to determine the signal that is measured from residues of the ointment on the skin. The tissue
samples are frozen at -80°C until they are cut into 18-25 µm thick slices on a cryotome, both in vertical and in horizontal direction.
The samples are kept at -80°C until DESI analysis. DESI imaging is performed on a Thermo XL Linear Ion Trap mass spectrometer,
equipped with a custom-built imaging stage, as described in［ 2 ］. Images are recorded with 100 µm resolution. Light micrographsof the samples are recorded before imaging and adjacent tissue slices are H&E stained for comparison with the DESI images.
PRELIMINARY DATAIn negative ionization mode DESI images show the heterogeneous distribution of lipids in the different compartments of the skin.
The distribution of hair follicles in horizontal cuts could be pictured in negative ionization mode by following the distribution ofcholesterol sulphate which is associated with the hair follicles. Optical images confirmed the localisation of hair follicles. Several
drug substances were topically applied to pig ear skin for different application periods, and the distribution of the substances inpositive and negative ion mode was recorded. This way the skin penetration behaviour of e.g. ibuprofen and lidocaine wasimaged.
REFERENCES
1. Trauer,S., et al., Permeation of topically applied caffeine through human skin - a comparison of in vivo and in vitro data. British
Journal of Clinical Pharmacology, 2009. 68（ 2 ）： p. 181-186.
2. Thunig, J., S.H. Hansen, and C. Janfelt, Analysis of Secondary Plant Metabolites by Indirect Desorption Electrospray Ionization
Imaging Mass Spectrometry. Analytical Chemistry, 2011. 83（ 9 ）： p. 3256-3259.

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 4: Imaging I

PMo-062

13:30 － 14:40

Platinum Vapor Deposition Surface-Assisted Laser Desorption/Ionization
for Mass Spectrometry Imaging of Small Molecules

Tomoyuki Ozawa1,2, Hirotaka Hisatomi2, Tetsuro Higashiisokawa2, Hideya Kawasaki2, Ryuichi Arakawa2
1

Nissan Chemical Industries Ltd., Funabashi, Japan, 2Kansai University, Osaka, Japan

Keywords:
SALDI-MS, Mass Spectrometry Imaging , Platinum Vapor Deposition
Novel aspects:
Effectiveness of Pt vapor deposition SALDI-MSI on the direct detection of small analytes for printed papers and TLC plates.
Abstract:
Introduction
Recently, a method of coupling MALDI/MS with mass spectrometry imaging（MALDI/MSI）has attracted considerable interest in
tissue imaging. However, MALDI/MSI has some problems,（ 1 ）the spatial resolution depends on the dispersion size of organic
matrix crystals and inhomogeneous crystallization of organic matrix may give poorly reproducible ion peaks at each point,（ 2 ）thesubstrates need to be electrically conductive in order to allow for desorption of the ions from the plate. To eliminate these
problems in MALDI/MSI, a method of coupling surface-assisted laser desorption/ionization mass spectrometry with imaging mass
spectrometry（SALDI/MSI）using nanoparticles and a nanostructured substrate has been proposed as an organic-matrix-free
approach. In this study, we first report Pt vapor deposition SALDI/MSI of small molecules as a solvent-free, homogenous, organicmatrix free and not depend on substrate method.
Methods
The Pt vapor deposition MSI experiments were performed by using a MALDI-TOF MS, AutoFlex III, Bruker equipped with a YAG
laser（355 nm）. The Pt deposition onto the target imaging sample was performed by a commercially available magnetron
sputtering device（E-1030, Hitachi）. The Pt-deposited sample was mounted onto a standard stainless steel MALDI plate using
electrical conductive tape. MSI was conducted in a point-to-point scanning fashion, where the laser beam rasters across a sample
surface. Thus, a mass spectrum was collected at each point. Later reconstruction of the 2 -D ion maps of the molecules of interest
revealed their spatial distributions by using Bruker software（flexImaging ）.
Results
We demonstrate the effectiveness of the Pt vapor deposition SALDI/MSI（Pt-MSI）on the direct detection of small analytes
without extraction or concentration for printed paper, as well as of various analytes（saccharides, pigments and drugs）separated
by glass-backed thin-layer chromatography（TLC）plate.
1. Inkjet printing
The Pt-MSI method was applied to inkjet-printed papers in order to profile the distribution of ink colorant and ink components
using a custom inkjet ink formulation of known composition（magenta）on both printed and unprinted papers. The degradation of
ink components via UV light was also examined. The distinct optical images were obtained from inkjet printing with magenta on apaper and with the exposure of UV light. We observed several ion peaks in the mass spectra of the printed paper； magenta ink
colourant（m/z = 603）, inkjet ink component（m/z = 172）from the printed region, the UV degradation product（m/z = 188）from
the printed region and a paper component（m/z = 393）from an unprinted region. These ion signals on the printed papers were
not detected without the Pt vapor deposition.
2. TLC-MS
We compared Pt-MSI with Au-MSI and LDI-MSI（no deposition）for pigments（crystal violet, Rhodamine 123, and Rhodamine B）on
the TLC plate. The peak intensities of the pigments are much lower in LDI-MS than when using metal deposition. And using Pt
deposition is greater than when using Au deposition.
Pt-MSI can be successfully applied to the imaging analysis of saccharide mixtures（D（+）-glucose, maltose and maltotriose）
separated on the TLC plate. In the photograph of a TLC silica plate after the TLC separation, we could not visually confirm theposition of the separated saccharides because we did not use colouring reagents. However, the Pt-MSI image can clarify thelocation of separated saccharides on the TLC plate. In the mass spectra of these spots separated by TLC, the components are
sodium adduct ions at m/z 203 from glucose, m/z 365 from maltose and m/z 527 from maltotriose.
This method would be useful in imaging analyses of various insulating materials such as polymers and biological materials.

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 4: Imaging I

PMo-063

11:10 － 12:20

Comparison of MALDI Imaging modalities using a Peptide Gel matrix forstem cell implantation

Duncan M Forster1, Adam Mcmahon1, Catherine Merry1, Omar Belgacem2
1

The University of Manchester, Manchester, United Kingdom, 2Shimadzu, Manchester, United Kingdom

Keywords:
MALDI Imaging, PET
Novel aspects:
This preliminary feasibility study will compare two MALDI imaging modalities, as well PET, to be used together in this application
to stem cell culture
Abstract:
Introduction
Peptide gels are being investigated as a medium for the location and growth of stem cells in vivo. We are developing approaches
based on MALDI-MS imaging and positron emission tomography to map the distribution of these gels at the intended site of stem
cell growth and to monitor migration of the peptide elsewhere in the organism. The current study aims to assess detection limits
for the peptide in tissue samples and to optimise sample preparation to monitor peptide distribution following administration. The
peptide is to be radiolabelled for PET imaging, slightly altering its structure. Migration of the modified peptide relative to theunmodified peptide is to be assessed by MS imaging. This provides an excellent opportunity to compare two different MALDI MS
instruments, along with testing prototype MALDI imaging target slides.
Methods
Detection limits were measured by applying known concentrations of the peptide to tissue sections, coating with matrix（CHCA）
and Imaging with MALDI MS. Fragmentation patterns during MS/MS were determined by analysis of MS/MS peaks. Direct imagingof the peptide（M+H）+ ion and MS/MS imaging were compared to assess their dynamic range and which is more appropriate to
mapping the high concentrations in the original gel deposited and low concentrations at remote sites. Initial peptide distributions
have been mapped by spiking tissues from newly sacrificed mice, flash freezing in liquid nitrogen, sectioning and observing
apparent distribution in tissue sections prepared for MALDI imaging . Imaging experiments were performed using a Shimadzu
Axima CFR+, along with a prototype Shimadzu MALDI MS, samples were coated with CHCA matrix using a TLC sampler. 3
different target slides were assessed - steel target, ITO coated glass and prototype Shimadzu target slides.
Preliminary Data
Distribution of the injected peptide in murine heart has been measured directly using the（M+H）+ peak and MS/MS experiments
performed using the 1121 Da parent and observing the fragment at 974 Da. PET labelling of the peptide will use［18F］
fluoroacetaldehde which forms a covalent adduct at lysine residues. Activities of up to 287 MBq of the［18F］fluoroacetaldehde
labelling agent have thus far been generated in low-activity trials. Limits of detection will be determined and data from MS/MS
experiments along with images showing peptide distributions at high and low concentrations will be presented.

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 4: Imaging I

PMo-064

13:30 － 14:40

NALDI-IMAGING OF TUMORS: PHOSPHOETANOLAMINE EFFECT ON THE
LIPID PROFILE OF A MELANOMA MODEL

Alessandra Tata1, Anna Maria A P Fernandes1, Vanessa G Santos1, Rosana M Alberici1,
Carlos A Parada2, Wellington Braguini2, Luciana Veronez2, Gabriela Silvia Bisson2, Felippe H Z Reis3,
Luciane C Alberici3, Marcos N Eberlin1

ThoMSon Mass Spectrometry Laboratory, Institute of Chemistry, University of Campinas- UNICAMP, 13084-971,
Campinas, SP, Brazil, 2Departamento de Enfermagem Materno-Infantil e Saude Publica, Escola de Enfermagem deRibeirao Preto, SP, Brazil, 3Departamento de Fisica e Quimica, Faculdade de Ciencias Farmaceuticas de Ribeirao Preto,
Universidade de Sao Paulo, Ribeirao Preto, SP, Brazil.
1

Keywords:
NALDI-imaging, phophoethanolamine, melanoma, phospholipids
Novel aspects:
NALDI- imaging mass spectrometry demostrated to offer the advantages of little or no sample preparation and to show thechanges in the phospholipid metabolite of the tumor after drug treatment.
Abstract:
Phospholipids are the main components of biological membranes and there is a special interest in elucidating their role in thetreatment of cancer. In fact, as observed before, the tumor progression is accompanied by an adaptive metabolic change showing
high levels of phosphatidylcholines（PCs）and phosphatidylethanolamines（PEs）. A class of potential synthetic antineoplastic
phospholipids such as alkylphospholipids, generally termed as“ether lipids“, acting on phospholipid metabolism, represents
promising treatment for cancers. In fact, ether lipids, such as phophoethanolamine（PHOS-S）, has been shown to be able toreduce the synthesis of phosphatidylcholines（PCs）in cancer cells interfering with turnover of phospholipids. The aim of this workis the evaluation of changing in the lipid profiles of an animal model of melanoma treated with phosphoethanolamine（PHOS-S）bynano-assisted laser-desorption- ionization imaging mass spectrometry（NALDI-IMS）. For the first time, commercially available
nano-structured surfaces are used as substrates for imaging tumor tissue sections of mice.
The implantation of B16/F10 melanoma cells was done in the dorsal of the mice. At the 10th day from the inoculation, the animals
were randomly divided into 2 groups： the control group received 0.1 mL water（placebo）and the treated group received 0.1 mL
phosphoethanolamine（PHOS-S）solution（ 5 mg/mL）daily during 10 days. After the treatment, control and treated mice weresacrificed and tumor mass was removed. The tumors were frozen in liquid nitrogen, stored at -80 °C , and subsequently cut in 30
μm sections using a cryo-microtome（CM1850, Leica, Germany）. After cutting each slice was transferred on a NALDI plate
provided by Bruker Daltonics. The NALDI plate was heated from its other side by the operator’s finger. The quick heating,“thawmounting“, resulted in an imprinting of the section. While the rest of the tissue was washed off by water rinse（approximately 5ml）, the imprinting of phospholipids stayed on the NALDI surface. It is worth noticing that any matrix application was needed.
The NALDI-imaging mass spectrometric experiments were performed using a MALDI-TOF Autoflex III Smartbeam（Bruker
Daltonics, Germany and USA）equipped with a 337 nm laser desorption ion source. The spatial（ 2 D）resolution was set to 150 μm. The imaging experiments were replicated trice on consecutive tissues. The images were created and normalized by
FlexImaging software and processed by Biomap software. The identification of the phospholipids was done by LIFT-MS and thelipid searches were performed through the on line database, www.lipidmaps.org. For the first time, it has been demonstrated how
a commercially available NALDITM chip can be used as support for desorption/ionization imaging experiments to differentiate thelipid profile of a human melanoma its fatty capsule. NALDI-MS allowed to identify and map the distribution of the lipid tumor
biomarkers. Compared with the fatty tissue, the tumor is mainly characterized by an high abundance of monounsaturated andpolyunsaturated phospholipids such as［PC（30： 1 ）+H］+ at m/z 706.6,［PC（32： 1 ）+H］+ at m/z732.6,［PC（34： 1 ）+H］+ at m/z
760.6  and［PC（36： 2 ）+H］+ at m/z 786.6. According with the literature［ 5 ］, not only an high abundance of phospholipids wasobserved in this study but also a significant low presence of triacylglycerols（TAGs）at m/z 895.7 and 897.7. Then, the lipid profile
of a melanoma was also compared with the one of the same cancer grown in mice fed with PHOS-S.
The PHOS-S-treated tumor slices were imagined and the phospholipid biomarkers mapped. A significant decreasing in abundanceof the lipid tumor biomarkers was observed. The treated tumor also presence different stages of tumor development that can beclearly observed in the NALDI images. The results confirmed the PHOS-S’s capability to inhibit the phosphatidhylcholines’s
turnover.

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 4: Imaging I

PMo-065

11:10 － 12:20

An optimized organ fixation technique for imaging and quantitative mass
spectrometry for high energy phosphate-metabolites

Sugiura Yuki1,2, Makoto Suematsu1
1

Keio University, Tokyo, Japan, 2Precursory Research for Embryonic Science and Technology (PRESTO)

Keywords:
metabolomics, imaging mass spectrometry, postmortem degradation, microwave irradiation
Novel aspects:
Employing a microwave fixation technique is essential for precise imaging and quantification of metabolites by mass spectrometry.
Abstract:
Previous studies have shown that MALDI-Imaging MS（IMS）can visualize distributions of diverse metabolic molecules in the
biological tissues. On the other hand, a highly comprehensive/quantitative analysis of metabolites by capillary electrophoresisMS（CE-MS）can efficiently complements the IMS technology. However, optimization of animal organ sampling protocol for thesemetabolomic analyses has remained as a quite critical issue, because major degradation of the metabolites occurs in tissues
within a couple of ten seconds after death, especially of high energy phosphate-metabolites. In this study, we have evaluatedseveral sample preparation techniques, namely, a focused-microwave irradiation, in situ freezing（ISF）method, and a postmortem
freezing fixation（PMF）with decapitation.
As results, we found that the PMF caused unacceptable autolytic reduction in ATP and increases in ADP and AMP； absolute
quantification by CE-MS revealed that 90% of ATP was broken into downstream metabolites, and therefore, AMP level represents
a ten times increase. Furthermore, we found that the micro-wave fixation as the best way for our purpose； extents of the ATP
reduction, AMP elevation, lactate elevation as a results of enhanced anerobic respiration, were much lower in the micro-waved
samples compared to in-situ frozen brains. Finally, we revealed that the microwave irradiation of brain samples improved IMS data
quality in terms of the number of effective pixels and the image contrast（i.e., the sensitivity and dynamic range）

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 4: Imaging I

PMo-066

13:30 － 14:40

A novel method to observe cell surface protein GRP78 expression by timeof flight-secondary ion mass spectrometer (TOF-SIMS)

Hsiao-Ting Hsieh2,3, Tsui-Yun Lo1,3, Fu-Der Mai1,2,3

Department of Biochemistry, School of Medicine, Taipei Medical University, Taipei, Taiwan, 2Graduate Institute ofMedical Science, Taipei Medical University, Taipei, Taiwan, 3Biomedical Mass Imaging Research Center, Taipei Medical
University, Taipei, Taiwan

1

Keywords:
HepG2, Rotenone, GRP78,scanning electrochemical microscopy（SECM）, time of flight-secondary ion mass spectrometer（TOF-SIMS）
Novel aspects:
The cell surface protein GRP78 translocation can be observed by TOF-SIMS imaging technology.
Abstract:
The protein expression level play an important role of living organisms. Traditional detection methods include western blot,
enzyme-linked immunosorbent assay, fluorescence resonance energy transfer and flow cytometry. But these methods havesome limitations and defect. In this study, we used rotenone which is a specific inhibitor of mitochondrial complex I on humanhepatocellular liver carcinoma cell line (HepG2). Rotenone induced cell apoptosis progression and endoplasmic reticulum stress.
After 48 hours we observed the endoplasmic reticulum stress associated protein GRP78 expression location change by scanningelectrochemical microscopy (SECM). We found that HepG2 cells mitochondria membrane potential were signicant decreases asrotenone concentration increased. The concentration of rotenone in 300nM can induce 5% HepG2 cell apoptosis and 500nM
can induce about 10% cell apoptosis. Using confocal microscope to observe GRP78 protein expression location, we found some
GRP78 translate to cell membrane and some GRP78 in the endoplasmic reticulum. We built the mass image of GRP78
expression by time of flight-secondary ion mass spectrometer (TOF-SIMS) analysis. All the preliminary results showed that thecell surface protein can be observed by TOF-SIMS imaging technology.

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 4: Imaging I

PMo-067

11:10 － 12:20

Imaging Mass Spectrometry of Plant Tissue Slices: Comparison of MALDITOF MSI and MALDI-FTICR MSI

Katsutoshi Takahashi1, Daisuke Miura2
1

Institute of Advaned Industrial Science and Technology, Tsukuba,Japan, 2Kyushu University, Fukuoka, Japan

Keywords:
MALDI-FTICR MSI, MALDI-TOF MSI, Imaging mass spectrometry, plant tissue, Arabidopsis thaliana
Novel aspects:
We tried to map small molecules in plant tissue slice using ultra-high-resolution and high-accuracy MALDI-FTICR MSI equippedwith in-house ion-source. Also we compared the map with convensional MALDI-TOF MSI results.
Abstract:
Recently MALDI mass spectrometry imaging（MSI）has been a powerful tool to map spatial distribution of molecules on thesurface of biological materials. Frequently MSI is applied to animal tissue slices to map various biological molecules on the slice,
such as functional lipid molecules, drug metabolites and so forth. However, it’s still been difficult to apply MSI techneques to planttissue because of the difficulties in sample preparation and the existence of secondary or tertiary metabolites. In general, planttissue is highly water rich and cell wall exits between plant cells, so special sample preparation technique was required to makethin slice of plant tissue. Ultra-high-resolution and ultra-high-accuracy in mass spectrometry are also necessary to identify
metabolites contained in plant tissue.
We have developed in-house MALDI ion-source, which was originally developed as a microscopic MALDI ion-source, and it was
equipped with Bruker-Daltonics commercial FTICR-MS（Apex-Qe-94T）. In our ion-source, small transparent ITO-coated slide glass
was inserted into the original ion-funnel horizontally and tightly focused UV-LASER beam was irradiated vertically from upper long
working distance Cassegrain mirror objective while sample specimen can be observed by lower zooming video microscope inreal-time. High-precision sample stage was also equipped inside the vacuum chamber and the exact position was able to becontrolled by in-house software. All of the MSI experiment sequence, including sample specimen observation with videomicroscope, sample stage motion control and triggering MALDI-FTMS run, was also controlled by in-house-build software writtenwith LabView.
We tried to make thin（40 micrometer）slice of quick frozen leaf of Arabidopsis thaliana and the frozen slice was vacuum dried
under freezing temperature followed by matrix application using sublimation or neblizer spraying, before dedicated for MALDI MSI
experiments. A leaf of Arabidopsis thaliana was first quickly frozen and ice-embedded. The ice-block was then embedded in OCT
compound followed by micro-slicing using cryo-microtome under freezing temperature. 40 micrometer thick thin slice ofArabidopsis thaliana leaf was successfully obtained by Kawamoto’s method utilizing special adhesive support film during thinslicing. The thin slice laid on adhesive support film was then put on the cold metal block inside vacuum chamber to be dried
under freezing temperature. The dried thin slice on the support film was glued on the small ITO-coated slide glass followed bymatrix application using sublimation or neblizer spraying.
We tried to use 2,5-dihydroxy-benzoic acid（DHBA）as matrix and MALDI-FTICR MSI experiment was done in negative-ion mode.
Also we tried to use another matrix substance 9 -amino-acridine（ 9 -AA）because 9 -AA was reported to work as a MALDI matrixof small acidic compounds in negative-ion mode, including typical plant hormones such as Auxin. We performed a lot of try-anderror experiments varying thickness of matrix layer on the dried slices, to find optimized experimental parameters to map small
molecules on the plant tissue slices.
Also, the samely prepared specimens were MS imaged using commercial MALDI-TOFMS,（Shimadzu AXIMER performance）, in
order to discuss the advantages and disadvantages of MALDI-FTICR MSI against convensional MALDI-TOF MSI in plant
physiological studies.

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 4: Imaging I

PMo-068

13:30 － 14:40

MALDI-MS Imaging Atlas of Mouse Development

Takashi Nirasawa1, Masaya Ikegawa2
1

Bruker Daltonics K.K., Yokohama, Japan, 2Kyoto Prefectural University of Medicine, Kyoto, Japan

Keywords:
MALDI-TOF MS, Imaging-MS, mouse development
Novel aspects:
MALDI imaging mass spectrometry is an effective new technology for the discovery of molecular signatures of mouse
development.
Abstract:
Background: Imaging mass spectrometry（IMS）using matrix-assisted laser desorption ionization（MALDI）is an effective tool formolecular studies of complex biological samples such as human and experimental animal tissue sections. Distribution maps ofmultiple analytes directly obtained from tissue sections can be correlated with histological and experimental features. Targeting
whole mouse embryos by IMS will cover spatial and temporal molecular arrangements without the need for target-specificreagents, allowing the discovery of novel and diagnostic markers of mouse development.
Materials and Methods: From E12 to E18 days post coitus（d.p.c.）mouse embryos were sacrificed for this experiment. Ten micron
thick frozen sections of whole mouse embryos were cut on a cryostat. The sections were transferred to precooled conductive
Indium-Tin-Oxide coated glass slides for sagittal and transverse orientation. The ImagePrep device was used for this experiment
which utilized vibrational vaporization of the matrix with a piezo-electric spray head. The MALDI measurement and image analysis
was carried out on a linear Autoflex instrument equipped with a smartbeam-laser and with the flexImaging 2.1 and ClinProTools 2.2
software packages（Bruker Daltonik GmbH, Bremen）. MALDI measurements were done in linear mode in a mass range of 1,00040,000 Da. The lateral resolution for the MALDI imaging was set to 100μm. Statistical analyses were carried out using the
ClinProTools 2.2 Software. For the statistical analyses, the mass spectra were internally recalibrated on common peaks andnormalized on the total ion count. An average spectrum created from all single spectra was used for a peak picking and to define
integration ranges. The data set was further analyzed by multivariate analysis such as principal component analysis（PCA）and
hierarchical clustering. For the PCA analysis and the clustering across different tissues, the mass spectra were selected on thetissue by assigning representative areas of developing tissue in each stage. Following the MALDI analysis, the matrix was washed
off the slides with 70% ethanol and the sections were stained with a modified nile blue staining, scanned at low resolution, andco-registered with the MALDI imaging results. This was also referred by The Atlas of Mouse Development（Kaufman MH, 1992）.
Results and Discussions: MALDI imaging in combination with hierarchical clustering allows the comprehensive analysis of the insitu mouse development proteome. On the basis of this cluster analysis, classification of complex and dynamic distribution oftissue proteome was well comparable to histological distribution. It is possible to trace development of brain, spinal cord, heart,vascular system, lung, bone, liver, kidney, gut, pancreas, bladder, upper and fore limbs, thymus, pineal gland, and so on.
Comparing the IMS atlas of mouse development with the above mentioned histology, it is highly efficacious in understanding
embryonic development. Therefore, it must be of particular interest to apply this knowledge into phenotypic screening ofgenetically mutant mouse to find out aberrant molecular organization in the early stage of the mouse development.

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 4: Imaging I

PMo-069

11:10 － 12:20

MALDI imaging of desalted rat brain sections reveals the up- and downregulation of phospholipids by ischemia

HAY-YAN J WANG1, HSUAN-WEN WU1, PING-JU TSAI1,2, CHENG BIN LIU1,3

Department of Biological Sciences, National Sun Yat-Sen University, Kaohsiung, Taiwan, 2Department of Surgery, Yuan’s General Hospital, Kaohsiung, Taiwan, 3Department of Obstetrics and Gynecology, Veterans General HospitalKaohsiung, Kaohsiung, Taiwan
1

Keywords:
MALDI mass spectrometry imaging, in situ desalting, brain ischemia, lipidonics.
Novel aspects:
Matrix sublimation/deposition combined with in situ desalting for MALDI-MSI successfully revealed the subtle lipidomic changes in
the ischemic rat brain parenchyma.
Abstract:
Conventional MALDI mass spectrometry imaging（MALDI-MSI）studies of ischemic brain sections revealed the changes ofphospholipids mostly in their alkalinated form as a result of the ischemic assault. However, the massive change of alkali metal ionsin the brain parenchyma following ischemic stroke likely masked the underlying changes of phospholipids following brain
infarction. To overcome the interference of massive cation change and to successfully reveal the subtle lipidomic changes byischemia, MALDI-MSI studies were carried out on the ischemic rat brain sections in which the matrix was applied by vacuum
sublimation/deposition following a simple in situ desalting process.
Brain ischemia was surgically induced by permanent middle cerebral artery occlusion（pMCAO）in male Sprague-Dawley rats.
Ischemic rat brains were collected 24 hours after pMCAO surgery and cut into 14 µm coronal sections, then collected onto theindium-tin oxide（ITO）-coated slides. After the initial vacuum drying, each brain section was drip-washed with 600 µL of 150 mM
ammonium acetate solution to remove the endogenous alkali metal ions. Thereafter, the brain sections were vacuum-dried foranother 20 min before applying the MALDI matrix 2,5-dihydroxybenzoic acid（DHB）onto the brain section by matrix sublimation/
deposition under vacuum.
MALDI-MSI result showed that the distribution of lysophosphatidylcholine 16： 0 （LPC 16： 0 ）in the desalted brain section andthat of sodiated LPC 16： 0 in the neighboring undesalted section was essentially identical, confirming the suitability of thedesalting method for MALDI-MSI application. MALDI-MSI of phosphatidylcholines（PCs）containing both saturated and saturated
plus monounsaturated fatty acyl（MUFA）residues showed slight increase over the ischemic brain parenchyma. However, thedistribution of PC 16： 0 /18： 1 showed a consistent reduction in the infarcted area. The distribution of polyunsaturated fatty acyl
（PUFA）residue-containing PCs revealed laminated patterns in the normal cerebral cortex. Ischemia disrupted the laminatedpattern of PC 16： 0 /20：4, and reduced its abundance as well as the abundance of PC 18： 0 /20：4. However, ischemia led to thesubcortical increase of PUFA-containing PCs containing stearoyl residue, and confined their cortical increase to limited areas. In
addition, ischemia caused the consistent decrease of sphingomyelin 18： 0 （SM 18： 0 ）with the corresponding increase ofceramide in regions showing moderate to high abundance of sphingomyelin. Together, the results showed that other than thedecrease of PCs and the corresponding increase of LPCs, ischemia also heterogeneously increased the abundance of PCs
containing mostly stearoyl residue over the ischemic areas. Beyond the changes of PCs and LPCs, the consistent decrease of SM
may also serve as a marker for brain injury. The combined imaging results revealed the subtle yet potentially important lipidomic
changes through which the effective management of brain ischemia may be based upon.

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 4: Imaging I

PMo-070

13:30 － 14:40

Visualization of Transdermal Drug Delivery by 2D-DESI Mass
Spectrometry ----- a Novel Approach to Generate Pseudo Cross-sectional
Skin Images

Akira Motoyama1,2, Keishi Kihara1, Yuichiro Mori1, Mitsuo Takayama2
1

Shiseido Co., Ltd, Yokohama, Japan, 2Yokohama City University, Yokohama, Japan

Keywords:
DESI, Transdermal Drug Delivery, Mass Spectrometry Imaging, Tape Stripping Technique, Stratum Corneum
Novel aspects:
A novel mass spectrometric visualization method has been developed that allows the generation of pseudo cross-sectional skin
images for fast and non-invasive transdermal drug delivery investigations.
Abstract:
Visualization of transdermal drug delivery is important for cosmetic companies that provide topical products containing mild
active ingredients. Fluorescent microscopy, CLSM（confocal laser scanning microscopy）, and TOF-SIMS（time-of-flight secondary
ion mass spectrometry）have been typically used for those purposes. The conventional methods, however, have a distinct
disadvantage in which biopsy samples are required to obtain a cross-sectional drug penetration image across skin. We present inthis study a novel mass spectrometric visualization method to generate pseudo cross-sectional skin images for fast and noninvasive transdermal drug delivery evaluations. After applying an aliquot of a topical cream containing 1 % of 4 -methoxy salicylic
acid potassium salt（ 4 MSK）to the inner forearm of a healthy male volunteer, skin samples were collected by the tape-stripping（TS）technique. Using the standardized TS procedure, stratum corneum（SC）, the most outer barrier layer of epidermis, wascollected as a sheet on an adhesive tape. After collecting several SC layers by repetitive TS operations, two-dimensionaldesorption elestrospray ionization / mass spectrometry（ 2 D-DESI MS）was utilized to obtain planer distribution information of
4 MS（K）penetrated into SC. The collected data was processed by a patent pending method to create a pseudo cross-sectional
skin image showing 4 MS（K）penetration through SC layers. Potentials and limitations of the approach will be discussed on thepresentation.

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 4: Imaging I

PMo-071

11:10 － 12:20

Chemical sensor arrays for application in mass spectrometry on surfaces
and tissues

Martina Lorey1, Rabah Soliymani1, Jari Yli-Kauhaluoma2, Marc Baumann1

1
Protein Chemistry Unit, University of Helsinki, Helsinki, Finland, 2Division of Pharmaceutical Chemistry, University of
Helsinki, Helsinki, Finland

Keywords:
MS-Imaging, mass-tag, microchip
Novel aspects:
We aim to develop diagnostic microchips, utilizing targeted secondary detection MS-imaging. These chips would allow rapid
diagnosis for various purposes, regardless the size or abundance of target molecules.
Abstract:
MALDI-imaging mass spectrometry has become one of the most promising analysis technologies, allowing measurements ofhundreds of different molecules on surfaces at the same time without disruption of sample integrity. However, like all techniques
it has limitations： Very hydrophobic proteins, membrane proteins, high mass proteins and low abundant molecules are hard tomeasure.
We try to overcome those limitations by applying a new approach which was lately published by Thiery et al. (1) . TArgeted
multiplex MS IMaging（TAMSIM）is a secondary detection method, which allows the concurrent detection of a basically unlimited
number of different target molecules on a tissue surface at the same time. The surfaces are treated with probes containing
antibodies, which bind to their particular targets. These antibodies carry chemical tags of a defined mass, which arephotodissociated by MS laser irradiation and detected via the time-of-flight analyzer in MALDI-TOF. Thiery et al. showed thatsimultaneous measurement of Chromogranin A and Synatophysin as markers for Langerhans islets on human pancreas tissues by
TAMSIM has a very good analogy with the immunohistochemistry staining of the same tissue section(1). To make this approach
multiplex, a library of photocleavable tags with different, distinct masses is synthesized. The mass tags contain an activated acidgroup which randomly binds to all available amino groups.
We aim to apply the same principle for the development of diagnostic microchips by creating two different microarrays for fluid
samples.
The first chip is designed to give solely a yes / no answer for e.g. biomarker detection. Specific antibodies against the desired
biomarkers are immobilized in an array on the chip surface, every position with another antibody. The fluid sample is treated withthe mass tag and applied on the chip. After incubation and washing a fast MS analysis will show on which spots the tag can bedetected, thus stating whether the tagged substrate is bound on that spot.
For the second kind of chip the samples are spotted on the chip surface and immobilized. A mixture of differently tagged
antibodies, which are specific against the targets of interest, is applied on the chip surface. After incubation and washing, the chipis screened via MS analysis, and each detected tag is stating a different target molecule on which the specific antibodies havebound.
Due to the specific nature of the mass tags no matrix is needed for desorption and ionization, which dramatically increases thesignal to noise ratio. Additionally every antibody can bind several tags, resulting in signal amplification. It would be possible todetect virtually every molecule for which a specific antibody can be synthesized, regardless of the size of the target molecule or
its abundance compared to other molecules in the same sample.
These chips would allow rapid diagnosis for various purposes： from research and drug testing to differentiation between cancer
types to provide optimal,“personalized“ medication.
（ 1 ）Thiery et al., Proteomics, 2008, 8, 3725-3734.

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 4: Imaging I

PMo-072

13:30 － 14:40

The Sausage Omelette: Breakfast of (IMS) Champions or a Novel
Quantitation Tool for Small Molecule Imaging?

Shannon Cornett1, Katherine Kellersberger1, Mark R Groseclose2, David S Wagner2, Stephan Castellino2
1

Bruker Daltonics, Billerica, MA, USA, 2GlaxoSmithKline, Research Triangle Park, NC, USA

Keywords:
Quantitation in MALDI Imaging, drug imaging, FTMS
Novel aspects:
A novel model for quantitation in small molecule IMS studies
Abstract:
Introduction
Imaging Mass Spectrometry（IMS）has proven to be a powerful technique for the analysis of drugs and their metabolites in tissue.
In IMS, success is dependent upon the ability to extract the molecule of interest from the tissue efficiently as well as the suitabilityof that molecule for IMS analysis. We previously introduced a model for assessment of drug suitability and sample preparation
optimization based on the“sausage“ model. Here we present the refinement of this approach（sausage omelette）and discuss
the application of this model to quantitation in IMS studies.
Methods
Bulk tissue was homogenized then aliquots were spiked with varying levels of a small molecule target and mixed thoroughly withminimal addition of water. Spiked homogenates were used to fill wells formed in an embryonic protein-based support matrix, thenfrozen at -80 until solid and sectioned at 12 um thickness. Following matrix coating with DHB（30 mg/mL in 50% MeOH）, sample
sections were analyzed on a Bruker solariX FTMS（Bruker, Billerica, MA）and data analyzed with the flex Imaging software.
Results:
While determination of relative levels of parent drug and metabolites in tissue is valuable to the understanding of distribution andmetabolism, the ability to use these data in the context of drug development, including determination of the efficacy and safety ofa drug requires a quantification step.
Due to the nature of the MALDI process, particularly in regard to the ionization of analytes directly from treated tissue, the issueof quantitation is problematic. Here, we show the refinement of an approach previously proposed for optimization of sample
preparation（including matrix application）and its value in developing a method for quantitation of target molecules directly fromtissue sections.
To assess this model, a number of drugs such as reserpine, verapamil, sunitinib, and terfenadine as well as several early-stage
development drugs were spiked into homogenized liver aliquots then injected into a support matrix, frozen and sectioned as
tissue. Analysis of these sections shows drug signal from homogenate plugs with no evidence of bleeding into the matrix orseparation of the plugs from the matrix during sectioning, indicating that this model nicely mimics dosed tissue. It was also
observed that the MS signal response is linear with respect to concentration over more than 2 orders of magnitude. If ion preselection and accumulation is performed（CASI）, more than 3 orders of magnitude linearity is observed.
Based on our results, this model shows great promise as a simple method for quantitation in small molecule IMS studies.

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 4: Imaging I

PMo-073

11:10 － 12:20

To Digest or Not to Digest, That’s the Question

Martin Schuerenberg1, Christine Luebbert1, Rainer Paape1, Janine Rattke1, Axel Walch2, Sandra Rauser2,
Michael Becker1
1

Bruker Daltonik GmbH, Bremen, Germany, 2Helmholtz Center Munich, Neuherberg, Germany

Keywords:
Biomarker identification from Imaging, spatioal proteomics, tryptic digest of tissue, LC-MALDI-TOF/TOF, software to link LC-MS
with Imaging data
Novel aspects:
New MALDI imaging method that provides identities plus distributions of more than 100 proteins in tissue sections simultaneously.
Abstract:
Introduction:
MALDI-Imaging Mass Spectrometry（IMS）of proteins in tissue sections combined with Top-Down proteomics represents a
powerful approach to biomarker discovery. However, lack of direct identification strategies is preventing its broader use inProteomics studies.
We have developed a new workflow that combines the spatial information obtained by IMS with the routine identification ofproteins from tissue sections by LC-MS. Protein digests were generated by trypsinization on two subsequent tissue sections
maintaining the spatial distribution of the peptides. One of the trypsinized sections was analyzed by IMS. Peptides from the othersection were identified by routine LC-MALDI-MS/MS. The new“Spatial Proteomics workflow“ allows routine identification of morethan 100 proteins from tissue sections as well as their spatial distribution at the 50 um level.
Methods:
Fresh frozen rat brain, testis samples and human breast cancer biopsies were sectioned to 10 um slices. Each section was
analyzed in parallel by analyzed with and without tryptic digestion. For the digests, two subsequent slices were placed onto one
glass slide and a trypsin solution was applied to both of them by supersonic vibration. Tryptic peptides were imaged from one
section in a reflector mode MALDI-TOF. The section was coated with CCA matrix. Tryptic peptides were identified by nanoLCMALDI-TOF/TOF analysis and Mascot searching（Matrix Science）after elution from the second slice. A software tool wasdeveloped that grouped the peptide masses by their associated protein and linked it to the peptide masses in the bottom-up
image for visual inspection of their co localization.
Results:
We analyzed rat organs using the new Spatial Proteomics approach as model systems, at the 50 um spatial resolution level. In
both organs, more than 200 peptides present in the images（i.e., 80 %）were identified by the parallel LC-MALDI analysis andapproximately 120 proteins localized. As primary validation tools for the assignment of protein distributions in the tissue sections,the intensity and co-localization of 2 or more peptides and the specificity of mapping the peptide molecular weights to 1 ormore proteins were used.
As an extension of the established top-down imaging strategy, this bottom-up Spatial Proteomics approach facilitates theidentification and simultaneous localization of a much greater number proteins than was previously possible. Proteins of biologicalrelevance were identified that were previously identified by top-down imaging and top-down sequencing such as［ 1 ］thymosin- 4
and LCFA-CoA from rat testis and［ 2 ］Cystein-rich intestinal protein 1 （CRIP 1 ）from breast cancer biopsies - a novel marker formetastatic cancer.
HER 2 +/CRIP 1 + specified human breast cancer biopsies were analyzed using the established Spatial Proteomics workflow. CRIP 1
was identified as one in 150 proteins. Arg-68 was present predominantly in the methylated form and to a lower extent in the nonmodified form. Arginine methylation heterogeneity present in CRIP 1 was in agreement with the unexplained top-down imaging
peak pattern［ 2 ］in breast cancer biopsies.
Globally, the Spatial Proteomics workflow provided protein distributions that are typically invisible in top-down images. These
included large（ 4 MDa titin）and membrane associated proteins such as MARCKS； however, mostly cytosolic, nuclear andcytoskeleton associated proteins were identified. The method may also be useful for protein imaging and identification from FFPE
tissue in which top-down analysis is made impossible by protein crosslinks and which is largely accessible from tissue banks.
1. Lagarrigue 2011 MCP 10：M110.005991
2. Rauser 2010 JPR 9 ：1854-63

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 4: Imaging I

PMo-074

13:30 － 14:40

Rapid assessment of drug and metabolite distribution in whole animal
tissues via accurate mass imaging

Arnd Ingendoh1, Cristine Quiason2, Katherine Kellersberger1, Brian Dean2, Sheerin Shahidi-Latham2
1

Bruker Daltonik GmbH, 2Genentech Inc., South San Francisco, CA

Keywords:
Imaging, MALDI, FTMS, metabolite, accurate mass
Novel aspects:
Identification of drug and metabolites in whole body tissue sections by rapid accurate mass full scan imaging.
Abstract:
Traditional imaging approaches relying on accurate mass measurements typically require long scan speeds that result in lengthy
acquisition times and hefty file sizes. For this reason, applications toward the assessment of whole body drug distributions havebeen limited. New methods allowing for ion accumulation prior to FTICR detection, along with online data reduction algorithms,
have significantly decreased unnecessary overhead, thus considerably improving the overall approach. In this study, rapid imaging
of whole body tissues with accurate mass, high resolving power and high dynamic range by MALDI FTMS in broadband mode
were successfully obtained in a significantly shorter time frame and allowed for the rapid assessment of drug and metabolite.
Male Sprague-Dawley rats（n= 3 ）were administered a single 30 mg/kg PO dose of Olanzapine euthanized at 2, 6, and 12 hrs postdose, and flash frozen. Whole-animal carcasses were sectioned at 20 micron thickness and whole-body sections were transferred
to MALDI target plates using double-sided tape. A solution of alpha-cyano- 4 -hydroxycinnamic acid（CHCA）matrix（ 5 mg/ml,
50/50（v/v）Acetonitrile/H 2 O）was spray coated onto tissues using the automated HTX Imaging sprayer. Full MS spectra from m/z
140-1000 were acquired on a 7.0T SolariX FTMS system（Bruker）equipped with a dual ESI-MALDI source employing smartbeam-II
technology. Data were processed and images were extracted using FlexImaging software（Bruker）.
High resolution（resolving power of 150,000 @400 m/z）and exact mass（< 2 ppm）imaging data were obtained in full scan MS
mode over the m/z range of 140 to 1000 using the 7 Tesla FTMS（Bruker）. Exact mass（+/-0.002 Da） 2 D images of drug showed
unique distributions across the whole-body sections. Each whole body section collected comprised of roughly 24,000 pixels andwith the acquisition rate of 0.8 seconds/pixel took 5 hours to acquire. The large sample introduction plate and fast acquisition
capabilities of the 7 Tesla FTMS made it an ideal platform for rapid whole body mass spectrometric imaging（WB-MSI）. This
study demonstrated the advantages of a fast acquisition approach for whole body imaging because it allowed for the acquisitionof a full MS spectrum within several hours, effectively enabling a full data set representing a PK time course to be obtained over a
course of a few days rather than weeks by traditional approaches.

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 4: Imaging I

PMo-075

11:10 － 12:20

Development of MALDI-MS Imaging of Metabolite and Protein in a Single
Tissue Section

Ayumi Yamaguchi, Daisuke Miura, Yoshinori Fujimura, Hiroyuki Wariishi
Kyushu University, Fukuoka, Japan

Keywords:
mass spectrometry imaging, metabolite, protein, MALDI-MS
Novel aspects:
Our proposed MALDI-MSI system could visualize spatial distributions about both proteins and metabolites on the same tissue
section.
Abstract:
Background
Matrix-assisted laser desorption/ionization（MALDI）mass spectrometry imaging（MSI）is a remarkable new technology thatenables us to determine the distribution of biological molecules in tissue sections by direct ionization and detection. This
technique is now widely used for analysis of biomolecules such as proteins, lipids and drugs, with high sensitivity. Previously, we
attempted to apply this technique to low-molecular-weight metabolite imaging using 9 -aminoacridine（ 9 -AA）as a matrix. As
metabolites represent the compound-level phenotype of information via gene expression and translation, the metabolite imaging
technique is expected to be a potential tool to systematically understand the biological processes. On the other hand, anintegrated analysis of multiple layers of biomolecules has received much attention to get a better understanding of the entire
biological system. Since expression levels of enzymes in some tissues reflect metabolite pattern there, integrated information of
spatial distributions of both metabolites and proteins in the same tissue section will be a breakthrough for unraveling complex
biological systems. In this study, we developed a MALDI-MSI technique for simultaneous analysis of the multiple biomolecules,
proteins and metabolites, in a single tissue section.
Results
Normal rat brain tissues were coronal sliced at 10-µm thickness with a cryostat-microtome and thaw-mounted onto a stainlessplate. First, we analyzed the distributions of metabolites in the tissue section. A matrix solution（10 mg/mL 9 -AA in 100 %methanol）was sprayed to the section using an airbrush. Single reflectron-type MALDI-TOF-MS（AXIMA Confidence, Shimadzu）
was used in negative ion mode for metabolite imaging. More than 100 metabolite peaks were detected, and such peaks could be
visualized with their unique distributions. By the MS/MS analysis using quadrupole ion trap-type instrument（AXIMA QIT,
Shimadzu）, we identified about 30 metabolites, including nucleotides, cofactors, phosphorylated sugars, amino acids, lipids, andcarboxylic acids. After metabolite imaging, the tissue section was washed twice in 70 % ethanol for removing 9 -AA, lipids, salts
and other contaminants, and once in 100% ethanol for fixing protein. The washed sample was subjected to direct tryptic digestionof proteins on tissue sections（in situ digestion）. Trypsin solution（0.2 µg/µL in 10 mM ammonium hydrogen carbonate）was
spotted onto the tissue section using chemical ink-jet printer CHIP-1000（Shimadzu）, and then 2,5-dihydroxy benzoic acid（25 mg/
mL in 0.1% TFA/60% methanol）was spotted on the same point. The tissue sample was analyzed by MALDI-MS（AXIMA QIT）in
positive ion mode for peptide MSI, and detected peptides were identified by MS/MS analysis. The MS/MS spectra obtained were
submitted to a MASCOT（Matrix Science）database search engine. Spatial distributions of 30 peptide peaks were successfully
visualized by the sequential MALDI-MSI after metabolite analysis and several peaks were identified.
Conclusions
The present multistep MALDI-MSI technique enabled to visualize the distribution of both metabolites and proteins on the samebrain tissue section for the first time. Overlay of the data from a serial MALDI-MSI allowed the comparison of their relative
abundance or spatial localization unambiguously. Although it is still primitive, we believe that our proposed strategy integrating
the spatial information of biomolecules in two different layers will strongly contribute to the advance in biological andpharmacological researches.

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 4: Imaging I

PMo-076

13:30 － 14:40

Automated Tissue State Assignment for High Resolution FTMS MALDI
Imaging Data

Paul Speir1, Jens Fuchser2, Soeren Deininger2, Michael Becker2, Katherine Kellersberger1
1

Bruker Daltonics, Inc., 2Bruker Daltonik GmbH, Bremen, Germany

Keywords:
Imaging, MALDI. FTMS, hierarchical clustering
Novel aspects:
First report of hierarchical clustering of MALDI-FTMS imaging data
Abstract:
Interpretation of MALDI imaging data can be tedious, especially if many peaks are present. Hierarchical clustering is a techniquethat allows clustering of mass spectra based on their overall similarity. The technique is already used for the fast and concise
semi-supervised segmentation of MALDI-TOF imaging data（Deininger et. al, J. Proteome Res 2008, 7 （12）：5230- 6 ）. Compared to
MALDI-TOF spectra of proteins the clustering of FTMS data is more challenging, mainly because FTMS spectra have many more
peaks. In this proof-of-concept study several parameters for the hierarchical clustering such as distance and linkage methods andmissing value treatment were evaluated and their results compared in respect to computational performance and analyticalresults.
MALDI imaging data from several model tissues such as mouse kidney, brain, and pancreas were recorded on a FTMS mass
spectrometer（Solarix, Bruker Daltonics）. For each mass spectrum a peak list was generated. The peak lists were binned into
one bucket table with a custom C# program. Clustering was calculated with R. Different parameters for the distance（such as
Euclidean, Correlation, Manhattan）and linkage（Ward, Complete, Average）were used for the calculation, for the Euclidean
distances data were also pre-treated with Principal Component Analysis（PCA）. Segmentation maps were reconstructed from theclustered data and compared to the anatomy of the tissue sections.
The data suggest that different distance and linkage methods lead to overall similar segmentations of the dataset, which were ingood agreement with the anatomy of the samples. In the kidney, the cortex, medulla, and renal pelvis were clustered as the main
tissue structures by all approaches. The same was true for the anatomical regions in the brain. This suggests that the differencesin the anatomical regions were significantly larger than random pixel-to-pixel variations, thus leading to a robust clustering result.
Differences were observed in the details： Ward linkage led to dendrograms that were easiest to interpret, since they resulted inlarger clusters at the top end of the dendrogram. While ward linkage resulted in the clearest segmentation it was computationallynot feasible to use this on larger datasets. Correlation was found to be a very appropriate distance method that could be applied
without normalization of the data. The preliminary data suggests that since the clustering was robust in the observed cases, it isfeasible to select the parameters according to the computational requirements, which can be different for datasets of different
sizes. Generally the clustering was applicable to FTMS data and allowed a concise segmentation of the datasets.

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 4: Imaging I

PMo-077

11:10 － 12:20

An Investigation of Spatiotemporal Metabolic Behaviors in Response to
Pathological Progress using Multiple Mass Spectrometry Techniques

Miho Irie, Yoshinori Fujimura, Daiki Setoyama, Mayumi Yamato, Daisuke Miura, Hiroyuki Wariishi
Kyushu University, Fukuoka, Japan

Keywords:
LC-MS, mass spectrometry imaging, metabolomics
Novel aspects:
Combination of MALDI-MS imaging and LC-MS-based matabolomics would visualize drastic changes in spatiotemporal metabolite
distribution of MCAO brain.
Abstract:
Background
Metabolomics, the measurement of global endogenous metabolite profile from a biological sample under different conditions, canlead us to an enhanced understanding of disease mechanisms and the discovery of diagnostic biomarkers. Mass spectrometry（MS）coupled with preseparation techniques such as liquid chromatography（LC-MS）or gas chromatography（GC-MS）is a
conventional strategy for metabolomics. These techniques analyze hundreds of metabolites simultaneously； however, they havedrawbacks in analysis of tissue samples due to a requirement of metabolite extraction, which causes the loss of information on
spatial localization of the metabolites. To better understand the complex biochemical processes in living organisms requires not
only the elucidation of the molecular entities involved in these processes but also their spatial distribution within the organism.
Recently, we developed in situ metabolomic imaging technique by using matrix-assisted laser desorption ionization（MALDI）-MS
that can visualize the localization of a broad range of metabolites simultaneously. The MS imaging（MSI）enabled to trace
spatiotemporal metabolic dynamics in the brain of transient middle cerebral artery occlusion（MCAO）rat during infarct formation
after ischemia-reperfusion. Although several distinctive changes during pathological progress were observed, detailed metabolic
dynamics still remained unclear because of a low coverage of detectable metabolites. Herein, we performed additional LC-MS
analysis to understand more comprehensive metabolic behaviors in the brain of MCAO rat. This data together with MSI data wasanalyzed for characterizing detailed metabolomic response to pathological alterations.
Results
To investigate metabolic variance after reperfusion following 1 h of MCAO, we used rat brains at different time intervals after
ischemia-reperfusion（0, 3 and 24 h）. For comparing the metabolic state in ischemic and contralateral hemispheres of MCAO
brain, tissues were coronally sliced at 10-µm thickness with cryostat and then subjected to MSI for visualizing the spatial
distribution of metabolites. On the other hand, metabolites extracted from three different regions such as whole cortex（CTX）,
hippocampus（HI）and corpus striatum（CPu）, were measured by LC-MS. Our previous MSI data of MCAO rat brain indicated thatdrastic spatiotemporal metabolic changes were occurred in the central metabolic pathway. Holistic evaluation of metabolic profile
in each region was firstly investigated by principal component analysis using LC-MS datasets. These results suggest that metabolicstates were clearly different between contralateral and ischemic hemispheres or among three representative compartments（CTX,
HI and CPu）. In ischemic hemisphere, significant metabolic changes at CTX and CPu were observed after reperfusion, while
metabolic state of HI was relatively stable. These observations indicate that metabolic profiling with spatial information isindispensable to better understand the mechanism of disease progress in MCAO brain. Indeed, region-specific metabolic
behaviors in amino acid metabolism, TCA cycle and nucleotide metabolism were shown by hierarchical clustering analysis. In eachspecific region（three compartments and two hemispheres）, data correlations between MSI- and LC-MS-based metabolite
analyses were slightly low（averaged R 2 =0.24）, but remarkable changes such as citrate and N-acetylaspartate were fully
appreciated in both analytical platforms（R2=0.96 and R2=0.89, respectively）. These findings raise the possibility that only a single
LC-MS analysis in small tissue compartments may cause an underestimation of metabolic changes in the tissue. A combination ofdifferent analytical platforms（MSI： spatial information but low coverage； LC-MS： high coverage but loss of spatial information）
enabled us to visualize more comprehensive metabolic behaviors in the tissue micro-regions. Details about metabolic changes
and its biological significance in MCAO brain will be discussed in the session.
Conclusion
Our proposed multiple mass spectrometric platforms revealed for the first time detailed metabolic behaviors after reperfusion in
MCAO brain. Although this is still primitive, further development of systematic analytical methodology for data integration willcontribute to the advances in biological and pharmacological researches.

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 4: Imaging I

PMo-078

13:30 － 14:40

Imaging Hair Analyses for Drugs: A New Perspective to Investigate Drug
Use History

Tooru Kamata1, Noriaki Shima1, Akihiro Miki1, Kei Zaitsu1, Munehiro Katagi1, Michiaki Tatsuno1,
Toyofumi Nakanishi2, Takako Sato2, Hitoshi Tsuchihashi2, Koichi Suzuki2
1

Forensic Science Laboratory, Osaka, Japan, 2Osaka Medical College, Osaka, Japan

Keywords:
imaging, hair, drug, MALDI, forensics
Novel aspects:
Images of drug-positive bands on the longitudinal section of hair shafts, which can provide chronological information of drug use,
were obtained by MALDI-TOF imaging mass spectrometry.
Abstract:
Amphetamine-type stimulants（ATS）represent the most prevalent and potent drugs of abuse. Such drugs ingested into the body
circulate in the bloodstream, and trace amounts of the drugs are deposited in the hair follicles and thus incorporated into the hairshafts. This paper presents the direct detection of ATS in hair and its imaging by MALDI-TOF-MS, with careful optimization ofsample preparation. Preliminary experiments were performed using human hair shafts spiked with the over-the-counter drug
methoxyphenamine（MOP）, as an ATS analog, at various concentrations. Method development was further carried out using hairspecimens from volunteers who took MOP（e.g., 50 mg/day x 5 ）. A longitudinal section of a lengthwise-cut single human hairshaft was directly analyzed by using the MALDI-TOF mass spectrometer autoflex speed（BrukerDaltonics）which utilized a
Smartbeam TM -II solid-state laser, after deposited with α-cyano- 4 -hydroxycinnamic acid matrix using an ImagePrep TM（Bruker
Daltonics）. Although lengthwise-cutting of a single hair shaft was laborious, this was necessary for effectively detecting ppm-level
drugs incorporated in hair. Images of drug-positive bands on the longitudinal section of hair shafts, which can provide
chronological information of drug use, were obtained by monitoring analyte-specific product ions in the selected reaction
monitoring mode. To further confirm the identity of drugs detected by MALDI-TOF-MS, the analyses of drugs and metabolites in
1 -cm segments（from the root side to the tip side）of these hair specimens were carried out by a conventional LCMS-MS
procedure.
Thus, imaging mass spectrometry can offer a new perspective in the analyses of drugs in hair：“imaging hair analyses for drugs“.

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 4: Imaging I

PMo-079

11:10 － 12:20

Easy ambient sonic-spray ionization mass spectrometry imaging (EASIMSI).

Dragos Lostun1, Mario F Mirabelli1,2, Tanam S Hamid1, Alexander Chramow1, Demian R Ifa1
1

York University, Toronto, ON, Canada, 2University of Calabria, Cosenza, Italy

Keywords:
Mass spectrometry imaging, Spray-based, Ambient ionization.
Novel aspects:
Initial characterization of easy ambient sonic-spray ionization mass spectrometry imaging（EASI-MSI）is presented.
Abstract:
Ambient ionization methods（AIM）allow for the examination of surfaces in their native conditions at atmospheric pressure withminimal or no preparation. Spray-based AIM such as desorption electrospray ionization（DESI）and desorption atmosphericpressure photo-ionization（DAPPI）have been successfully applied to mass spectrometry imaging（MSI）. In DESI, a spray of
charged droplets creates a thin film on the surface which dissolves the analytes. Subsequent primary droplets hit this film forming
secondary microdroplets which then undergo the classic electrospray mechanism of ionization. In DAPPI, a heated jet of mixed
nebulizer gas and spray solvent vapor is directed to the sample and promotes thermal desorption of the analytes which thenundergo the classic gas-phase mechanism of photoionization. Easy ambient sonic-spray ionization（EASI）is also a spray-based
method. It has been applied to direct analysis of inks for document examination and in characterizing organic compounds
separated on thin-layer chromatography plates. No voltage or heating is required by EASI. The ionization of the sample is achieved
via sonic spray ionization（SSI）assisted by a high-velocity gas flow. Although chemical images have not been presented by EASI,
its potential for MSI is implied. We report the initial characterization of EASI-MS for mapping the distribution of（i）inks in printed
patterns on paper,（ii）biomolecules from tissues, and（iii）drugs separated by thin-layer chromatography. Preliminary results
showed that chemical images can be easily obtained using no voltage DESI. However, the high-velocity gas flow and the solvent
flow rate required by EASI represent challenges to attain stable signals and good lateral resolution.

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 4: Imaging I

PMo-080

13:30 － 14:40

Optimization of conditions for acetylcholine detection in the nerve tissue
sections by imaging mass spectrometry

Ikuko Yao1,2, Yuki Sugiura2,3, Nobuhiro Zaima3, Mitsutoshi Setou3, Seiji Ito1

1
Kansai Medical University, Osaka, Japan, 2Precursory Research for Embryonic Science and Technology (PRESTO),Tokyo, Japan, 3Hamamatsu University School of Medicine, Hamamatsu, Japan

Keywords:
imaging, brain, spinal cord, acetylcholine
Novel aspects:
We successfully reconstructed acetylcholine distribution in mouse brain and spinal cord tissue sections by MS/MS imaging.
Abstract:
Matrix-assisted laser desorption/ionization mass spectrometry（MALDI-MS）provides the means for analysis of a wide variety of
biomolecules. This technology known as imaging mass spectrometry（IMS）provides us a novel opportunity to visualize diverse
types of molecules directly on tissue surfaces without the need for target-specific reagents such as antibodies. As most previous
IMS studies analyzed the relative abundances of larger metabolite species, it is important to expand its application to smallermolecules, such as neurotransmitters. Here we applied IMS technique to visualize neurotransmitter distribution in the central
nervous system（CNS）.
We chose acetylcholine（ACh）and examined both single MS imaging and MS/MS imaging for ACh. Monitoring ion transition by
MS/MS measurement improved the S/N ratio of the ACh-derived signal by eliminating matrix interference. Previous studies
reported that collision-induced dissociation of ACh yields a major fragment ion at m/z 87 arising from the loss of trimethylamine.
Therefore, we compared the S/N ratio and sensitivity of ACh-derived signals between single MS measurements（ion at m/z 146）
and MS/MS measurements（ion at m/z 146 > 87）. For both measurement results of 1 nM ACh standard measurements by MS/MS
and single MS measurements, the spectra obtained from the sample containing spots exhibited clear ACh-derived signals, whereas those from blank DHB spots only exhibited matrix-derived background peaks. In particular, in the single MS measurement, a
DHB-derived peak observed at m/z 146 interfered with the detection of ACh in the MS mode. Conversely, there was no observablematrix interference at m/z 87 in the product ion spectrum of m/z 146. As expected, this advantage offered an excellent S/N ratiothat is superior to that of the single MS detection, even for the measurement of a trace sample concentration.
We also tried to directly detect neurotransmitters and visualize their localization in the tissue samples. In MS/MS imaging, thetransmitter could be visualized with a high S/N by elimination of matrix interference. In the MS analyses of mouse spinal cord,reconstructed ion intensity maps for ACh ion showed that the ion which corresponds to ACh distributed both inside and outside
tissue section, suggesting that containing matrix derived chemical noise. Tandem MS solved the first problem of the sensitivity. In
the tandem MS-imaging analysis of ACh in the mouse spinal cord, the signals were detected around the region containing richmotor neurons. Similarly, we analyzed mouse brain sections with tandem MS imaging. In comparison with the previous reports, it
was revealed that ACh was localized in the region abundant in acetylcholinestelase.
These results show that ACh is the suitable molecular species to detect in positive ion detection mode by IMS, and that tandem
MS imaging is useful for the analyses of tissue distribution of ACh in the CNS tissues.

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 4: Imaging I

PMo-081

11:10 － 12:20

MALDI-IMS - The challenge of on-tissue digestion, a comparison ofdifferent protocols to improve imaging experiments

Hanna C Diehl, Julian Elm, Judith Baronner, Helmut E Meyer, Corinna Henkel
Medizinisches Proteom Center, Ruhr University Bochum, Germany

Keywords:
MALDI-IMS, Imaging, on tissue-digest, ImagePrep
Novel aspects:
Preliminary results reveal interesting differences concerning the spatial resolution between different drying times of tissue samples.
Abstract:
Abstract
Objective： Matrix-assisted laser desorption ionization（MALDI）-Imaging mass spectrometry（IMS）has become a powerful andsuccessful tool for biomarker detection1 and drug development. Depending on the research question investigators are trying to go
beyond the exclusive visualization of the spatial distribution of potential markers or areas of interest. Their focus lies on theidentification of the unknown components and hence increased efforts have been made to develop and improve existing
methods.
Current on tissue trypsin digestion techniques in MALDI-IMS are either done via spray coating or droplet deposition of trypsin
solution2,3 and/or matrix solution respectively. Both can be performed either in a manual or automated manner. The latter allows
greater confidence of inter-run reproducibility, however the entire process from sample collection to data analysis is influenced bya number of more or less controllable and uncontrollable factors.
Design and Method： The overall goal of this study was to test and compare various protocols of tissue digestion in order toimprove the quality and reproducibility of MSI data. Frozen and freshly prepared mammalian tissue samples were automaticallysprayed with trypsin and matrix using the ImagePrep device from Bruker Daltonics. Subsequent measurements were carried out
with an UltrafleXtreme instrument（Bruker Daltonics）. The different experiments were carried out varying the trypsin incubation
times/drying phases, the matrix, the raster width of the laser and the setting of the laser attenuator.
Results： Preliminary results reveal interesting differences concerning the spatial resolution. But due to the fact that theexperiments are still ongoing at the time of abstract submission, a final conclusion cannot be drawn.

References
1
Schwamborn, K., Krieg, R. C., Reska, M., Jakse, G. et al., Identifying prostate carcinoma
by MALDI-Imaging. Int. J. Mol. Med. 2007, 20, 155159.
2
Groseclose MR, Andersson M, Hardesty WM, Caprioli RM. Identification of proteins directlyfrom tissue： in situ tryptic digestions coupled with imaging mass spectrometry. J Mass
Spectrom. 2007 Feb；42（ 2 ）：254-62.
3
Baluya, D. L., Garrett, T. J., Yost, R. A., Automated MALDI matrix deposition method withinkjet printing for imaging mass spectrometry. Anal. Chem. 2007, 79, 68626867.

Acknowledgement
The work was supported by P.U.R.E.（Protein Unit Ruhr within Europe）.

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 4: Imaging I

PMo-082

13:30 － 14:40

Molecular detection of breast cancer-related phosphatidylinositol by highresolution imaging mass spectrometry

Masahiro Kawashima1, Noriko Fukao2, Minoru Suzuki2, Taka-aki Sato2, Koichi Tanaka2,
Nobuko Kawaguchi-Sakita1, Masahiro Sugimoto1, Takayuki Ueno1, Kazuya Terasawa3, Gozo Tsujimoto3,
Kazuharu Shimizu3, Masakazu Toi1
Department of Breast Surgery, Graduate School of medicine, Kyoto University, Kyoto, JAPAN, 2Koichi Tanaka
Laboratory of Advanced Science and Technology, Shimadzu Corporation, Kyoto, JAPAN, 3Department of Cancer Omics
Research, World-Leading Drug Discovery Research Center, Graduate School of Pharmaceutical Sciences, Kyoto
University, Kyoto, JAPAN1

Keywords:
imaging mass spectrometry, breast cancer, phosphatidylinositol, PI 3 K pathway, palmitic acid
Novel aspects:
This high-resolution imaging mass spectrometry analysis for breast cancer tissues revealed the PIs reconstruction in breast cancer
cells which would affect the PI 3 K or arachidonic acid pathway.
Abstract:
Breast cancer is the common cause of death among Japanese women and its incidence is rapidly growing for the last few
decades. Several studies showed the high-fat diet and obesity increase the risk of breast cancer, and suggested that palmitic acid
（16： 0 ）and a-linolenic acid（18： 3 ）could affect the breast cancer risk. Furthermore, the phosphatidylinositol- 3 kinase（PI 3 K）
pathway is frequently mutated in the breast cancer. Its abnormal activation is related to the growth, survival, and resistance oftumors to conventional chemo and radiation therapy. Thus, the blockade of this pathway has a great concern as an emerging
target for breast cancer therapy. Although, the composition of phosphatidylinositols（PIs）altered in cancer tissues, its significanceor impact on PI 3 K pathway remains poorly understood. The objective of this study is to investigate the phospholipid distribution
in breast cancer tissue by the imaging mass spectrometry and find out the new pathological and biological features in terms of
the PI and their metabolism.
In this study, we obtained breast cancer tissues from two patients who were diagnosed as the postmenopausal triple-negative（ER-, PgR-, Her 2 -）breast cancer. The tissues were obtained during surgery, quickly frozen, and stored at -80°C until analysis. The
serial sections were prepared from each sample and attached onto the indium tin oxide coated glass slides. Every section wascoated with 9 -aminoacridine（ 9 -AA）, which was used as the matrix for matrix-assisted laser desorption/ionization mass
spectrometry（MALDI/MS）. The 9 -AA was mechanically evaporated for positive and negative imaging MS analysis. The imaging
mass spectrometry was performed by the high-resolution mass spectrometer（AP-MALDI, 10um spacial resolution, Shimadzu Corp.,
Kyoto）. All imaging data were analyzed by the BioMap（Novartis, Basel, Switzerland）.
The phospholipid distribution obtained showed three patterns； 1 ）cancer cell-specific localization, 2 ）stroma and stromal cellspecific localization, and 3 ）uniform distribution. Intriguingly, the distribution of PIs showed a unique localization. PI（18：0 /20：4 ）,one of the major molecular species of PI, was localized in stromal cells while it was almost undetectable in cancer cells. In
contrast, PI（18： 0 /18： 2 ）, PI（18： 1 /16： 0 ）and PI（16： 0 /16： 0 ）were mostly observed in only the cancer cells. PI（18：
0 /22： 6 ）, PI（18： 1 /20： 4 ）, PI（16： 0 /20： 4 ）, and lysoPI（18： 0 /OH）were uniformly distributed while the signal intensitiesof the first three PI were dramatically lower than that of lysoPI（18： 0 /OH）. PA（16： 0 /18： 1 ）, a precursor of PI, was detectedin cancer cells. From these findings, we could speculate that this replacement of acyl-chain or de novo PI synthesis occurred in thebreast cancer and regulate the PI 3 K or arachidonic acid pathway in the cancer cells, although we didn’t compare with the normal
ductal and stromal cells. At least, majority of PIs in cancer cells could be a palmitic acid or stearic acid containing PIs. In addition,
The palmitic acid containing phosphatidylcholine（PC）, PC（16： 0 /16： 0 ）and unknown molecular species of PC, which werelocalized in cancer cells changed their sn- 1 acyl-chain from palmitic acid（16： 0 ）to palmitoleic acid（16： 1 ）in accordance withthe distance from the stromal region. Although this was observed only in one case, we thought it was because each tumor cellcluster was very small in another case.
In summary, we observed the unique localization of PIs and PCs in breast cancer tissue using high-resolution imaging mass
spectrometry. We will confirm these findings by measuring the tissues containing normal ductal and stromal cells and investigate
the key factors related to the PIs and PCs reconstruction.

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 4: Imaging I

PMo-083

11:10 － 12:20

Oxygen defect chemistry proposed by zinc and antimony images in thedense tin dioxide ceramics by means of NanoSIMS 50

Isao Sakaguchi, Ken Watanabe, Hajime Haneda, Naoki Ohashi, Shunichi Hishita
National Institute for Materials Science, Tsukuba, Japan

Keywords:
Transparent oxides, high-resolution imaging, defect, tin dioxide
Novel aspects:
We studied the oxygen defect chemistry through zinc and antimony distribution in tin dioxide ceramics.
Abstract:
Transparent oxides such as zinc oxide and tin dioxide, an n-type semiconductor with a wide band gap, has been extensively
studied as a useful material for application to solar cells, gas sensors, and oxidation catalysis. It is considered that the origin ofn-type conduction is oxygen vacancy. Especially, the study on oxygen defect in tin dioxide is not enough, because it is difficult to
produce dense ceramics and single-crystal. In this study, we propose new defect chemistry on the oxygen vacancy through the
distribution of zinc and antimony in the dense tin dioxide ceramics.

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 4: Imaging I

PMo-084

13:30 － 14:40

MALDI Imaging of Metabolites Reconstructed by CE-MS Based
Quantitative Analysis

Tsuyoshi Nakanishi1, Takako Hishiki2,3, Shigeki Kajihara1, Kiyoshi Ogawa1, Makoto Suematsu2,3

Shimadzu Corporation, Kyoto, Japan, 2School of Medicine, Keio University, Tokyo, Japan, 3JST ERATO Suematsu Gas
Biology Project, Tokyo, Japan

1

Keywords:
MALDI, imaging mass spectrometry, CE-MS, hypoxia
Novel aspects:
Evaluation of the distribution and the content of various metabolites by using metabolite maps reconstructed from the CE-MS
based quantitation
Abstract:
Matrix-assisted laser desorption/ionization（MALDI）imaging mass spectrometry has been widely used to display the spatial
localization of endogenous metabolites and administered pharmaceutical drugs in various tissues. This technique is currently used
to the wide variety of animal and plant systems. In MALDI imaging mass spectrometry（IMS）technique, to keep a high sensitivity
and a good reproducibility, a homogeneous matrix coating on tissue slices is required. As a typical matrix coating method, a spraymethod, a droplet method using a robotic spotter and a vapor deposition technique are known and each method is chosen
according to the aim（a spatial resolution, the reproducibility, an extraction efficiency of molecules, a cost, etc.）. To keep the
reproducibility between multiple samples on IMS experiment, we performed matrix deposition by usage of a robotic spotter. In
this study, we performed IMS of endogenous metabolites in a mouse brain tissue using 9 -aminoacridine as a MALDI-matrix. As aconsequence of IMS, these molecular images displayed a unique distribution of each metabolite between multiple samples andthese molecular images suggested homogenous matrix deposition between many spots led to acquire reproducible results among
different slices.
Subsequently, to reflect the concentration of metabolites in tissues to the molecular images, the quantitative data by CE-MS wastried to couple to imaging of metabolites. Quantitative analysis by CE-MS was achieved using a sequential tissue slice next to atissue slice for IMS measurement. First, a ratio of the peak intensity of a target metabolite at nth spot（Intn）/ the median of thepeak intensities of the metabolite of the all the spots（Int ave ）was calculated from the IMS data and next, the apparent
concentration（Cn）of each metabolite at the nth position of tissues was determined by multiplying the ratio by the metabolite
concentration（C’）of tissue quantified by CE-MS. Thus metabolite maps（AMP, ADP and ATP）were reconstructed to evaluate thechanges of energy metabolism among the multiple samples. Furthermore, energy charge（EC）was also calculated using
reconstructed molecular images from AMP, ADP and ATP. Using this IMS technique linked to the quantitation, we displayed the
distribution of ATP and its degradation metabolites, ADP and AMP in mouse brain tissues from both wild type and transgenic mice
deficient with a target enzyme under the normoxia/hypoxia. Molecular imaging reconstructed by CE-MS based quantitative data
could become a powerful tool to evaluate the fluctuation of metabolites between multiple tissue slices from different mice,
reproducibly and reliably.
References（ 1 ）Hattori K, et al.（2010）Paradoxical ATP elevation in ischemic penumbra revealed by quantitative imaging mass spectrometry.
Antioxid Redox Signal 13：1157-1167.
（ 2 ）Morikawa T, et al.（2012）Hypoxic regulation of the cerebral microcirculation is mediated by a carbon monoxide-sensitivehydrogen sulfide pathway. Proc. Natl. Acad. Sci. U.S.A. 109：1293-1298.

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 5: Advances in Spray Ionization Techniques

PMo-085

11:10 － 12:20

New nano ESI source development for increased performance nanoLC-MS
with plug-and-spray configuration

Reiko Kiyonami2, Peter Soendergaard1, Christian Ravnsborg1, Vlad Zabrouskov2
1

Thermofisher Scientific Odense Denmark, 2Thermo Fisher Scientific San Jose CA

Keywords:
nano ESI-source, column heater, column/emitter integration
Novel aspects:
Newly developed nano-electrospray source allows non-LC-MS experts to get optimized nanoLC-MS results by using an integrated
column-emitter design.
Abstract:
Introduction
Nano-flow LC-MS is widely used for qualitative and quantitative proteomics studies due to its high sensitivity and specificity；
however, it is frequently observed that irreproducible and poor results are obtained owing to imperfect connections of tubing,column, high voltage electrode, and the emitter. Improper connections often result in leaks and large swept volumes that cause
substantial peak broadening and thus poor sensitivity while a poor high voltage connection will yield poor data because of
unstable spray. In order to address these common issues, we have developed a new nano-electrospray source and emitterconcept in which, a column, column heater, high voltage electrode and an emitter are combined in one ready-made assembly.
The performance of this new source concept was evaluated.
Methods
A prototype of new developed nano-electrospray source was installed on the Orbitrap Elite mass spectrometer and connected toan Easy-nLC 1000 nanoLC pump. Three column（50 µm id x 15 cm, 2 µm）assemblies were used for evaluating column-to-column
and run-to-run reproducibility. Both complex peptide mixtures and simple digest mixture were used. The retention time
reproducibility, peak shape, resolution and peak capacity in different temperature ranges were evaluated. In order to test thedevice flexibility, several flow rates from 150 nl/min to 1000 nl/min were used. The mobile phases were 0.1% FA/H 2 O and 0.1% FA/
MeCN. A 60 min linear gradient was used for the complex sample separation. A 15 min linear gradient was used for the simple
digest sample separation
Preliminary Data
The column assembly is positioned in the source without the need to adjust the X,Y, or Z positions of the emitter. A high spraystability was achieved over all LC runs with three different columns. The obtained chromatographic resolution, sensitivity, andreproducibility matches other state-of-the-art data without need for adjustments（or expert intervention）beyond plugging in thecolumn/sprayer assembly. More than a thousand proteins were identified with increased component detection from the complex
protein digest mixtures. The reproducibility of the retention time from column to column for the targeted peptides from the simple
standard mixtures was less than one percent.

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 5: Advances in Spray Ionization Techniques

PMo-086

13:30 － 14:40

Electrostatic Spray Ionization and Microchips Emitter for On-Line MS
Monitoring of Chemical and Electrochemical Reactions

Liang QIAO1, Romain SARTOR1, Elena TOBOLKINA1, Dmitry MOMOTENKO1, Mathieu ODIJK2,Pekka PELJO3, Hubert GIRAULT1

1ECOLE POLYTECHNIQUE FEDERALE DE LAUSANNE, SWITZERLAND, 2UNIVERSITY OF TWENTE, ENSCHEDE,NETHERLANDS, 3DEPARTMENT OF CHEMISTRY, AALTO UNIVERSITY, ESPOO, FINLAND

Keywords:
Electrostatic Spray Ionization, Microfluidic chip, Mass Spectrometry, Nitration
Novel aspects:
The ESTASI provides an alternative strategy to ionize a sample from a surface as compared to laser desorption ionization anddesorption electrospray ionization.
Abstract:
An electrostatic spray ionization（ESTASI）method has been developed for mass spectrometry（MS）analysis of samples in or on
an insulating substrate.1 The principle is based on the capacitive coupling between an electrode and the sample. In practice, a
metallic electrode is placed close to the sample and isolated by an insulator. Upon application of a high voltage pulse to theelectrode, an electrostatic charging of the sample occurs leading to a bipolar spray pulse. When the voltage is positive, thebipolar spray pulse consists first of cations and then of anions. This method has been applied to a wide range of geometries toemit ions from samples in a microchip, in a silica capillary, in a disposable pipette tip or from samples on a polymer plate.
Specifically, proteins or peptides separated by an isoelectric focusing gel electrophoresis can be directly profiled by the ESTASIMS without staining and extraction procedures. Indeed, with the ESTASI-MS, proteins with an amount of several nanograms canalready be detected, indicating a strategy more sensitive than the classical Coomassie Brilliant Blue staining and providing themolecular weight or even fragmentation information at the same time. This protein profiling strategy with ESTASI-MS has a brilliant
future in top-down proteomics.
We have also developed multi-functional polymer microchips as both microreactors and electrospray emitters for on-line analysisof reaction products by MS. Typically, the microchips include several microchannels to introduce reagents to a main reaction
channel, electrodes to apply high voltage for electrospray or apply voltages for on-line electrochemical reactions, and a
microchannel to provide sheath flow to stabilizing electrospray ionization（ESI）. When the electrode providing high voltage is incontact with the main channel for spray, the microchip is used for regular ESI. When the electrode for high voltage is isolated fromthe main channel by polymer, the microchip is used for capacitive ESTASI.
In a first application, the microchip-MS was used to investigate the nitration of tyrosine.2 With the advantages of on-line reaction,
we have found new mechanisms for nitration on tyrosine catalyzed by copper（II）. In general, micromolar concentrations ofcopper catalyze the nitration process by nitric oxide（NO）oxidation, whilst millimolar concentrations catalyze the nitration
process by nitrite oxidation. Comparing to batch reaction, the on-line microchip-MS system is useful to detect reaction productswith short lifetime produced in seconds, such as radicals. When different solvents are introduced via different microchannels, themicroreactor can be used for the research of liquid-liquid interface reactions. We have investigated the mechanism of ferrocene
methanol oxidation at the 1,2-dichlorobenzene-water interface.
Since sheath flow of a buffer suitable for ESI is provided, the microchip can be used as an emitter to connect with various
microfluidic devices for efficient and stable ESI of analytes. In one example, we have connected the microchip emitter with a pushpull probe developed in our group for scanning electrochemical microscopy（SECM）.3 Therefore, surface chemical component
was firstly characterized and imaged by SECM and then extracted for microchip-ESI-MS imaging. In another example, the ESI
emitter microchip was connected with a microfluidic chip for electrochemical reaction, similar as the one reported recently in
BIOSENSORS&BIOELECTRONICS.4 Drug metabolites generated by electro-oxidation was analyzed on-line by ESI-MS.
Reference
1. Electrostatic-Spray Ionization Mass Spectrometry, L. Qiao, et al., Chemical Science, SC-EDG-02-2012-020213, revised
2. L. Qiao, et al., J. Am. Chem. Soc.,2011, 133, 19823-19831.
3. D. Momotenko, et al., Anal. Chem., 2011, 83, 5275-5282.
4. M. Odijk, et al., Biosens. Bioelectron., 2010, 26,1521-1527.

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 5: Advances in Spray Ionization Techniques

PMo-087

11:10 － 12:20

Miniaturized Peptide Sample Pretreatment Platform for Indirect Spray
Analysis

Hoi Sze Yeung, Tak-Wah Dominic Chan

The Chinese University of Hong Kong, Hong Kong SAR, China

Keywords:
indirect spray, solid phase extraction, sample pretreatment, ambient ionization, peptides
Novel aspects:
Miniaturized peptide sample pretreatment platform that only required nano-liters of sample. Subsequent indirect spray mass
spectrometric analysis can be carried out using same platform.
Abstract:
The presence of salt or matrix components is known to suppress analyte signal1 in the mass spectrometric analysis of complex
mixtures. Therefore, solid phase extraction（SPE）is an important and widely used sample pretreatment technique 2 topreconcentrate the target analyte and remove interfering components prior to the mass spectrometric analysis. However, theminimal volume of sample required for SPE clean-up is typically one to a few micro-liters.
Our group has developed a sensitive ambient ionization technique, named indirect spray 3 . Using membrane funnel, 1 fmol of
peptide is sufficient to provide detectable signal in a Fourier-Transform Ion Cyclotron Resonance mass spectrometer. Sample
volume requirement for an indirect spray analysis can be as low as 50 nL. To couple the advantages of SPE and indirect spray, SPEsorbent attached membrane funnel is prepared. Preliminary results showed that SPE in sorbent attached membrane funnel format
was feasible. Angioteinsin II could be adsorbed on the C18 sorbent efficiently by simply loading the sample onto the surface of thesorbent film. After the washing step, angiotensin II signal could remain essentially the same as before washing. Desalting was
feasible for peptide sample prepared under physiological concentration of salt（150 mM NaCl）. Determination of salt tolerance,
reproducibility and sensitivity of indirect spray using this miniaturized peptide sample pretreatment platform are underway. We
will also explore the applicability of other sorbent materials for specific peptide analysis.
Buhrman, D. L.； Price, P. I.； Rudewicz, P. J. J. Am. Soc. Mass Spectrom.1997, 7, 1099-1105.
Majors, R. E. LCGC North Am. 2002, 20, 1098-1113..
3
Yeung, H. S.； Chan, T.-W. D. In 58th Annual Conference on Mass Spectrometry and Allied Topics； Salt Lake City, UT, May 23-27,
2010； The American Society for Mass Spectrometry： Santa Fe, NM, 2010； p 912.
1
2

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 5: Advances in Spray Ionization Techniques

PMo-088

13:30 － 14:40

Novel off-line electrospray ion source with a disposable needle

Hideki Hasegawa, Hiroyuki Satake, Masao Suga, Yuichiro Hashimoto
Hitachi Ltd., Central Research Laboratory, Kokubunji, Japan

Keywords:
ElectrosprayIonization Off-lineESI DisposableNeedle
Novel aspects:
Novel off-line electrospray ion source with a disposable needle for liquid sample analysis.
Abstract:
Introduction
Electrospray ionization is a useful tool for analyzing liquid samples, such as environmental, pharmaceutical, and biological samples.
Usually, a liquid sample is pumped through a metal capillary of the electrospray ion source. The capillary is contaminated andthen is sometimes clogged by dirt that is in the sample.
Off-line ion sources such as probe electrospray1 and ESI chip2 were reported to reduce contamination and blockages. For further
improvement, we have developed a novel off-line electrospray ion source（off-line ESI）using a disposable needle that is available
commercially and is easy to exchange. We will show the preliminarily results obtained using an off-line ESI with a disposableneedle.
Methods
Our off-line ESI consists of a disposable needle. The disposable needle consists of a metal capillary and a plastic reservoir with avolume of about 100 uL. A liquid sample was introduced from the end of the metal needle into the sample reservoir using a syringethat was connected to the reservoir. After supplying the sample, the syringe was separated and a high voltage of 3 - 5 kV wasapplied to the metal needle. The liquid sample was continuously sprayed from the end of the disposable needle by gravity（without pumping）.
Preliminary data
Reserpine was used as a measurement sample, and the following three solvents were used： methanol, methanol/water： 1 /1,
and water. The ion sensitivity of reserpine ions using the off-line ESI with the disposable needle was about half that of the normal
ESI. The signal lasted for over 60 seconds with an inner diameter of 0.19 mm.
We investigated the flow rate by gravity dependence on the inner diameter of the capillary for several solvents. For methanol, theflow rates with an inner diameter of 0.13, 0.19, 0.26, and 0.33 mm were 20, 50, 200, and 600 uL/min, respectively. The flow rateincreased proportionally to the 3.7th power of the capillary’s inner diameter, which is consistent with the conductance of acapillary being proportional to the 4 th power of the capillary’s inner diameter. The flow rate of the methanol /water： 1 / 1 wasreduced to 40-50 % of that of the methanol solvent, and the flow rate of the pure water was reduced to 70-80 % of that of themethanol solvent. This result shows that the flow rate is approximately inversely proportional to the viscosity of the solvent.
References［ 1 ］Hiraoka, K.； Nishidate, K.； Mori, K.； Asakawa, D.； Suzuki, S.； Rapid Commun. Mass Spectrom. 21, 3139-3144（2007）［ 2 ］Zhang, S.； Van Pelt, C.K.； Henion, J.D.； Electrophoresis, 24, 3620-3632,（2003）

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 5: Advances in Spray Ionization Techniques

PMo-089

11:10 － 12:20

Movement of Ions and Charged Droplets from Atmospheric Region into
the Gas Dynamic Interface

Natalia Fomina1, Nikolai Gall2, Sergey Masyukevich1, Alexandra Kretinina1, Lidiia Gall1,
Alexander Bazhenov3, Sergey Bulovich4

1
The Institute for Analytical Instrumentation RAS, Saint-Petersburg, Russia, 2A.F.Ioffe Physico-Technical Institute of RAS,
St.Petersburg, Russia, 3All Russian Scientific Research Institute for Geology and Mineral Resources named of
I.S.Gramberg, St. Petersburg, Russia, 4Saint-Petersburg State Polytechnical University St.Petersburg, Russia

Keywords:
Transport capillary, gas-dynamic Interface, ESI, APCINovel aspects:
direct separate measurement of ion and droplet losses in their movement through a transportation capillary from atmosphericregion into the interface
Abstract:
Mass-spectrometry with ambient ionizations requires ion transfer into the high vacuum region. A nozzle or transportation capillary
is often used in modern instruments； a number of vacuum steps are used before an ion enters the high vacuum. Physicalprocesses in ion movement from the atmospheric region to the gas dynamic interface, i.e.the low vacuum part of the instrument,
are very important as they affect strongly on main analytical features： the mass range, detection limit, and precision.
The transport capillary is a cylindrical channel with the diameter D and length L. The nozzle can be treated as a capillary withextremely small length. Droplets can be evaporated in their movement through the capillary, and the charged particles are able todiffuse to its walls； this results in loss of signal and instrument discriminations.
The presented work discusses dependence of the capillary transmission on the L/D relation, of a sort of charged particles, and amagnitude of the through the capillary. Capillaries have been used with L/D ratios of 0.8, 32.5, and 420, and lengths L0.3, 30, and 150
mm. Studies were carried out by a double focusing mass spectrometer MX-3303 with a Kontorovitz-Grey gas-dynamic interface inthe positive ion mode.
Atmosphere pressure corona ionization（APCI）and electrospray ionization（ESI）have been used to get various types of thecharged particles： APCI generates only ions； they are mostly water clusters and provide a steady and high ion current. On thecontrary, ESI gives both ions and charged droplets, and their relative role in the total current depends on the spraying details.
It was shown experimentally that the capillary transmission decreases with the increase in its relative length. In the APCI mode ofionization with the input current of 1500 nA, the current transmission drops by a factor of three when the capillary with L/D =0.8
has been substituted by the one with L/D = 32.5； for the capillary with L/D = 420, the transmission was decreased by a factor of
50. In the ESI mode, the input current was ~ 60 nA, and the drop in transmission was 3 times for L/D =32.5, and ~150 times for L/D= 420.
To determine the charged particle type entering the gas dynamic interface, we applied retarding potential to the skimmer： theelectric and gas dynamic forces work oppositely. The voltage of 20- 80 V was enough to repulse ions as they have rather highmobility； however, it was too low to affect droplets. This made it possible to evaluate the efficiency of droplet evaporation inside
the transportation capillary. To validate the above ideas, detailed simulation of ion and droplet movement have been done using
SIMION 8.0 and ASXIM.
The output current consisted of droplets for more than 99% for a nozzle or short capillary； as for the capillary with L/D = 420, the
30 % of output current were ions. This means that droplets are evaporated actively in the capillary, and this effect can not beignored when one estimates the relative effectiveness of different methods of introducing the charged particles into a gas-dynamic
interface. We have simulated the gas dynamic movement inside all three types of the capillary using Fluent 6.0 and calculated thecharged particle diffusion to the capillary wall. The results are in good agreement with the experimental findings.

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 5: Advances in Spray Ionization Techniques

PMo-090

13:30 － 14:40

An introduction of a novel atmospheric pressure Micro/Nano-ESI ion
sourceReza Javahery1, Hui Qiao1, Brett Larsen2, Lorne E Taylor2, Lisa Cousins1

1
IONICS Mass Spectrometry, Toronto, Canada, 2Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto,
CanadaKeywords:
Electrospray, Sources, Micro/Nano-ESI
Novel aspects:
A novel micro/nano-ESI ion source provides ease of use, improved ion signal stability, and better S/N ratio.
Abstract:
Introduction
The advent of micro/nano-ESI has considerably extended the applications of ESI in analytical mass spectrometry. It offers some
remarkable benefits such as lower sample consumption and higher ionization efficiency. However, conventional micro/nano-ESI
systems can suffer from unstable Taylor cone effects, clogging of emitters, and incomplete droplet desolvation, making them moredifficult to operate. In the present study, a novel source was developed to accommodate nano to micro LC flow ranges andoperate atatmospheric pressure. Compared to the conventional micro/nano-ESI ion source, this new ion source offers ease of use,
2 fold improvement in signal stability, and higher S/N ratio with more complete droplet desolvation.
Methods
The experiments were carried out on an IONICS EP10+ with IONICS HSID TM interface. Comparisons of signal stability,
reproducibility, and S/N ratios were made for the new ion source and a conventional microspray source. Flat-cut silica capillaries：
25 um（ID）were used as sprayer emitters. Reserpine（100pg/uL）in H2O：MeOH（ 1 ： 1 ）with 0.1% acetic acid was used to study
the signal stability with both syringe and nano-HPLC pumps. Both BSA（tryptic digest）（50fm/uL）in H2O（100%）with 0.1% formic
acid and testosterone（10pg/uL）in serum were run with nano LC columns（75um（ID）, C-18）to study the S/N ratio of the MRM
spectra. The LC gradient was 5 % to 95%in the organic solvent in a 15-minute run.
Preliminary data
The ion source conditions such as sprayer emitter position, gas flow, and sprayer voltage, were fully optimized. For this new ion
source, the optimization process was simple and straightforward with good reproducibility, offering ease of use. However, for theconventional microspray ion source, the optimization process was very time consuming with poor reproducibility. There were also
larger ion signal variations at different ion source conditions for the conventional microspray source. After the ion source
optimization, the ion signal stability was measured with a reserpine sample using both syringe and nano-HPLC pumps.
The variation in the total ion counts from Q 1 scans were recorded. The results consistently showed that the new ion source had
2 -fold improvement in stability compared to the microspray ion source for both syringe and nano-HPLC pumps. Using a nanoHPLC pump with 75umID columns, the MRM spectrum from multiple injections of reserpine also showed that the new ion source
provided two times higher signal reproducibility than that of the microspray ion source. The MRM spectra from a BSA digest were
examined to compare the S/N ratio for both ion sources. The results clearly showed reductions of chemical noise level of the newion source due to the more complete droplet desolvation. Although the absolute ion intensity of the microspray ion source wasbetter than that of the new ion source, the resulting S/N ratio of the new ion source was better. A similar trend was also found in
the MRM spectra of testosterone in serum. Since the MRM spectrum itself can greatly reduce the chemical noise level, the Q 1
multiple ion（MI）scan of a BSA digest was also used to examine the background noise and S/N ratios. The result clearly showed
improved S/N ratio with the new ion source.

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 5: Advances in Spray Ionization Techniques

PMo-091

11:10 － 12:20

Electrospraying, Movement and Evaporation of Charged Droplets Using
Rotating Gas Flows and Transverse Ion Extraction

Sergey Masyukevich1, Nikolai Gall2, Natalia Fomina1, Mihail Lapushkin2, Alexandra Kretinina1

1
Institue for Analitical Instrumentation, Saint-Peterburg, Russia, 2A.F.Ioffe Physico-Technical Institute of Russian Academyof Sciences, St.Petersburg,Russia

Keywords:
Electrospraying, Rotating Gas Flows
Novel aspects:
new ESI design with rotating gas flow and transversal ion extraction
Abstract:
The mass-spectrometer sensitivity are highly determined by droplet eletrospraying, their movement by gas and electric forces,
evaporation, and ion collection to the sampler. The most significant signal loss occurs in the ambient region because of spray
inefficiency, imperfect droplet evaporation and spatial repulsion of the charged particles. The signal losses due to each of thesereasons can be of 2 to 3 orders of magnitude； the total loss in signal compared to its maximal theoretical value is often as highas 105 even for effective commercial instruments.
We have examined the sequence of physical processes that leads to losses in ions at the atmospheric region, and indicated themutual relationship between the named mechanisms. Generally, electrospray proceeds under the influence two electric fields：
the outer electric field created by the counter electrode and the space charge field created by droplets emitted from the Taylor
cone. Space charge field plays an important role, suppressing the spraying at high flow rates, and causing high and low frequency
oscillations of the emitted current.
Evaporation of droplets is limited by the drift time of their movement, by heat flux, and significant readsorbing of solvent
molecules. Droplet move much more slowly than ions, and are the main source of space charge to cause an effective repulsion of
the particles.
We have studied experimentally, in simulation and theoretically the possibility of electrostatic focusing of ions at the atmosphericpressure by electrostatic lens system. It was shown that the nature of particles and the space charge density are the main factors
determining focusing efficiency. Thus for ions got from corona ionization, the focusing efficiency dropped from ~ 15% at 50 nA down
to 0.1% at 1 mA. Focusing almost did not work at the first situation： the current part reaching the sampler entrance was nearly
the same as ratio the geometric orifice area and the area of the ion cone. In the case of ESI, the situation was much more
complicated and depended on the temperature and solvent composition
To overcome these difficulties, we have developed a novel spray system, based on a combination of droplet evaporation in arotating heated gas and electrostatic ion extraction in the transverse direction. The measurements showed a significant increase
in sensitivity of the instrument, as high as two orders of magnitude for favorable conditions, and sufficient increase in spraying
effectiveness for high sample flow rates

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 6: Novel Approaches in Proteomics Analysis

PMo-092

13:30 － 14:40

A reference method for the SI-traceable quantification of somatropin inhuman serum using an isotopically labeled protein internal standard

Caroline E Pritchard1, Milena Quaglia2, Alison E Ashcroft3, Gavin O’Connor2

1
LGC, London, UK & University of Leeds, Leeds, UK, 2LGC, London, UK, 3Astbury Centre for Structural and Molecular
Biology, University of Leeds, Leeds, UK

Keywords:
protein quantification, SI-traceable, IDMS
Novel aspects:
Use of isotopically labelled protein internal standards to perform SI-traceable quantification in human serum samples.
Abstract:
The need for certified reference materials is well recognized within the clinical community and by the International Federation for
Clinical Chemistry and Laboratory Medicine（IFCC）. Use of suitable reference materials supports results that are accurate,
specific, and most importantly comparable between laboratories. Although methods for the traceable quantification of standard
protein solutions have now been developed, such standards are rarely commutable for use with routine clinical or biochemical
immunochemical measurement techniques. Solution standards behave differently during the analytical process than the complex
biological samples they are being used to standardise. Consequently, there is a need to develop methods that give traceable
results for protein standard materials in complex matrices such as human serum.
Using somatropin（recombinant human growth hormone, rhGH）as a model, a method for the SI-traceable quantification of a
specified protein inhuman serum has been developed. Isotopically labelled rhGH was used as an internal standard to perform
isotope dilution mass spectrometry（IDMS）. This should account for any losses encountered during sample preparation,
incomplete digestion due to the presence of the remaining matrix proteins and any modifications that occur during the method. Inorder to remove any structural differences between the natural rhGH and its labelled analogue, denaturing and reducing
conditions were used prior to sample clean-up. Removal of the large（>30 kDa）proteins within serum and subsequent reversephase fractionation was performed using a restricted-access type media. The resultant simplified matrix was digested using trypsin
and quantification of the tryptic peptides released was performed by mass spectrometry.
In order to achieve traceability, a rhGH solution standard previously quantified using internal labelled peptide standards was used
to spike blank human serum. A serum sample was prepared at 10 ng/g and quantified using the method described to give results
traceable to the SI with a total measurement uncertainty of <20 %.

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 6: Novel Approaches in Proteomics Analysis

PMo-093

11:10 － 12:20

Large-scale LC-MS-based Profiling of Soluble Protein Complexes

Cuihong WAN1, Pierre C Havugimana1, Andrew Lugowski1, Kathy Foltz2, Andrew Emili1
1

University of Toronto, Toronto, Canda, 2University of California Santa Barbara, California, USA

Keywords:
protein-protein-interactions； LC-MS/MS； HPLC； co-elution； seaurchin
Novel aspects:
Global examination of protein complexes in sea urchin development using a novel high-through screening approach based onhigh-performance HPLC separation and LC-MS/MS analysis
Abstract:
Macromolecular complexes are essential for all aspects of cell biology, including development. Generating comprehensive cellular
protein-protein interactions maps is therefor eessential for understanding the basic molecular biology of organisms. While
progress has been made in the large-scale isolation and identification of stable protein complexes simple model organisms likeyeast, existing high-throughput assays for mapping physical interactions like affinity purification are difficult to apply to nontraditional classical model organisms such as sea urchin. Here we describe the application of an integrative proteomic profiling
approach for systematically enriching and identifying the components of soluble protein complexes during early stages ofdevelopment in Strongylocentrotus purpuratus. Soluble protein was extracted from lysates of eggs prior to fertilization, 5 min
post-fertilization, at 2 -cell cleavage stage, and from hatched blastulas, and subjected to extensive non-denaturing fractionation
with mix-bed ion-exchange high pressure liquid chromatography（IEX-HPLC）. Proteins belonging to the same complex that coelute during HPLC are then identified and quantified by tandem mass spectrometry（LC-MS/MS）using a high precision Orbitrapvelos instrument. SEQUEST and PepQuant（homemade software）were used for MS/MS database searching and label-free MS 1
precursor ion-based quantification, respectively. Interaction scores were assigned based on the similarity in fractionation profiles（ie Pearson correlation score）of co-eluting protein pairs based on the recorded spectral counts and peak intensities. Next, an
integrative machine-learning procedure using supporting biological evidence（e.g. co-evolution, functional annotations, coexpression, co-localization, conservation）was used to filter the protein protein interactions to generate a high-confidence
PPInetwork, while clustering was applied to predict protein complexes. This work provides an unprecedented perspective into theglobal molecular organization and conservation of protein complexes in sea urchin cells during early development, and should
facilitate further mechanistic insights into animal gene function.

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 6: Novel Approaches in Proteomics Analysis

PMo-094

13:30 － 14:40

Mass spectrometry-based sequencing of protein C-terminal peptide usingalpha-carboxyl group specific derivatization and COOH capturing

Chihiro Nakajima, Hiroki Kuyama, Koichi Tanaka

Koichi Tanaka Laboratory of Advanced Science and Technology, Shimadzu Corporation, Kyoto, Japan

Keywords:
C-terminal sequencing of protein
Novel aspects:
The C-terminal enrichment was successfully performed by double digestion using GluC and AspN, and COOH selective capture
with tosylhydrazide glass material.
Abstract:
The C-terminus of protein is of great importance in determining various cellular functions. Presently, mass spectrometry（MS）has
been the method of choice for analyzing the protein C-terminus, but even with the cutting-edge technology, it is still difficult toanalyze the C-terminus of a protein of interest, and it is not too much to say that there are only a few methods for the analysis.
The conventional methods of peptide mass fingerprinting and peptide fragment fingerprinting can often not give any informationof C-terminal structure, in particular when the protein is C-terminally processed or posttranslationally modified. Hence, an efficientand practical technique for characterizing protein C-terminal variation has strongly been needed.
We here present an approach to MS-based sequence analysis of selectively enriched C-terminal peptide from protein. Thisapproach employs a combination of the specific derivatization of alpha-carboxyl group（COOH）, proteolytic double digestion
using endoproteinase GluC and AspN, and enrichment of C-terminal peptide through the use of COOH-capturing material. Highly
selective derivatization of alpha-COOH was achieved by a combination of specific activation of alpha-COOH through oxazolone
chemistry and amidation using 3 -aminopropyltris（2,4,6-trimethoxyphenyl）phosphonium bromide（TMPP-propylamine）. This
amine component was used to simplify fragmentation in MS/MS measurement, which facilitated sequence determination. The
peptides produced after GluC/AspN digestion were then treated with a COOH scavenger to enrich the C-terminal peptide thatlacks COOH group, and the obtained C-terminal peptide was readily sequenced by MALDI-MS/MS due to the TMPP mass tag.
In the current status, there is no general method for C-terminal sequencing analysis of protein, partly due to its highly varied
nature, hence versatile techniques for such purposes have been required. The method presented in this session can be usedcomplementarily with previously reported protocols for protein C-terminal analysis.

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 6: Novel Approaches in Proteomics Analysis

PMo-095

11:10 － 12:20

Identification of proteins with different expression profiles related to
Nasal Polyps in Aspirin Intorelant Asthma(AIA) using MALDI-TOF/MS

Yukie Tanaka1, Dai Susuki2, Shigeharu Fujieda2, Yutaka Fujii1

Department of Molecular Biology and Chemistry,University of Fukui,Fukui,Japan, 2Department of Otorhinolaryngology
Head & Neck Surgery,University of Fukui,Fukui,Japan

1

Keywords:
Proteomics analysis,MALDI-TOF/MS,Nasal polyps,Aspirin Intolerant Asthma（AIA）
Novel aspects:
Proteomics analyasis is one of usefull methods for investigate mechanisms of AIA.
Abstract:
Introduction: The mechanism of aspirin intolerant asthma（AIA）remains unclear. AIA patients have a character with intractablenasal polyps. We performed proteomics analysis of nasal polyps to identify proteins related to AIA using different expression
profiles.
Methods: Proteomics analysis by nasal polyps had been performed in AIA and Chronic Rhinosinusitis（CRS）. Polyp samples weretaken from 8 patients with AIA and 8 patients with CRS. Proteins extracted from polyps were labeled with Cy 3 or Cy5. Samples
were run 2 D-DIGE（ 2 dimensional differential gel electrophoresis）. The target protein spots were found with Progenesis PG240
software. And then these were subjected MALDI-TOF/MS analysis. The database search for peptide mass fingerprinting
candidates was performed using a Mascot Search Program. Immunohistochemistry and western blotting by using antibodies
against these proteins were followed in nasal polyps for AIA and CRS. Finally, in vitro, we studied regulation of these proteinsusing EoL- 1 which is human eosinophilic cell lines.
Results and Discussion： We identified 122 diffrentially expressed protein by 2 D-DIGE. Compared with CRS, there were 79 proteins
up-regulated and 43 proteins down-regulated. Among these, 4 proteins including protein X（temporary name）were highly
overproduced in AIA patient’s nasal polyps. To validate the results obtained in this study, we selected one candidate protein（protein-X）related to AIA and assesed more detail. The differential expression of protein-X in cytoplasm of eosinophil was
confirmed by immunohistochemical analysis. Aspirin and lipopolysaccharide（LPS）has not changed protein-X mRNA in EoL-1.
CysLT 1 R mRNA was up-regulated by LPS and decreased by transfected siRNA for protein-X.
Proteomics analyasis is one of usefull methods for investigate mechanisms of AIA. Ptrotein-X is overexprresed in AIA patient’s
nasal polyps and have important role of AIA as LPS.

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 6: Novel Approaches in Proteomics Analysis

PMo-096

13:30 － 14:40

Specific Racemization of a Cysteine Residue in the Hinge Region of
Immunoglobulin Gamma 1 during Storage

Masato Amano1, Naoki Kobayashi1, Takashi Nakazawa2, Susumu Uchiyama3, Kiichi Fukui3
1

Daiichi Sankyo, Hiratsuka, Japan, 2Nara Women’s University, Nara, Japan, 3Osaka University, Osaka, Japan

Keywords:
Antibody Therapeutics, Hydrogen/Deuterium Exchange, Racemization
Novel aspects:
Finding of racemization in antibody therapeutics and investigation of analytical method for racemization in protein with hydrogen/deuterium exchange mass spectrometry.
Abstract:
Therapeutic antibodies often suffer from degradation due to various modifications during storage. We detected a novel
degradation of immunoglobulin gamma 1 （IgG 1 ）stored for 6 month at 40 degrees celsius, and identified the modification as theracemization of a cystein residue in the hinge sequence of heavy chain. The racemization was confirmed by mass spectrometry inconjunction with extracted ion chromatography of the tryptic digest of IgG 1 forced to degrade in D2O. To rationalize the possible
relationship between the racemization of cysteine, we suggest a new reaction mechanism that assumes a base catalyst to initiatethese reactions by activating the amide nitrogen of neighboring lysine residue.

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 6: Novel Approaches in Proteomics Analysis

PMo-097

11:10 － 12:20

Identification and affinity characterization of carbohydrate-binding
epitopes in human Galectin-9 and rat Galectin-5 by proteolytic ExcisionMS and bioaffinity analysis

Frederike Eggers1, Adrian Moise1, Sabine Andre2, Hans-Joachim Gabius2, Michael Przybylski1
1

University of Konstanz, Konstanz, Germany, 2Ludwig-Maximilians-University, Munich, Germany

Keywords:
CREDEX-MS, CRD, SAW-bioaffinity, Affinity-MS, SAW-ESI-Ion-Trap-MS-online-coupling-system
Novel aspects:
SAW-bioaffinity analysis of carbohydrate peptide/protein interactions.New clinically relevant carbohydrate binding epitopes.
Abstract:
The emerging physiological significance of carbohydrate（glycan）-protein（lectin）interactions direct increasing attention to thestructural analysis of this contact（ 1 ）. Using human adhesion/growth-regulatory galectins, we have developed a robust andreliable approach to identify sequence stretches in contact to the ligand. In this study we want to focus on Galectin- 9 and
Galectin- 5 because sequence homology analysis revealed that the C-terminal carbohydrate-binding domain of galectin- 9 has
extensive similarity to that of monomeric galectin-5. In contrast to galectin-5, which is mainly expressed in erythrocytes, galectin- 9
was found to be widely distributed, i.e. in liver, small intestine, thymus, kidney, spleen, lung, cardiac and skeletal muscle,
reticulocyte and brain. Galectin- 9 is also known to be a selective eosinophil chemoattractant and it was found in tumor tissue of
Hodgkin’s disease. Because of such features characterisation of galectin-carbohydrate interactions and identification of bindingsites is of crucial importance for drug design. Combination of proteolytic excision and affinity-mass spectrometry yielded peptides
reactive with cognate sugars, as ascertained by biosensor and cell assays.

Experimentally, galectins were adsorbed to an affinity resin bearing cognate sugar and proteolytically digested. After washing out
unbound galectin fragments, the fragments remaining on the column were eluted, and both fractions analyzed by mass
spectrometry. This procedure was termed CREDEX-MS（Carbohydrate recognition domain excision/extraction）. The identified
CRD-peptides were synthesized by Fmoc-SPPS and their binding properties characterized by affinity-MS. The interactions of lactosewith full-length galectins and with identified CRD-peptides were studied by SAW-bioaffinity-measurements and online-bioaffinitycoupling of SAW with ESI-MS. The lactose was immobilized on bioaffinity-sensor-chips in the form of a glycopeptide. SAW
measurements were performed with an S-Sens K 5 Biosensor instrument in PBS buffer and elution was performed with ACN ： 0.1%
TFA 2 ：1.
CREDEX-MS of galectins identified at least two carbohydrate-binding peptides for every galectin - in complete agreement with thebinding sites of the crystal - or molecular modelling structures. All identified carbohydrate-binding peptides were synthesized andtheir affinity for lactose demonstrated by affinity-MS. To get more detailed information about the affinity and interaction kinetics of
the CRD peptides and full galectins with lactose, SAW-bioaffinity measurements were performed. Lactosyl-glycoprobe wasimmobilized on the SAW chip and dissociation constants of galectin peptides and galectins with lactose determined to be in theµM range. For a real-time study of carbohydrate-lectin interaction a newly developed online-coupling of SAW with ESI-Ion Trap MS
was used. With this system the peptides interacting with on chip bound carbohydrate could be studied directly after the elution
and it could be proved which peptides interact with the carbohydrate on the chip. This system was tested with synthetic CRD
peptides and it was shown that online-coupling of mass spectrometry with the biosensor was successful.
The presented results document the validity of the concept to obtain bioactive peptides from lectins（ 2 ）. These bioinspired
peptides are becoming objects of detailed structural analysis and inspire their tailoring, e.g. by incorporating non-natural aminoacids, as medically useful mini-lectins or lectin blockers.
Surface acoustic wave measurements were shown as a working tool for determination of dissociation constants as well as incombination with ESI-Ion-Trap-MS for the study of carbohydrate-lectin-interactions.

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 6: Novel Approaches in Proteomics Analysis

PMo-098

13:30 － 14:40

Differentiation of isobaric residues in SPITC-derivatized tryptic peptides
using MS/MS technique in a novel Curved Field Reflectron.

yuzo yamazaki, Keisuke Shima

Shimadzu Corporation, Kyoto, Japan

Keywords:
MALDI, CID, SPITC, isoform, CFR
Novel aspects:
Specific fragment ions obtained by a novel CFR can discriminate isobaric residues in SPITC-derivatized peptides without impairing
interpretable sequence information.
Abstract:
Fixing a strong negative charge at N-terminus of tryptic peptide is a quite effective chemical derivertization for de novo sequencing
by using post-source decay（PSD）on MALDI-TOFMS. However, whereas the chemical derivertization causes interpretable y-ionsmainly, one cannot differentiate isobaric amino acid residues, for instance, Ile/Leu and α- /βAsp. We will report our study ofdifferentiation of these residues by using PSD and a high energy CID in a novel curved field reflectron.
PSD and high-energy CID-MS/MS in the curved field reflectron were performed in positive ion mode. For the high-energy CID
experiments, helium was used as collision gas, and a collision energy was 20 keV（laboratory frame of reference）. 4 -sulphopenyl
isothiocyanate（SPITC）was purchased from SIGMA. Tryptic digests of BSA and β-casein were provided to SPITC-derivatization, as
described in the previous report. Some peptides including Ile/Leu were analyzed by PSD and the MS/MS. Two synthesizedpeptides, which include α- and β Asp respectively, were also provided to the derivatization, and then analyzed by MS as well.
As reportedly, the derivertization of the tryptic peptides produced y-ions mainly in PSD measurement, which is useful to interpretsequences denovo easily. However, it was impossible to differentiate Ile and Leu due to the same residual mass. The high-energy
CID-MS/MS（HE-CID）of the derivertized peptide produced moderate intensities of w- and d-ions that were generated by sidechain fragmentation, while dominant y-ions were still observed in the MS/MS spectra. It is noted that, in an operation of CFR, one
can switch PSD to HE-CID so rapidly that it is easy to recognize d- and w-ions derived from Ile/Leu by comparison with both
spectra. For instance, y-59 between two y-ions, a mass difference of which is 113, indicates Leu, while y-45 does Ile. Namely, byusing HE-CID of the SPITC-derivertized peptide, it is easy to read an exact sequence from MS/MS spectrum, even though if thesequence includes Ile/Leu.
We also applied the derivertization to a differentiation of another isobaric residue, α- and βAsp. As we reported previously, PSD
is applicable to differentiate Asp isomerization, because the isomerization causes significant changes in the intensities of fragment
ions, which are responsible for cleavages at the N- and C-terminal sides of the isomeric residues. The SPITC derivatiztion induced
more cleavable N-terminal side of the residue than the one in a underivertized counterpart. Notably, the specific y-ion ratios of
Asp isomers were still retained after the derivertization, which is thought to be useful to perform further quantitative analysis.

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 6: Novel Approaches in Proteomics Analysis

PMo-099

11:10 － 12:20

Study of hydrogen-induced cleavage of peptides with MALDI-ISD andsonolytic hydrolysis

Motoshi Sakakura, Mitsuo Takayama

Yokohama City University, Yokohama, Japan

Keywords:
In-source decay, MALDI, Hydrogen radical
Novel aspects:
Hydrogen-induced cleavage of peptides with MALDI-ISD and sonolytic hydrolysis methods has been compared from the standpoint of the mechanism for both methods.
Abstract:
Introduction
Simple and efficient protein cleavage holds the key for the success of bottom up proteome analysis approach using mass
spectrometry. In-source decay（ISD）coupled with MALDI has been recognized as efficient ladder sequencing method. MALDI-ISD
is caused by hydrogen transfer from matrix to analyte, and can be successfully applied for sequencing protein and peptide. Here
we report an additional method for sequence analysis of peptides. The method is consists of an ultrasound sonolytic device andreductive hydrogen donors such as 1,4-benzenedithiol. The sequence informations of peptides obtained with MALDI-ISD andsonolytic hydrolysis were compared on the standpoint of the hydrogen-induced reactions.
Experimental
Mass spectra were acquired on an AXIMA-CFR MALDI time-of-flight mass spectrometer（Shimadzu, Kyoto, Japan）. The sonication
was performed with a Branson 450 sonifier（Danbury, CT, USA）. The frequency and power used were 20 kHz and 50 W,
respectively. Analytes used were horse myoglobin, glucagon, ACTH18-39, and phosphorylated peptide. Analytes were prepared as
an aqueous solution at 10pmol/ul. The ultrasound was applied to 1000ul of the aqueous solution with addition of hydrogen donors
such as 1,4-benzeneditiol. 100umol additives were added into 1000ul of the analyte solutions. Peptide solution was premixed in 1 ：1 ratio with serine doped CHCA； then a volume of 1 uL of sample solution was deposited onto a MALDI sample plate andsolvents removed by allowing evaporation in air room temperature.
Results and discussion
MALDI-ISD, which uses hydrogen transfer reaction, resulted in the formation of N-terminal side c-series ions of both peptide andprotein analytes without loss or degradation of amino acid side chains and phosphate group. On the other hand, ultrasoundsonolysis combined with hydrogen donors in aqueous solution was applied to cleave of peptide and proteins, and resulting
products were analyzed by MALDI-TOF MS. The hydrolytic characteristics of peptides were that Xxx-Pro bond was more
susceptible to the sonolysis than other amino acid residues, while the information originated from Xxx-Pro degradation never beobserved in MALDI-ISD, and that hydrolysis occurred without any side chain degradation. MALDI mass spectrometric analysis of
the products obtained with sonolytic hydrolysis gave information about amino acid sequence of the peptides without any
byproducts. The mechanism for the sonolytic hydrolysis of peptides would be presented on basis of the acid hydrolysis, while themechanism of the MALDI-ISD has been known as a laser-induced hydrogen attachment reaction to the carbonyl oxygen on the
peptide backbone.
The characteristics of chemical acid hydrolysis of peptides in solution have been reported that the peptide bonds at the
C-terminal side（Asp-Xxx）, the N-terminal side of serine and threonine（Xxx-Ser/Thr）, and both sides of glycine（Xxx-Gly and GlyXxx）are relatively sensitive to acid hydrolysis. Moreover, the acid hydrolysis of peptides often leads to the formation of
dehydration products as a result of side reactions. Furthermore, the peptide bond at the N-terminal side of proline（Pro-Xxx）is
insensitive to acid hydrolysis. From the degradation characteristics of peptides with sonolytic hydrolysis differing from chemicalacid hydrolysis, it was suggested that the sonolytic may not be arisen from protons, but hydrogen radicals.
Consequently, although the degradation characteristics of peptides with sonolytic hydrolysis quite differ from both the MALDI-ISD
characteristics of which the peptide bond at Xxx-Pro is insensitive to MALDI-ISD and the chemical acid hydrolysis, the use of bothmethods of MALDI-ISD and sonolytic hydrolysis was very useful for amino acid sequencing of peptides.

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 6: Novel Approaches in Proteomics Analysis

PMo-100

13:30 － 14:40

Electrochemically Assisted Reduction of Disulfide Bonds of Peptides andProteins With On-Line Mass Spectrometric Detection

Martin Eysberg, Agnieszka Kraj, Hendrik-Jan Brouwer, Nico Reinhoud, Jean-Pierre Chervet
Antec, Zoeterwoude, the Netherlands

Keywords:
disulfide bond reduction, electrochemistry
Novel aspects:
Efficient reduction of disulfide bonds using a novel electrochemical approach based on a new electrode type and square wave
pulsesAbstract:
Disulfide bonds are one of the most important post-translational modifications of proteins. They are stabilizing protein’s
3 -dimensional structure and are crucial for their biological function.
The reduction of intra- and intermolecular disulfide bonds is necessary for successful characterization and assignment of thebonding sites by MS.
Off-line reduction is performed using highly concentrated chemical agent（e.g. dithiothreitol（DTT））that needs to be removedprior LC/MS analysis. Alternatively, thiol - free reducing agents as TCEP（tris（ 2 -carboxyethyl）phosphine）can be used. However,the sample preparation remains laborious and difficult to combine with on-line LC/MS.
Moreover, the possibility of on-line disulfide bond reduction can be beneficial for the determination of disulfide bondarrangements or top down proteomics strategy, which relies on fragmentation of intact proteins without enzymatic digestion.
In this poster, we present electrochemically（EC）assisted reduction of biologically active peptides and proteins containing
disulfide bonds followed by on-line mass spectrometric detection. For the first time the reduction of disulfide bonds is performed
using a proprietary semi-precious metal working electrode. The unique properties of the working electrode allow for complete
reduction of all disulfide bonds of the tested proteins and peptides. Furthermore, a special electrochemical method based onsquare-wave potential pulses was developed and applied for efficient and stable reduction.
Insulin, a small protein of 5733 Da containing 3 disulfide bridges was used as model protein for system evaluation. The results arecompared with the previous approach exploiting a conductive diamond working electrode. Reduction of other peptides andproteins such as Somatostatin with one disulfide bond（1638 Da）and α-Lactalbumin with four bonds（14 178 Da）will be shown todemonstrate the power of electrochemical S-S bond reduction.
The data were acquired using high resolution Fourier transform ion cyclotron resonance mass spectrometer（FT ICR）that allowed
for accurate molecular weight determination of intact protein before and after reduction. Additionally, high mass resolving power
was necessary for unambiguous identification of a peak originated from Somatostatin and its reduced form.
The electrochemical cell can be positioned before or after the HPLC separation, i.e., in an EC/LC/MS or LC/EC/MS set-up, resulting
in a fully automated platform for fast characterization of protein/peptide based biopharmaceuticals.

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 6: Novel Approaches in Proteomics Analysis

PMo-101

11:10 － 12:20

Isotope dilution LC-ESI/MS/MS method for the determination ofphenylthiohydantoin (PTH)-amino acids derived from Edman degradation
products of proteins

Ryo Satoh, Takaaki Goto, Seon Hwa Lee, Tomoyuki Oe
Tohoku University

Keywords:
protein, absolute quantitation, Edman degradation
Novel aspects:
The first isotope dilution LC-ESI/MS/MS method for the determination of phenylthiohydantoin-amino acids derived from Edman
degradation products of proteins has been developed.
Abstract:[Objective]
Edman degradation reaction, reported in 1950, is a well-known method for amino acid（AA）sequencing in a peptide. It was usedalmost exclusively before mass spectrometry（MS）-based sequencing appeared. The methodology is based on the stoichiometriclabeling of N-terminal α-amino group by Edman reagent（phenylisothiocyanate, PITC）followed by the quantitative degradation torelease the N-terminal AA as the phenylthiohydantoin derivative（PTH-AA）. Since the methodology is robust, we have been
studying the possibility to quantitate not only peptides, but also proteins as the PTH-AAs. That could enable to perform absolute
quantitation of proteins without use of protein standards. To prove this idea, we have already confirmed the concept using matrix
assisted laser desorption ionization（MALDI）-time of flight（TOF）/MS［ 1 ］. Here, we demonstrate the first isotope dilution
liquid chromatography（LC）-electrospray ionization（ESI）/tandem mass spectrometry（MS/MS）method for the determination of
PTH-AAs derived from Edman degradation products of proteins. The long-term stability experiments of twenty PTH-AAs and theapplications for model proteins/peptides have been also performed.
[Experiments]
High-performance liquid chromatography（HPLC）was carried out using an UltiMate 3000 HPLC series（Dionex Co.）with an Inertsil
ODS- 4 column（150 × 2.1 mm i.d., 3
μm, 100 &#8491；, GL Sciences, Inc.）. A TSQ Vantage triple stage quadrupole mass
spectrometer（Thermo Fisher Scientific Inc.）with an ESI source was used for selected reaction monitoring（SRM）. The long-term
stability of PTH-AAs was examined in four different storage conditions（25%（v/v）aqueous trifluoroacetic acid, pH3, pH7, and pH11
buffers）at 4 &#8451； followed by monitoring with HPLC/UV. A mixture of twenty AAs was allowed to react with PITC and［13C6］-PITC to obtain standards and the corresponding internal standards, respectively. Human serum albumin（HSA）, angiotensin II, α-casein, lysozyme, ribonuclease A, pepsin, and insulin were used as model proteins/peptides.
[Results & Discussion]
The LC conditions were optimized in terms of effective ESI and chromatographic separation of the isovalent mass amino acids,
such as Leu vs. Ile. Using acidic mobile phase（0.1% formic acid/water-acetonitrile）, all PTH-amino acids were well ionized andthe base line separation between PTH-Ile and PTH-Leu was also achieved. Since the optimal fragment ion for SRM was selected toeach PTH-AA/［ 13 C 6 ］-PTH-AA, simultaneous analysis of twenty PTH-AAs was attained as stable isotope dilution method. The
calibration curve of each PTH-AA showed good linearity（r2 = 0.995-1.000）throughout the range of 1 -100 pmol/injection. Also, thestorage condition of each PTH-AA（at least 90 % within a week）was optimized for long-term storage as solutions. From theexperiments using the seven model proteins/peptides, corresponding PTH-AAs released from the N-terminal amino acids wereobserved selectively. The yields of PTH-AAs derived from proteins were almost quantitative even from the largest model protein（77%, HSA, 66 kDa）. Since this stable isotope dilution LC-ESI/MS/MS method is robust and selective enough for the determinationof PTH-AAs, it could be used for absolute quantitation of proteins even without the use of protein standards.
［ 1 ］Oe T., Maekawa M., Satoh R., Lee S.H., and Goto T., Rapid Commun. Mass Spectrom.24, 173-179（2010）.

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 6: Novel Approaches in Proteomics Analysis

PMo-102

13:30 － 14:40

Mechanistic Study on Non-enzymatic Isomerization of N-terminal Amino
Acids by Endogenous Aldehydes

Ryo Kajita, Takaaki Goto, Seon Hwa Lee, Tomoyuki Oe
Tohoku University, Sendai, Japan

Keywords:
isomerization, N-terminal amino acid, ketimine
Novel aspects:
The mechanism of non-enzymatic isomerization of N-terminal amino acid mediated by endogenous aldehydes has been revealed
to involve a ketimine intermediate.
Abstract:[Objective]
The aldehydes can easily react with other biomolecules containing a nucleophilic functional group. There have been a lot of
studies on the aldehyde-mediated biomolecular modifications because a large number of different aldehydes have been detectedin vivo and most of them have intrinsic bioactivities. In the reaction of the bioactive peptide, angiotensin II（Ang II： H-Asp-Arg-ValTyr-Ile-His-Pro-Phe-OH）with the lipid peroxidation derived aldehydes, we have recently identified a pyruvamide-Ang II formed via
oxidative decarboxylation of N-terminal Asp as the most abundant product along with other modified Ang IIs. It was suggested
that the pyruvamide-Ang II was produced by deamination of N-terminal Asp after the formation of Schiff base intermediate andsubsequent decarboxylation. In addition, the non-enzymatic stereo-isomerization of N-terminal amino acid was observed in thereaction between model peptides and various endogenous aldehydes under a physiological condition. We reasoned that the
ketimine formed by tautomerization of the Schiff base intermediate can add a hydrogen atom non-stereo-specifically, leading to
the isomerization of N-terminal amino acid. To clarify this mechanism, the model peptide was reacted with aldehyde in theabsence or presence of heavy water and the reaction mixture was analyzed by LC/ESI-MS and MS/MS. The possibility ofN-terminal amino acid isomerization of the protein was also examined.
[Experiments]
The model peptide（L-FMRF-amide： H-Phe-Met-Arg-Phe-NH2 ）was incubated with pyridoxal 5 ’-phosphate（PLP）in phosphatebuffer（pH7.4）at 37 o C. LCQ-DECA（Thermo Fischer Scientific Inc.）was employed for the analysis of the reaction mixture. The
chromatographic separation of reaction products was carried out on an Inertsil ODS- 3 （150 × 2.1 mm i.d., 3 μm； GL sciences
Co. Ltd）with water and acetonitrile containing 0.1% formic acid as mobile phases at 0.2 mL/min. The same reaction was also donein the heavy water. As for the protein model, human serum albumin（HSA）and its N-terminal tetra-peptide（L-DAHK： H-Asp-AlaHis-Lys-OH）were used. HSA was incubated with PLP, followed by a trypsin digestion. The tryptic peptide was analyzed by LC/ESIMS after derivatization with dansyl chloride.
[Results and Discussion]
LC/ESI-MS analysis of the reaction between L-FMRF-amide and PLP revealed the presence of five products together with D-FMRFamide which was formed by isomerization of N-terminal Phe. Four products had an identical protonated molecule（MH+）whichcorresponded to an increase in mass of 229 Da（+［PLP-H2O］, Schiff base）. The MH+ of another product was consistent with thedeamination of L-FMRF-amide. MS/MS and（MS）3 analyses further revealed that both modifications occurred at N-terminal Phe,and that the intermediate of N-terminal isomerization could be Schiff base. Results of the reaction in the heavy water showed that
deuterium was added to D-FMRF-amide and two Schiff bases, which was confirmed by their isotopic compositions（ratios ofheavy to light isotopes）. This suggests the formation of ketimine intermediate during the reaction. Thus, the isomerization of
N-terminal amino acid occurred through a mechanism involving non-stereo-specific addition of a proton to ketimine. The detectionof deaminated product generated by an irreversible hydrolysis of ketimine intermediate also supports this isomerization
mechanism. The isomerization of N-terminal Asp was subsequently observed in the reaction of L-DAHK, equivalent to N-terminal
sequence of HSA, with PLP. Furthermore, D-DAHK fragment was detected when HSA was reacted with PLP, followed by tryptic
digestion. These results suggest that the isomerization of N-terminal amino acid can occur in the protein through the samemechanism.

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 6: Novel Approaches in Proteomics Analysis

PMo-103

11:10 － 12:20

Peptide fragmentation upon low and high energy CID in ESI and MALDI
mass spectrometry: A statistical approach

Christine Enjalbal, Mathieu Dupre, Sonia Cantel, Jean Martinez
University of Montpellier 2

Keywords:
Peptide, fragmentation, low and high energy dissociation, MS/MS, CID, gas-phase rearrangement, De Novo sequencing
Novel aspects:
Statistical approach to question peptide fragmentation upon CID activation in ESI and MALDI mass spectrometry from an homemade non biaised peptide set covering a wide structural diversity .
Abstract:
Proteomics has emerged as a major tool in understanding protein functions as well as protein-protein interactions at the cellularlevel. Mass spectrometry, through“top-down“ or“bottom-up“ strategies, constitutes one of the best method to identify protein
primary sequences. In the course of these analytical protocols, MS/MS experiments are conducted to induce fragmentations of
the polyamide skeleton. Such data, recorded upon high or low energy dissociation conditions, ideally enable to decipher theamino acid sequence. However, the extent of the gathered information varies strongly according to structural and instrumental
conditions（nature of the peptide chains, type of mass spectrometer, activation method, fragmentation tuning,...）. Following astatistical approach, we investigated the low and high energy collision induced dissociation（CID）behaviors in ESI and MALDI
mass spectrometry（ESI-Q-q-Tof and MALDI-Tof-Tof instruments）of various synthetic peptides（300 sequences of variable
compositions and lengths mimicking proteolytic peptides obtained by the action of various enzymes such as trypsin, Lys-N, Glu-C,...）. 1,2 Our attention was particularly focussed on singly charged peptides that often failed to be identified by sequencing
algorithms. Only half of the studied compounds showed charge directed dissociations in accordance with the mobile protonmodel producing fragment ions directly related to the primary sequence. For the peptides that did not exhibit the expectedfragment ion series, alternative dissociation behaviors issued from complex rearrangements were evidenced thus providing a morecomprehensive fragmentation model suitable for more efficient automated MS/MS spectra interpretation. Such remarkable gasphase behaviors were mainly attributed to the presence of basic residues（Arg, His, Lys）and to their relative localisation within
the peptide sequences.3,4
1. L. Mouls, J-L. Aubagnac, J. Martinez, C. Enjalbal, J. Proteome Res. 6, 1378-1391（2007）
2. N. Shenar, N. Sommerer, J. Martinez, C. Enjalbal, J. Mass Spectrom. 44, 621-632（2009）
3. M. Dupre, S. Cantel, P. Verdié, J. Martinez, C. Enjalbal, J. Am. Chem. Soc. Mass Spectrom, 2011, 22, 265-279.
4. M. Dupre, S. Cantel, J. Martinez, C. Enjalbal, J. Am. Chem. Soc. Mass Spectrom, 2012, 23, 330-346.

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 6: Novel Approaches in Proteomics Analysis

PMo-104

13:30 － 14:40

Rapid Asp-selective Acid Hydrolysis for Proteomic Workflows

Joe R Cannon, Catherine Fenselau

University of Maryland, College Park, MD United States

Keywords:
Middle-down, Middle-out, Orbitrap, Acid Digestion
Novel aspects:
Presented is a novel method for enhanced analysis of middle-out and bottom-up sized peptides using high resolution accuratemass on a hybrid mass spectrometer.
Abstract:
Microwave-supported acid hydrolysis exploits kinetically selective cleavage at Asp residues to provide very rapid protein cleavage,
10sec to 20 min, depending on sample complexity. We report the evaluation of this technique in three different proteomic
workflows. First, its utility is demonstrated for digestion in solution, using a system poorly suited for tryptic digestion, the humanribosome. The high abundance of Lys and Arg residues required to interact with the phosphate backbone of ribosomal RNA
makes the average peptide length following tryptic digestion less than 5 residues； too small for effective data dependent analysis（Cannon et al, JPR 2010, 9 ： 3886-90）. Second, the digestion method is shown to be effectively applied“in-gel“ to bands of anelectrophoretically fractionated whole cell lysate from human multiple myeloma cells. From eight excised bands, a total of 1124
peptides were identified representing 604 proteins. Finally, the same mixture was used to demonstrate increased efficiency inanalyzing high and low mass peptide fractions partitioned about a 3000Da molecular weight threshold.
Longer peptide products are produced, on average, by this single residue cleavage reaction. This is illustrated in silico and in thelaboratory by comparison of acid cleavage products from multiple myeloma with tryptic peptides from the same cancer cells. We
and others have reported that many advantages are offered by longer peptides in middle-out proteomics strategies. A workflow
has been designed and demonstrated to enrich peptides with masses exceeding 3000Da and increase their successful analysis by
LC-MS/MS. Products of microwave-supported acid cleavage are fractionated into larger and smaller peptides using a 3 KDa
molecular weight filter. Subsequent LC-MS/MS analysis of the larger peptides is further biased by setting data dependent scanningparameters to reject charge states less than z = 4. Successful implementation of this middle-out strategy also requires the use of
optimized HPLC and an upgraded search program such as ProSight. In addition to the more informative higher mass peptides, thefraction enriched in lower mass peptides can be analyzed in classical high throughput bottom-up fashion.

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 6: Novel Approaches in Proteomics Analysis

PMo-105

11:10 － 12:20

Optimization of matrix preparation methods for MALDI-ISD of protein

Ryunosuke Iimuro, Mitsuo Takayama

Yokohama City University, Yokohama, Japan

Keywords:
MALDI-ISD, Matrix, Preparation, Protein
Novel aspects:
The optimization of matrix preparation methods for MALDI-ISD of protein was performed to obtain the abundant and steady ISD
fragment peaks without sweet spots.
Abstract:
Introduction
The intensity and sharpness of MALDI-ISD fragment peaks of peptide and protein depend on the matrix used and co-crystallizationof sample（matrix and analyte）solution. The procedures for analyte-matrix solution preparation are shown to greatly influence on
the co-crystallization of sample solution. Depending on the co-crystallization, the ISD spectra were obtained with or without the“sweet spots“. In order to get the reproducibility of ISD fragment ions, the ISD spectra have to be obtained by automatically andrandomly scanning the sample crystal surface. Here we introduce 1 -naphthol derivatives as novel matrix material to optimize thematrix solution preparations, by changing the fractional compositions of solvent and with or without added trifluoroacetic acid
（TFA）. Here we report the preparation methods of matrix solution for the MALDI-ISD of protein to attain co-crystallization
without“sweet spots“. The methods obtained would be applied to several kinds of protein and phosphorylated peptide.
Methods
The chemicals used as matrix were 5 -amino- 1 -naphthol（5,1-ANL）and 5 -hydroxy- 1 -naphthol（5,1-HNL）, and were obtained
from Tokyo Chemical Industry Co., Ltd.（Tokyo, Japan）. In order to determine the optimum matrix solvent compositions, different
fractional compositions of solvents such as acetone, acetonitrile（ACN）and water（H 2 O）were prepared at 10 mg/mL in acetone/
H 2 O（ 1 ： 1 ）, ACN/H 2 O（ 1 ： 1 ）, ACN/H 2 O（ 7 ： 3 ）, and ACN. In order to determine the influence of acid on the cocrystallization, matrix solution was prepared with and without added 0.1% TFA, i.e., the matrix solution at 10 mg matrix/mL solventwith added 0.1% TFA was prepared. Aqueous analyte solution of 10uL was premixed in a tube with the same volume of a matrix
solution of 10uL. A 1 μL of sample solution was deposited onto a MALDI sample plate and the solvents were removed by drying
in air at room-temperature. Analyte used for the optimization was horse cytochrome c（Mr12360）.
Results and discussion
Of the different matrix solvent compositions tested, the composition of acetone /H 2 O（ 1 ： 1 ）for whichever matrix was notsuitable for MALDI-ISD, because the abundance of ISD fragment peaks was very low. The composition of ACN/H 2 O（ 7 ： 3 ）was
suitable for MALDI-ISD, while ACN/H 2 O（ 1 ： 1 ）was not suitable for MALDI-ISD. The solubility of matrix into solvent has been
demonstrated to be important in MALDI sample preparation. Both matrices 5,1-ANL and 5,1-HNL have better solubility in the ACN/H 2 O（ 7 ： 3 ）than ACN/H 2 O（ 1 ： 1 ）. As a result, ACN/H 2 O（ 7 ： 3 ）was most suitable for automatically and randomly
scanning the sample surface. It was found that addition of TFA into the matrix solution showed a remarkable influence on thequality of the ISD spectrum. The matrix solution of 5,1-ANL prepared with TFA was not suitable for MALDI-ISD of protein, because
the abundance of ISD fragment peaks was absolutely lower than when the solution without added TFA was used, while［M+H］+
peak was absolutely high in the matrix solution with added TFA. On the other hand, the solution of 5,1-HNL prepared with TFA
was suitable for MALDI-ISD. The abundance of both［M+H］+ and ISD fragment peaks was absolutely high compared with thesolution without TFA. In conclusion, we have presented optimized methods of matrix preparation procedures for MALDI-ISD ofprotein, i.e., ACN/H 2 O（ 7 ： 3 ）without added TFA for 5,1-ANL and ACN/H 2 O（ 7 ： 3 ）with added 0.1 % TFA for 5,1-HNL. The
optimized matrix preparation methods obtained here would be applied to several different proteins and a tetra-phosphorylated
peptide. The MALDI-ISD spectra obtained with the optimized matrix preparation methods showed the informative ISD fragment
ions for the analysis of sequencing and secondary structural characteristics.

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 6: Novel Approaches in Proteomics Analysis

PMo-106

13:30 － 14:40

Comparative Proteomic Analysis of Virulence Variations in Xanthomonas
Campestris pv. Campestris Strain 17, 11 and P20H

Tao-Shan Chang, Chien-Chen Lai

National Chung Hsing University, Taichung, Taiwan/Institute of Molecular Biology College of Life Science

Keywords:
Xanthomonas campestris pv. campestris / black rot / 2 D electrophoresis / LC-MS/MS
Novel aspects:
To our knowledge, this is the first report of using the proteomic strategy to analysis of virulence variations in xanthomonas
campestris pv. campestris strain 17, 11 and P20H.
Abstract:
Xanthomonas campestris pv. campestris（Xcc）is a Gramnegative plant pathogenic bacterium causing black rot in crucifers,
resulting in tremendous loss in agriculture. The ability of Xcc to infect plants successfully depends on certain factors including
extracellular enzymes（such as protease, endoglucanase, pectinase and mannanase）, exopolysaccharides（xanthan gum）and
biofilm production. A newly isolated pathogenic Xc17 from an infected cabbage leaf for some phenotypic characteristics havepreviously been implicated as factors contributing to pathogenicity. A wild-type strain of X. campestris,Xc11, was found to havelost its pathogenicity spontaneously after frequent subculturing for years. This non-pathogenic derivative, designated Xc11A. X.
campestris P20H was a non-mucoid mutant previously isolated from Xc11A by mutagenesis with nitrous acid. Recently theproteomics have been developed more widespread, the techniques can offer insights into the quantity and quality of the final
gene products. In this study, we aim to elucidate the physiological and molecular mechanism response to virulence variations inxanthomonas campestris pv. campestris strain by using proteomics method. Total proteins of intracellular and extracellular wereextracted for comparative proteomics analysis via 2 D electrophoresis and LC-MS/MS analysis. The result shows several
differentially expressed proteins spots of bacterial proteome in Xc17, 11 and P20H. These identified proteins may be helpful inelucidating the molecular basis of virulence variations in Xcc.

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 6: Novel Approaches in Proteomics Analysis

PMo-107

11:10 － 12:20

Proteomic Evidences for Starch Biosynthetic Pathway in Rice Endosperm

Chih-Wei Liu, Chien-Chen Lai

Institute of Molecular Biology,National Chung Hsing University, Taiwan

Keywords:
Rice / Starch biosynthesis / Proteomics / Gluconeogenesis
Novel aspects:
Novel starch biosynthesis pathway in developing rice endosperm has proven and demonstrated in our study.
Abstract:
Rice（Oryza sativa）is one of the most important cereals in the world. The starch reserves of the rice grains generally contain morethan 75% amylopectin； however, the abundance of amylose can affect the properties of starch in the grains. In this study, we use
the Tainung 67（TNG67）and its sodium azide-induced mutant SA0419 as the investigated materials. The low amylose content in
SA0419 grains（ 8 %）is apparently different from TNG67 grains（20%）. Moreover, the superior efficiency of starch biosynthesis andshorter developing period are observed in SA419. We want to resolve the regulated mechanisms of starch biosynthesis in riceendosperm from protein expression level. Endosperm proteome of the grains of 14 days after anthesis（DAA）were extracted andseparated by 2 -DE. MS-based protein identification strategy was used to analyze the total proteome and differential-expression
proteome of TNG67 and SA0419. The higher abundance of enzymes in SA0419 was used to produce the more α-D-glucose- 1 P, andhigh level of starch biosynthesis enzyme were generated to produce the ADP-glucose and to accelerate the accumulations ofstarch. Furthermore, lack of granule-bound starch synthasein SA0419 is the major key factor to cause the low amylose content.
Overall, the starch biosynthesis model in the rice endosperm was presented in this study. The increased in gluconeogenesis
pathway and reduction of the other metabolism pathway in SA0419 elevated the substrate contents for starch biosynthesis.

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 6: Novel Approaches in Proteomics Analysis

PMo-108

13:30 － 14:40

MALDI-TOF-MS-based proteomics for urinary biomarker discovery in
gamma-irradiated mouse

Keita KIRIYAMA, Susumu YOSHIOKA, Daisuke IIZUKA, Hidehiko KAWAI, Shunsuke IZUMIHiroshima University, Hiroshima, Japan

Keywords:
Biomarkers, Radiation, Hepcidin, Adipsin, Deglycosylation
Novel aspects:
1 ）Hepcidin 2 as potential biomarker for radiation exposure. 2 ）Adipsin was deglycosylated with radiation injury.
Abstract:
On March 11, 2011, a series of disasters at Fukushima in Japan killed thousands of people, left hundreds of thousands of people
homeless and nuclear accidents exposed large numbers of people to ionizing radiation. It is now necessary to develop biomarkers
for radiation dose assessment that can be used for mass screening in the event of a radiological mass casualty incident. Highthroughput appliances that could evaluate such incident are expected as being employed to guide triage and subsequent
therapeutic choices. Our aim of this study is to develop a high-throughput, sensitive biodosimetry technique which can be applied
for the early triage of a radiation emergency medical system.
We employed a proteomic approach to analyzing urinary proteins, using mice as an animal model. The urine samples werecollected from the mice which were exposed to total-body radiation（4.0-6.0Gy）produced by the 137Cs gamma-ray source. The
urine was separated by HPLC and/or SDS-PAGE and analyzed by MALDI-TOF mass spectrometry using Ultraflex TOF/TOF（Bruker
Daltonics）and AXIMA-QIT（Shimadzu）.
We identified hepcidin 2 as potential biomarker for radiation exposure. The urinary concentration of hepcidin 2 was increased
at 24 hours after gamma-irradiation in a dose-dependent manner, which coincides with the previous observation. The blood
hepcidin 2 levels was also increased at 8 hours after gamma-irradiation, this is the first observation of the increase of urinary
hepcidin 2. In addition, a 27-kDa protein was increased at 24 hours after gamma-irradiation in a dose-dependent manner. The 27kDa protein（SDS-PAGE）was identified by peptide-mass fingerprinting and MS/MS analysis as adipsin. Adipsin is a glycoprotein of
44- and/or 37-kDa, 1) and a serine protease homolog, which is synthesized and secreted by adipose cells and is found in the
bloodstream. These results indicate that the sugar chain of urine adipsin was cleaved by radiation exposure.
Thus, urinary metabolites may be a potential biomarker for acute ionizing radiation exposure. Further studies may be warranted
for the development of new biodosimetry methods for radiation exposure.
1 ）B. S. Rosen et al., Science, 244, 1483-1487（1989）.

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 6: Novel Approaches in Proteomics Analysis

PMo-109

11:10 － 12:20

LDI-MS on nanostructured materials for sensitive and efficient protein
digest characterization

Mathieu Dupre1, Sonia Cantel1, Yannick Coffinier2, Rabah Boukherroub2, Jean Martinez1,
Christine Enjalbal1
1

University of Montpellier, Montpellier, France, 2University of Lille, Lille, France

Keywords:
LDI-MS, Nanostructured silicon, Tryptic peptides, PMF, Peptide sequencing
Novel aspects:
Nanostructured silicon platforms for direct high-throughput robust protein digest characterization.
Abstract:
Laser desorption/ionization mass spectrometry（LDI-MS）using specific inert surfaces to promote ion formation has been widely
investigated the last decade［ 1 ］. In addition to porous silicon through the original DIOS technique, different materials were
tested as potent LDI-promoting agents, such as metals, carbon-based structures, porous particles, nanomaterials and more
recently, ordered three-dimensional silicon architectures. All of these matrix-free methods are now referred as Surface Assisted
Laser Desorption Ionization-Mass Spectrometry（SALDI-MS,http：//www.iupac.org）.
Up to now, disposable ready-to-use target plates（MassPREPTM, QuickMassTM and NALDITM）can be purchased for SALDI analysis
of small molecules. In order to overcome the limitations encountered with the use of such commercially available probes（surface
pollution affecting detection sensitivity and reproducibility）, we explored a variety of inert silicon-based UV-absorbing materials
that were presenting different physico-chemical properties for the analysis of peptides［ 2 - 4 ］. Both material architecture（amorphous powders, structured particles, structured surfaces）and material hydrophilic/hydrophobic character tuned by specificchemical derivatization（oxidation, silanization）were probed as crucial parameters for achieving efficient and robust detection ofan home-made array of model peptides covering a wide structural and mass diversity（50sequences, m/z 500-2000 Da）. Throughthis set of experiments, we were able to compare the performances of all investigated silicon-based supports, especially taking
into account peptide detection sensitivity（down to femtomolar concentrations）and reproducibility/repeatability（intra-spot/interspot signal variations）as well as the method robustness（easy sample preparation, need for ionization additives, straightforward
sample deposit depending on material wetting properties, ionization source tuning using conventional MALDI-TOF/TOF
instrument）. Having illustrated the capability to achieve both peptide detection and sequencing on these ionizing surfaces in thesame run, high-throughput identification of protein tryptic digests by a rapid MS profiling and subsequent MS/MS analyses wasundertaken. Tryptic digests from Cytochrome C, Beta-casein, BSA and Fibrinogen were analyzed in the femtomolar range（forinstance, from 50 fmol for Cytochrome C down to 2 fmol for Fibrinogen for a nanostructured silicon surface）.Comparison of the
MS and MS/MS data with those obtained with sample conditioned in organic matrix［5,6］and on commercial target（NALDITM）
demonstrated a great behavior for low mass responses.
We thus demonstrated the capability of LDI on nanostructured silicon supports to be a complementary method to MALDI Peptide
Mass Fingerprinting（PMF）ensuring determination of peptide molecular weights and sequences for more efficient protein
database searches in proteomic workflow.
［ 1 ］Peterson DS. Mass Spectrom. Rev. 2007；26：19-34.
［ 2 ］Shenar N, Martinez J, Enjalbal C. J. Am. Soc. Mass Spectrom. 2008；19：632-44.
［ 3 ］Shenar N, Cantel S, Martinez J, Enjalbal C. Rapid Commun. Mass Spectrom. 2009；23：2371-9.
［ 4 ］Dupre M, Coffinier Y, Boukherroub R, Cantel S, Martinez J, Enjalbal C. J. Prot. 2012； 75:1973-1990.
［ 5 ］Dupre M, Cantel S, Verdie P, Martinez J, Enjalbal C. J.Am. Soc. Mass Spectrom. 2011；22：265-79.
［ 6 ］Dupre M, Cantel S, Martinez J, Enjalbal C. J. Am.Soc. Mass Spectrom., 2012；23：330-46.

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 6: Novel Approaches in Proteomics Analysis

PMo-110

13:30 － 14:40

Evaluation of peptide adsorption-controlled liquid chromatography-mass
spectrometric (PAC-LC-MS) method to quantify peptides in biologicalfluidsRyoya Goda1, Ryo Yokoyama2, Nobuhiro Kobayashi1
1

Daiichisankyo, Co., Ltd., Tokyo, Japan, 2KK AB Sciex, Tokyo, Japan

Keywords:
peptide adsorption-controlled liquid chromatography, tandem mass spectrometry, biological fluids, simultaneous quantitation
Novel aspects:
PAC-MS/MS method has the potential for highly sensitive and quantitative analysis of peptides in biological fluids.
Abstract:
Introduction
Endogenous peptides in biological fluids have actively been investigated as biomarkers for discovery of clues to underlying causeof disease, for early and correct diagnostics, and for effective therapy against various diseases. However, both discovery offunctional peptides and analysis of their functions are difficult because many peptides exhibit their functions only atconcentrations in the low picomolar range. Recently, there is a compelling need for a methodology based on high-performanceliquid chromatography-mass spectrometry（LC-MS）for the quantitation of peptides in biological fluids, because LC-MS methods
possess considerably higher selectivity based on the mass-to-charge（m/z）ratios in addition to chromatographic separation, andbetter reproducibility in comparison with immunological methods.
We found that the adsorption capacity of a peptide to the column packing changed abruptly and reversibly across a specificamount of organic solvent（the critical point, fn = 1 ）in the peptide solution. Circular dichroism spectral analysis suggested thatthe abrupt change in the adsorption capacity was caused by the conformational change induced by the organic solvent in the
peptide solution. This solvent-induced conformational change was found to be reversible across the critical threshold. Based on
this property, we developed a new gradient system, which is named peptide adsorption-controlled liquid chromatography（PACLC）, for quantitative analysis of peptides in the solution with a higher fn value than 1 （fn> 1 ）. PAC-LC enables quantitation of
peptide in such solutions without any loss of sensitivity due to peptide adsorption to various materials, and found to be superior
to a standard LC system in terms of precision, loading volume onto the system, sensitivity, and the simultaneous and quantitative
analysis of peptides with good precision. Then, we have evaluated the usefulness of PAC-LC coupled with tandem mass
spectrometer（4000 QTRAP and TripleTOF5600）for quantitation of peptides in biological fluids such as plasma and cerebrospinal
fluid（CSF）.
MethodUsing PAC-LC coupled with 4000QTRAP, quantitation of peptides in biological fluids was carried out using multiple reaction
monitoring（MRM）mode. In the case of TripleTOF 5600, quantitation of peptides was carried out using either TOF-MS scan or
TOF-MS/MS scan mode. During the evaluation, stable isotope-labeled peptides were also evaluated as the internal standards tominimize the influence of matrix on the reproducible quantitation. The calibration curves for endogenous peptides were drawn
from the standard calibration solutions.
Preliminary Data
The results of the study suggest that PAC-LCMS method is a powerful tool to quantify peptides in biological fluids. For example ofanalysis using MRM mode, PAC-LC-MS method enables simultaneous quantitation of amyloid β peptides（Aβ） 1 -38, 1 -40, 1 -42
and 1 -43 in dog CSF with good reproducibility. The basal concentration of Aβ 1 -38, Aβ 1 -40, Aβ 1 -42 and Aβ 1 -43 in dog CSF
is found to be approximately 300, 900, 200 and 30 pM, respectively. Also, stable isotope labeled Aβ used as the internal standard
can minimize the influence of CSF matrix on the reproducible Aβ quantitation. In the case of TripleTOF 5600, calibration curves
from the standard calibration solutions were successfully constructed in the concentration range from 10 pM to 1000 pM forvarious peptides using TOF-MS scan mode. For TOF-MS/MS mode, the lower limit of quantitation was expected to be lower than
10 pM. The obtained results suggest that PAC-LCMS coupled with TripleTOF5600 have easily applicability to the quantitation of
peptides in biological fluids.

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 6: Novel Approaches in Proteomics Analysis

PMo-111

11:10 － 12:20

Comparative proteomics of CD133-positive liver cancer stem cells

Sheng-Ta Tsai1,2, Chih-Chiang Tsou3, Wan-Yu Mao1, Wei-Chao Chang1, Wen-Lian Hsu3,
Chia-Ning Shen1,2, Chung-Hsuan Chen1,2,4,5

1
Genomics Research Center, Academia Sinica, Nankang, Taipei, Taiwan, 2Institute of Biochemistry & Molecular Biology,
National Yang-Ming University, Taipei, Taiwan, 3Institute of Information Science, Academia Sinica, Nankang, Taipei,
Taiwan, 4Department of Chemistry, National Taiwan University, 5Institute of Atomic & Molecular Sciences, Academia
SinicaKeywords:
Hepatocellular carcinoma / LC-MS/MS / Liver cancer stem cells / Label-free quantitation
Novel aspects:
The proteomes of CD133+ liver cancer stem cells, CD133- liver cancer cells and normal human hepatocytes were investigatedusing two label-free methods.
Abstract:
CD133-positive liver cancer stem cells, which are characterized by their resistance to conventional chemotherapy and their tumor
initiation ability at limited dilutions, have been recognized as a critical target in liver cancer therapeutics. In the current work, weinvestigated the proteome of CD133-positive liver cancer stem cells for the purpose of identifying unique biomarkers that can beutilized for targeting liver cancer stem cells. The subpopulation of Huh 7 hepatoma cells that express the glycosylated CD133
antigen was sorted and compared with normal hepatocytes and CD133-negative hepatoma cells. Label-free quantitation was
performed by ID-based Elution time Alignment by Linear regression Quantitation（IDEAL-Q）. The results showed that 151 proteins
were differentially expressed in the CD133-positive hepatoma cells when compared with CD133-negative cells. 21 up-regulated
plasma membrane proteins were identified in the CD133-positive hepatoma cells, suggesting potential surface biomarkers for livercancer stem cells. We validated the expression of some of the up-regulated membrane proteins either by MaxQuant software orby RT-PCR, immunoblot, and immunofluorescent analysis. We identified annexin A 1 and annexin A 3 were highly expressed in theCD133-positive hepatoma cells. These findings confirmed that mass spectrometry-based label-free quantitative proteomics can beused to gain insights into liver cancer stem cells.

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 6: Novel Approaches in Proteomics Analysis

PMo-112

13:30 － 14:40

Quantitative proteomics approach with isotope dimethyl labeling forprotein pathway analysis using high resolution mass spectrometry

Ping Sui, Konstantin Artemenko, Jonas Bergquist
Uppsala University, Uppsala, Sweden

Keywords:
Quantitative proteomics, Isotope dimethyl labeling, Mass spectrometry, Rat spinal cord
Novel aspects:
Our method provide a fast, simple and inexpensive way for protein pathway analysis in quantitative proteomics, especially forsmall animal sample, for example rat spinal cord.
Abstract:
Protein expression in the cell could be easily affected by, among other things, drugs, disease or stress. The change in protein
expression may activate a response bio-mechanism and influence expression of other related proteins, which makes
corresponding pathways up or down regulated. By quantifying the protein expression in biological tissues we can identify theactivated pathways, which are caused by or related to a certain disease or clinical symptoms. In this study, we compared theprotein expression level in contralateral dorsal and contralateral ventral parts of the spinal cord in naive rats. We use dimethyl
isotope labels to globally label the tryptic peptides for relative quantification. Proteins were extracted from homogenized rat
spinal cord tissues, then digested, labeled and analyzed by LC-MS/MS using FT-ICR instrument. Sample preparation protocol andthe acquisition parameters of LC-MS/MS were optimized, especially for relative quantitative proteomics analysis in spinal cordsample. Stability and repeatability of the method were evaluated as well. Related bio-pathway analysis was performed using IPA
from Ingenuity.
The results show that 17.2% of the mapped proteins are related with the nervous system development and functions and most ofthem have higher expression level in the contralateral dorsal region than in the contralateral ventral region. Relevant pathways in
the nervous system can be identified with a high confidence value, for example GABA receptor signaling and protein ubiquitination
pathways. The efficiency of the isotope dimethyl labeling is stable and data acquisition of FT-ICR is robust for relative quantitative
proteomics analysis. This method could be applied to the study of neuro-related dysfunctions e.g. neuropathic pain in the central
nervous system. Due to the complexity of biological samples and the high dynamic nature of proteome, large number of samples
and replicates are needed to enhance the confidence of the inferences from the experiment.

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 6: Novel Approaches in Proteomics Analysis

PMo-113

11:10 － 12:20

MS-based proteogenomics and comparative proteomics help to identify
novel key proteins in organisms with newly sequenced genomes

Rainer Cramer1, Laurence V Bindschedler1, Dana M Gheorge2, Liam J McGuffin1, Celia J Smith1,
Clara M Pliego Prieto2, Pietro D Spanu2
1

University of Reading, UK, 2Imperial College London, UK

Keywords:
proteogenomics, pathogen effectors, protein interaction, host-pathogen interaction, comparative proteomics
Novel aspects:
We will demonstrate the power of MS-based proteogenomics and comparative proteomics in annotating newly sequenced
genomes and identifying and characterizing novel proteins not seen before, analysing Blumeria graminis.
Abstract:
The barley powdery mildew（Blumeria graminis f.sp. hordei）is an economically important fungal obligate pathogen in temperateregions around the world. Large-scale MS-based proteomics has been successfully employed for a proteogenomic approach invalidating the annotation of its recently assembled genome. A comparative MS-based analysis of the proteomes of Blumeria sporeand hyphae cells to Blumeria haustoria cells（i.e. the cells involved in infection and direct interaction with the host through nutrient
uptake and elicitor delivery）allowed the identification of Blumeria effector candidate（BEC）proteins unique to haustoria cells.
These proteins are secreted, most have little or no sequence similarity to known enzymes and are likely to represent novel
structural folds as they have poor predicted 3 D models. Some of these proteins were shown to have a biological function in theinfection process. Their Blumeria and barley interactors are being sought by“pull down“ affinity chromatography using expressedtagged recombinant BEC proteins as baits.
We will demonstrate the power of MS-based proteogenomics and comparative proteomics in annotating newly sequenced
genomes and identifying and characterizing novel proteins not seen before.

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 6: Novel Approaches in Proteomics Analysis

PMo-114

13:30 － 14:40

MALDI msn analysis of protein oxidation in major diseases

Lina Sellami1, Claude Villard1, Kamel Mabrouk1, Therese Schembri1, Didier Gigmes1,
Matthew Openshaw2, Omar Belgacem2, Daniel Lafitte1
1

Aix Marseille Universite, Marseille, France, 2Shimadzu, Manchester, United Kingdom

Keywords:
MALDI, MSn, single isotope selection, protein modification, oxidation
Novel aspects:
MALDI MSn of complex protein patterns with single isotopic selection at various MS/MS stages
Abstract:
Introduction
Production of reactive oxygen species（ROS）is a characteristic feature of a variety of（patho）physiological processes. It is
recognized that ROS are makers of disease since they can induce deleterious effects, e.g. oxidation of biomolecules such as lipids
and proteins. Today, oxidative stress is thought to be involved in many pathologies ranging from neuronal（e.g. Alzheimer’s andParkinson’s disease）and metabolic disorders（e.g. hypercholesterolemia and diabetes）to chronic inflammatory diseases（e.g.
atherosclerosis and arthritis）and cancer.
In this context, protein carbonylation as the major form of protein oxidation is considered as universal indicator for oxidative
stress of biological samples and products. During oxidative damage carbonyl groups can be generated directly from amino acids（e.g. Arg, Lys, Pro, Glu, Asp）or indirectly via Shiff’s base（e.g. aldehydes）or Michael adducts（e.g. α,β-unsaturated carbonyls）ofadvanced lipid oxidation products（e.g. fragmented OxPLs）, which are oxidized to the carbonyl level.
The limited number of current data on the successful analysis of carbonylated proteins by mass spectrometry can be explained by
the difficulty of characterizing oxidized proteins by conventional proteomic approaches. We present in this work top down andbottom up strategies using MALDI MSn technologies that allow exhaustive characterization of protein and peptide modification.
Material and methods
For protein oxidation, protein and peptide stock solution were mixed with 5 mM NaOCl solution. The mixture was incubated for
15 min at 37 °C in a water bath. Following oxidation, the proteins and peptides were purified using a ZipTip microcolumn andeluted the matrix. 2 µl（approximately 3 µg）were applied either to the MALDI-TOF and MALDI-QIT-TOF target and allowed to dry
at ambient temperature. MALDI spectra were acquired on a MALDI-TOF and on a high vacuum MALDI-QIT-TOF instrument（Shimadzu, Manchester, UK）. The control software（Launchpad, version 2.9.2）was used for fragment annotation and analysis.
Protein identification was performed using mascot（http：//www.matrixscience.com）.

Results, discussion and perspectives
The MALDI-QIT-TOF technology was used to fragment oxidized peptides and proteins. Protein fragments were analyzed afterdigestion or directly in the source by in source decay. Ions were transferred to the ion-trap and submitted further to multiple
cycles of MS n using CID. The resolution for all the fragmentation stages（more than 15000 FWH）allowed selection of single
isotopes selection and up to five cycles. Protein carbonylation generates modifications on lysine（- 1 Da）, Threonine（- 2 Da）,
proline（+16 Da）or arginine（-43 Da）. Especially for lysine, the most abundant modification, selection of a single isotype in each
MS/MS step has been a real benefit for unambiguous characterization of protein modification.

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 6: Novel Approaches in Proteomics Analysis

PMo-115

11:10 － 12:20

Quantitative Proteomics Using a Quadrupole-Orbitrap Mass Spectrometer(Q-Exactive): Application to the Analysis of Clinical Samples

Bruno Domon, Sebastien Gallien, Elodie Duriez

Luxembourg Clinical Proteomics Center CRP-Sante Luxembourg

Keywords:
quadrupole-orbitrap, quantification, proteomics, clinical application, targeted experiments
Novel aspects:
Novel quantification method to analyses peptides in complexe biological samples, leveraging the quadrupole-orbitrap instrument.
Abstract:
A hybrid quadrupole-orbitrap mass spectrometer offers unique capabilities, in term of resolution, mass accuracy, and acquisition
speed that has enabled novel quantitative proteomics approaches. This instrument, combined with stable isotope dilution,
allowed developing new methods to detect and quantify large sets of peptides in complex mixtures, such as urine and blood
proteomic samples.
Quantification was performed either on the precursor ions, using the selection ion monitoring（SIM）mode, or on the fragment
ions resulting from collision dissociation（i.e. reaction monitoring mode）.
The high resolution capability in both MS and MS/MS modes allows a dramatic improvement of the measurement selectivity, byeffectively discriminating the signals of analytes from those of the background. In addition, the narrow m/z isolation window of thequadrupole, results in an increases of the dynamic range and the limit of detection, by gating only the ions of interest in thetrapping device. This mode of operation enables reliable quantification over a wide dynamic range.
The sample preparation consisted in the precipitation of the proteins present in the urine samples, followed by trypsin proteolysis.
The isotopically labeled synthetic polypeptide standards, comprising proteotypic peptides of the protein of interest and auniversal reporter peptide were spiked into the sample prior to the proteolysis. Analyses were performed on a nano-HPLC system
coupled with a hybrid quadrupole-orbitrap instrument（Q-Exactive, Thermo Scientific, Bremen）.
The MS/MS experiments were used to conduct in a targeted（SRM-like）mode, to quantitative reference peptides spiked in urine
and plasma samples, and thus precisely measure the corresponding endogenous peptides. Various combinations of quadrupole
isolation windows and orbitrap resolution settings were evaluated to determine their effect on the selectivity of themeasurements. Other parameters such as the collision energy were also evaluated. The actual quantification was carried out postacquisition by extracting the ion chromatograms of specific fragment ions. The co-elution of the traces was used to assess the“purity“ of the ions measured, and a reference library of MS/MS spectra was used to systematically confirm the identity of thepeptides analyzed.
The performance of SRM-like quantification was determined using urine samples, and benchmarked with the results obtained by
SRM technique on triple quadrupole instrument. Experiments were performed on ten clinical samples spiked with a defined
amount of isotopically labeled peptides representing four endogenous proteins. Time-scheduled experiments were performed on
both instruments using the same precursor ion isolation window. The selectivity improvement due to higher resolution allowed abetter discrimination of interferences in the MS/MS spectra. To scale-up the number of peptides targeted in one single LC-MS/MS
analysis, acquisition parameters were adapted to keep a low duty cycle. It included relaxing the resolving power, and selecting
several peptide precursors to generate a composite fragment ion spectrum（i.e. multiplexing capabilities）. The quality of themeasurements was assessed in a second study comprising 200 isotopically labeled reference peptides spiked into urine tryptic
digests. Experiments with different resolution（17k-70k）and quadrupole isolation windows（ 1 Th-100Th）were performed, including
the acquisition in data independent mode. The selectivity for each set of experimental parameters provided a baseline for theestablishment of acquisition methods generally applicable. The approach was applied to the precise quantification of potential
biomarkers of recurrence of bladder cancer in urine and serum samples.
These results demonstrate the benefit of using the high resolution, accurate mass to increase the selectivity of quantitative assays
in very complex matrices by a better discrimination of the interferences. The recent development of the acquisition software
enabled the very precisely analysis of more than 500 peptides in one single experiment, thus dramatically easing the verification
step of biomarkers studies.

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 6: Novel Approaches in Proteomics Analysis

PMo-116

13:30 － 14:40

Using Selected Reaction Monitoring to determine cell and drug specificprofiles of caspase cleaved substrates in apoptosis

Kazutaka Shimbo1,2, Gerald W Hsu2, Huy Nguyen2,3, Sami Mahrus2, Jonathan C Trinidad2,
Alma L Burlingame2, James A Wells2,31
Institute for Innovation, Ajinomoto Co., Inc., Kawasaki, Japan, 2Department of Pharmaceutical Chemistry, University ofCalifornia, San Francisco, San Francisco, CA, 3Department of Cellular and Molecular Pharmacology, University ofCalifornia, San Francisco, San Francisco, CA

Keywords:
apoptosis, caspase, proteomics, quantification, selected reaction monitoring
Novel aspects:
Quantitative proteomics, SRM coupled with N-terminomics, approach could determine caspase-cleaved peptide profiles indifferent cancer cell lines and inducers of apoptosis. These may aid in the development of specific biomarkers.
Abstract:
Introduction
Apoptosis is a conserved pathway and has important roles in all metazoans. Dysfunction in apoptosis is critical to cancer cell
survival and most cancer chemotherapies promote apoptosis. Apoptosis can be triggered by two pathways, extrinsic and/or
intrinsic pathways. Caspases, cysteine-class proteases, are activated during apoptosis and cleave substrates after aspartic acidresidues to deconstruct the cell. Different cancer drugs can induce apoptosis through different substrates and pathways, forinstance, inhibiting protein kinases, topoisomerases or inhibiting the proteasome to induce the cellular stress. Moreover different
cell lines show different response to these drugs for reasons that are often poorly understood. Here, we used a label-freequantitative proteomics approach called selected reaction monitoring（SRM）coupled with a positive enrichment N-terminomics
approach to determine caspase-cleaved peptide profiles in different cancer cell lines and different inducers of apoptosis.
Methods
Three hematologic malignancy cell lines, T-cell leukemia（Jurkat cell line）, B-cell lymphoma（DB cell line）, and plasma cell
multiple myeloma（MM 1 S cell line）, were used for these experiments. Cells were grown to uniform density and treated withstaurosporine（DB, Jurkat and MM 1 S）and either doxorubicin（DB and Jurkat）or bortezomib（MM 1 S）. Cell viability and caspase
activity were monitored during the cultivation to control for different rates of cell death. In the initial discovery phase, cells were
harvested at approximately 25% and 50% cell viability, and enriched for N-terminal peptides（Mahrus et al, Cell. 2008, 134, pp866876）. Enriched samples were pooled, fractionated and analyzed by conventional LC/MS/MS（QSTAR Elite, AB/Sciex）and proteinsand peptides were identified using ProteinProspector（University of California, San Francisco）. The putative SRM transitions weregenerated from MS/MS spectrum of identified caspase-cleaved peptides. For the relative quantification by SRM, cells were treatedwith three different drugs（staurosporine, bortezomib and doxorubicin or MG132）and harvested at multiple time points between
100% viability and near complete death（ 0 % viability）. SRM（QTRAP 5500, AB/Sciex）was performed and data were processedusing in-house perl scripts. Internal standards were included to control for experimental and analytical variability and permit
relative quantitative comparisons of peptide intensity.
Results and Discussions
From the discovery runs using pooled samples, we identified a total of 2083 α- amine containing peptides from 1256 proteins.
About one-third（674/2083）of cleavages followed aspartic acid, which were putative caspase cleavage sites, and 800 peptides（including different charge states, missed cleavages by trypsin, and methionine oxidations）were selected to generate SRM
transitions. Interestingly, 90% of peptides could be detected at some level in all three cell lines using SRM analysis of individual
apoptotic cell lysates from the discovery experiment. However, there are large differences in signal intensities of peptides
depending on cell type and inducer. For example, in MM 1 S cells treated with the proteasome inhibitor bortezomib, levels ofactivating transcription factor- 4 （ATF 4 ）increase dramatically early in drug treatment and then decrease upon cleavage byactivated caspases. Thus, caspase-derived cleavage products are a sensitive reflection of cell-type and drug-induced stress, andprovide useful fingerprints for mechanism of drug action and response.
In summary, these studies reveal the remarkable sensitivity of caspase cleavage profiles for drug and cell-type. These data may aid
in the development of specific apoptotic biomarkers for determining drug action, cell-type specific cell death, and possibly new
targets and pathways for cancer drug treatments.

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 6: Novel Approaches in Proteomics Analysis

PMo-117

11:10 － 12:20

Rapid enzyme digestion system consisting of microwave and air bubblemediated cooling.

Hyunjung Seo1, Dong Min Kim1,2, Chang Won Park1,2, Yangsun Kim1,2
1

HUDSON SURFACE TECHNOLOGY, FORT LEE, NJ, USA, 2APPLIED SURFACE TECHNOLOGY INC., SUWON, KOREA

Keywords:
microwave, cooling, proteomics, digestion, glycan
Novel aspects:
enzyme reaction in the microwave system combined with air bubble-mediated cooling can drastically reduce reaction time whilestill maintaining the efficiency of the enzyme digestion.
Abstract:
Efficient enzymatic digestion of proteins is critical in the analysis of protein samples, in particular when global proteomic analysis
is required. However, the traditional water-bath enzymatic digestion has always been the major rate-limiting step in the sample
processing for proteomic analysis. In recent years, microwave has been applied to protein digestions, and demonstrated very
rapid completion of most digestion reactions within several minutes. In this study, the new microwave-assisted enzyme digestion
system was developed, employing a reaction chamber equipped with water-bubbling system by air supply. Air bubbles in the
microwave chamber keep on slightly decreasing the temperature of the chamber, leading to the continual irradiation of thechamber by microwave pulses, which, in combination with air bubble-mediated cooling system, will maintain the repetitive
radiation of microwave pulses and constant temperature in the chamber. This combination of microwave pulses and coolingsystem by air bubbles controlled the water chamber temperature with variation less than 0.5 &#8451；. We tested in-solution andin-gel digestion of BSA and transferrin with several enzymes such as trypsin and chymotrypsin. The enzymatic release ofoligosaccharides（N-linked glycans）from glycolproteins was also attempted with PNGase F in the microwave system. All the
enzyme reactions were carried out with 400W of microwave power output & cooling air-bubble system at 37 &#8451； for 10 min.
The efficiency of trypsin or chymotrypsin digestion was determined by MALDI-TOF mass spectrometry（MS）with matrix prespotted plates. The homogenous and continual microwave radiation throughout the reaction time significantly reduced thenumber of miss-cleavaged peptides in protein digestion. The sequence coverage of more than 80 % by including one misscleavaged peptides of BSA has been achieved in most of repeated experiments with air-bubbling microwave water chamber. The
constant irradiation by microwave pulses significantly reduced the miss-cleavaged peptides from BSA and transferrin when
peptide mass fingerprinting by MASCOT search was used for the analysis. In contrast, microwave irradiation which was notconstant and uniform mostly led to the incompletion of reaction. We analyzed multiple mass spectrometry spots on the sample
plates, and the MS data were very similar among identical spots, indicating the consistency of data analysis. The digestion
efficiency from 10-min reaction with microwave system in combination with air bubble-mediated cooling reached almost identicallevel of efficiency obtained from conventional overnight digestion in regular water-bath. N-linked glycans released by PNGase F
were also analyzed by MALDI-TOF MS, demonstrating that just 10-min digestion reaction in the microwave chamber with bubblemediated cooling system was enough to result in the digestion efficiency similar to that acquired from overnight reaction using
conventional incubators. These results point out that enzyme reaction in the microwave system combined with air bubblemediated cooling can drastically reduce reaction time while still maintaining the efficiency of the enzyme digestion.

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 6: Novel Approaches in Proteomics Analysis

PMo-118

13:30 － 14:40

Biomarker Discovery and Validation with Nonglycosylated Tryptic
Peptides from N-linked Glycoproteins in Human Plasma

Ju Yeon Lee1,2, Jin Young Kim1, Gun Wook Park1,4, Kyung-Hoon Kwon1, Yeong Hee Ahn1,
Myeong Hee Moon2, Young Ki Paik3, Jong Shin Yoo1,41
Korea Basic Science Institute, O-chang Campus, Chungcheongbukdo, Korea, 2Department of Chemistry, Yonsei
University, Seoul, Korea, 3Yonsei Proteome Research Center and Biomedical Proteome Research Center, Department ofBiochemistry, Yonsei University, Seoul, Korea, 4Graduate School of Science and Technology, Chungnam National
University, Daejon, Korea

Keywords:
plasma, N-linked glycoproteins, label-free quantification, multiple reaction monitoring（MRM）quantification.
Novel aspects:
This work provides a targeted means of discovering and validating non-glycosylated tryptic peptides as biomarkers in humanplasma, without the need for complex enrichment processes or expensive antibody preparations.
Abstract:
A simple mass spectrometric approach for the discovery and validation of biomarkers in human plasma was developed bytargeting non-glycosylated tryptic peptides adjacent to glycosylation sites in an N-linked glycoprotein, one of the most importantbiomarkers for early detection, prognoses, and disease therapies. The discovery and validation of novel biomarkers requires
complex sample pre-treatment steps, such as depletion of highly abundant proteins, enrichment of desired proteins, or thedevelopment of new antibodies. The current study exploited the steric hindrance of glycan units in N-linked glycoproteins, whichsignificantly affects the efficiency of proteolytic digestion if an enzymatically active amino acid is adjacent to the N-linked
glycosylation site. Proteolytic digestion then results in quantitatively different peptide products in accordance with the degree of
glycosylation. The effect of glycan steric hindrance on tryptic digestion was first demonstrated using alpha- 1 -acid glycoprotein（AGP）as a model compound versus deglycosylated AGP. Second, nonglycosylated tryptic peptide biomarkers, which generally
show much higher sensitivity in mass spectrometric analyses than their glycosylated counterparts, were quantified in humanhepatocellular carcinoma（HCC）plasma using a label-free method with no need for Nlinked glycoprotein enrichment. Finally, themethod was validated using amultiple reaction monitoring analysis, demonstrating that the newly discovered non-glycosylated
tryptic peptide targets were present at different levels in normal and HCC plasmas. The area under the receiver operating
characteristic（ROC）curve generated through analyses of non-glycosylated tryptic peptide from vitronectin precursor protein was
0.978, the highest observed in a group of patients with HCC. This work provides a targeted means of discovering and validating
non-glycosylated tryptic peptides as biomarkers in human plasma, without the need for complex enrichment processes or
expensive antibody preparations.

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 6: Novel Approaches in Proteomics Analysis

PMo-119

11:10 － 12:20

The application of biomarker discovery for atopic dermatitis andnormalized spectral index based label free quantitation

Ki Na Yun1, Gun Wook Park1, Hye Kyoeng Min1, Jong Shin Yoo1, Seongmin Noh2, Chang Ook Park2,
Kwang Hoon Lee2, Jing Young Kim1

1
Korea Basic Science Institute, Ochang, Korea, 2Brain Korea 21 Project for Medical Science, Yonsei University College ofMedicine, Seoul, Korea

Keywords:
Proteomics, label free, Biomarker, atopy
Novel aspects:
We applied label free quantification to discover biomarker for the development of therapeutic agent in atopic dermatitis.
Abstract:
Quantitative proteomics measures abundance changes of many proteins among multiple samples in a high-throughput manner.
Results from such measurements provide information on how biological systems respond to environmental perturbations at a
genomic scale. A number of methods have been developed for quantitative proteomics to obtain high proteome coverage,
accurate quantification, and wide applicability to different types of samples. Here, we used normalized spectral index based label
free method for protein quantification. Label-free quantification can be achieved by correlating protein abundance with mass
spectrometric signal intensities of peptides and the number of MS/MS spectra matched to peptides and proteins. In terms ofaccuracy, precision, and reproducibility, quantification performance was tested by using standard proteins samples. We comparednormalized spectral index based label free quantitation with MS chromatogram based label free quantification. We applied thismethod to discover biomarker for the development of therapeutic agent in atopic dermatitis.

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 6: Novel Approaches in Proteomics Analysis

PMo-120

13:30 － 14:40

Multidimensional LC-MALDI Workflow for High Sensitivity Detection of
Low-Abundance Peptides from Complex Samples

Shinichi Iwamoto1, Yusaku Hioki1, Ritsuko Tanimura1, Yuki Ohta1, Takashi Shimada2, Koichi Tanaka1

Koichi Tanaka Laboratory of Advanced Science and Technology, Shimadzu Corporation, Kyoto, Japan, 2Life Science
Research Center, Shimadzu Corporation, Tokyo, Japan

1

Keywords:
Multidimiensional LC-MALDI, Spotting robot, Column-integrated probe, PTM analysis
Novel aspects:
A novel spotting robot for the MALDI target was developed for the high sensitivity nanoflow LC-MALDI analyses and applied tothe off-line multidimensional LC-MALDI workflow.
Abstract:
Introduction:
The discovery of protein biomarkers is important for early detection and elucidation of disease diagnosis. However, it is not easy
to detect extremely small quantity of peptides from highly complex samples such as serum or plasma without effective
fractionation and enrichment strategies. A proteomic strategy based on multi-dimensional liquid chromatography（MDLC）
combined with mass spectrometric analysis is essential approach for the effective detection of such kind of low-abundancebiomarkers. The purpose of our study is to construct an extremely high sensitivity MDLC and matrix-assisted laser desorption/
ionization mass spectrometry（MALDI-MS）workflow available as a proteomics platform for the discovery of protein biomarkers
and post-translational modifications（PTM）. Recently, we developed a prototype of a spotting robot on MALDI target, which issuitable for the high sensitivity nanoflow LC-MALDI analyses. In this presentation, we demonstrate and discuss the advantages ofthe off-line MDLC-MALDI workflow.
Methods:
The prototype of the novel MALDI spotter instrument was designed for the highly accurate collection of nanoliter fractions eluting
from nano-LC system on MALDI targets to support 1536 well format. This spotter is equipped with a laser sensor for high-precisionz-axis positioning with an overall error of 15 mm. The probes were constructed from 0.050 mm i.d. 0.150 mm o.d. x 150 mm length
fused silica capillary tubing. Cadenza ™ CD-C18 resin（Imtakt, Japan）was self-packed into the capillary with sol-gel frit. The
2,5-dihydroxybenzoic acid（DHB）precoated targets were prepared using piezoelectric inkjet device. MALDI-QIT-TOF MS（AXIMA
Resonance™； Shimadzu/Kratos, UK）measurement was performed in positive ion mode.
Results:
The newly designed MALDI spotter is fully compatible with nano-LC system. The outside of column probe tip coated with ahydrophobic material allows various kinds of LC eluents including an acetonitrile gradient to deposit stably on a MALDI target. The
performance of MDLC-MALDI system has also been evaluated in terms of a high selectivity and sensitivity detection of the PTMcontaining peptides. To optimize peptides separation, hydrophilic interaction chromatography（HILIC）mode was used because of
its orthogonality to 3 -dimensional-reverse phase mode. The ion-exchange phase was also used to separate other sets ofmolecules which have similar hydrophobic/hydrophilic properties. The final column eluents were directly spotted on DHB
precoated targets from capillary column-integrated probe using the nanoliter spotting robot to prevent post-column diffusion. The
peptides with PTMs spiked in highly complex sample were reproducibly detected using our MDLC-MALDI system. The
combination of MALDI-MS and MDLC is a powerful approach for the highly sensitive analysis of low-abundance peptides andtherefore this is an effective breakthrough in the sample complexity in the field of proteomics or PTM analysis.

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 6: Novel Approaches in Proteomics Analysis

PMo-121

11:10 － 12:20

LC-ESI/MS analysis of protein modifications derived from a mixture of
13-HPODE and [13C18]-13-HPODE

Ryo Takahashi, Seon Hwa Lee, Takaaki Goto, Tomoyuki Oe
Tohoku University, Sendai, Japan

Keywords:
protein, modification, lipid, hydroperoxide, LC-ESI/MS
Novel aspects:
We have employed a mixture（ 1 ： 1 ）of 13-HPODE and［13C18］-13-HPODE to facilitate the detection and characterization ofprotein modifications by LC-ESI/MS.
Abstract:[Objective] Many studies have implicated oxidative stress as a substantial contributor to the degenerative diseases of aging,cancer, and cardiovascular diseases. Oxidative stress can induce the production of reactive oxygen species（ROS）and theactivities of cyclooxygenases and lipoxygenases. They can oxidize polyunsaturated fatty acids （PUFAs） to form lipidhydroperoxides that undergo decomposition to highly reactive bifunctional electrophiles such as 4 -oxo- 2 （E）-nonenal（ONE）
and 4 -hydroxy- 2 （E）-nonenal（HNE）. These aldehydes are involved in the inflammation, apoptosis and other cellular signaling
through modification and cross-linking of the related proteins. Because of the difficulties in dealing with complex systems, mostprevious studies have focused primarily on either protein modification by a single electrophile or structural characterization of
specific modifications on a model amino acid/peptide. It is now important to conduct a comprehensive study of the protein
modifications results from various oxidation products of PUFAs. To develop the methodology of the global screening for lipidhydroperoxide-derived protein modifications, we have employed angiotensin II（Ang II： DRVYIHPF）as a model peptide. It is awell-known bioactive peptide in the renin/angiotensin system and has been found to be readily modified by reactive aldehydes
such as ONE and HNE [1]. As for the source of electrophiles, 13-hydroperoxy-（Z,E）-9,11-octadecadienoic acid（13-HPODE）was
prepared from the linoleic acid（LA）, the most abundant PUFA in mammalian tissue.［13C18］-13-HPODE was also prepared and a
mixture（ 1 ： 1 ）of 13-HPODE and［13C1  8］-13-HPODE was reacted with Ang II to facilitate the detection and characterization of
Ang II modifications by liquid chromatography（LC）-electrospray ionization（ESI）/mass spectrometry（MS）. Thus, any modified
Ang II derived from 13-HPODEs will be observed as a doublet（i.e. m：m+ 9 for ONE- or HNE-derived modification）in the mass
spectrum with identical retention times by LC-MS. This will allow to develop the pseudo neutral loss scan method for an effective
screening.
[Experiments] 13-HPODE and［ 13 C 18 ］-13-HPODE were prepared using soybean lipoxidase from linoleic acid and［ 13 C］-linoleic
acid, respectively. 13-HPODE and Ang II were reacted in the presence of ascorbic acid（AA）or AA/CuII and analyzed to optimize
the LC/MS condition. A mixture（ 1 ： 1 ）of 13-HPODE and［13C18］-13-HPODE was then reacted with Ang II in the presence of AA
or AA/CuII and analyzed using an optimized LC-ESI/MS conditions. LCQ-DECA（Thermo Scientific Inc.）equipped with ESI source
was employed for the analysis of the reaction mixture. The chromatographic separation of reaction products was carried out on a
SunFire C18 column（150 × 2.1 mm i.d., 3.5 μm； Waters, Milford, MA）with water and acetonitrile containing 0.1% formic acid as
mobile phases at 0.2 mL/min.
[Results & Discussion] The reaction conditions to generate 13-HPODE and［13C18］-13-HPODE from linoleic acid and［13C］-linoleic
acid, respectively, were optimized. They were isolated using normal phase HPLC system, and each concentration was determined.
The oxidative condition for the reaction between 13-HPODE and Ang II was then optimized. The LC/MS condition was also
optimized. It was revealed that each electrophiles-modified Ang II and the corresponding［13C18］-labeled one elute at the same
retention time. The modified AngIIs derived from 13-HPODEs were confirmed by doublets in their mass spectra. The
comprehensive pseudo neutral loss scan method will then be developed based on the current results for global screening ofprotein targets of electrophiles.
［ 1 ］Lee, S. H., Takahashi, R.,  Goto, T., and Oe, T., Chem. Res. Toxicol. 23, 1771-1785（2010）.

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 6: Novel Approaches in Proteomics Analysis

PMo-122

13:30 － 14:40

Systematic investigation of peptide fragmentation processes in HCD on an
Orbitrap instrument using a computer Expert System

Annette Michalski, Neuhauser Nadin, Jurgen Cox, Matthias Mann
Max-Planck-Insitute of Biochemistry

Keywords:
automated MS/MS annotation peptide fragmentation
Novel aspects:
Peptide fragmentation knowledge can be successfully applied to large-scale datasets in an automated manner providing a uniqueinsight into the composition of collision induced fragmentation spectra.
Abstract:
MS-based Proteomics projects often produce millions of MS/MS spectra containing information about tens of thousands ofsequence unique peptides and their modifications. Peptide and protein identification based on database searches are largely
automated and obtaining the peptide sequence from regular fragment ions seems straightforward. While the investigation ofdifferent peptide fragmentation mechanism has been of great interest to the mass spectrometric community for decades, thisknowledge has not been applied to large-scale studies and it is difficult to access for biological researchers using proteomics as atool. The latest generation of mass spectrometers that provides high resolution and high mass accuracy for both MS and MS/MS
scans（’high-high-strategy’）allows measuring the peaks in MS/MS spectra with high mass accuracy - for low m/z even revealing
the chemical compositions. Here we develop a computer-assisted Expert System that incorporates much of the knowledge about
peptide fragmentation pathways and we validate it by applying it to a statistical investigation into the nature of higher energy
collisional dissociation（HCD）and collision induced dissociation（CID）fragmentat ion spectra.
We acquired data from digested E.coli, yeast and HeLa cell lysate on LTQ Orbitrap Velos and LTQ Orbitrap Elite mass
spectrometers（Thermo Scientific）. Peptide identification was carried out with MaxQuant and the Andromeda search engine witha strict FDR of 0.0001. Our dataset initially contained more than 100,000 high scoring and unique tryptic peptides but was further
filtered to obtain greater than 16,000 very high-quality MS/MS spectra nearly free of co-eluting peaks. The MS/MS spectra werethen comprehensively annotated by an Expert System that was iteratively developed together with this study.
The major ion types that we found in our study can be classified into 6 different categories： regular ions that result fromcleavage of the peptide back bone（a-, b- and y-type ions）, neutral loses, internal fragments, immonium ions, side chain fragments
and the intact precursor mass. Strikingly, we find the amino acid coverage of our collection of tryptic peptides to reach 80% for a
peptide length of up to 20 amino acid in total. This demonstrates high fragmentation efficiency and the potential for de novo
sequencing. While HCD spectra in general feature highly complete y-ion series but only low mass b-ion series, in CID spectra theb-ion series also contributes substantially to the sequence information content. In contrast, there are many internal ions in HCD
spectra（10% of the intensity coverage）, but these are nearly absent in CID spectra. Likewise, in HCD spectra 6 % of the intensity
coverage is accounted for by immonium ions and side chain fragments are also specific and prominent in the low mass region.
Our study demonstrates that peptide fragmentation knowledge can be successfully applied to large-scale datasets by our
computer-assisted Expert System at a level of quality comparable or better than a human expert. This allows the expert focussing
on very specific problems such as the discovery of novel fragmentation pathways.

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 6: Novel Approaches in Proteomics Analysis

PMo-123

11:10 － 12:20

Intraocular Lens Adsorbome: a Proteomic Study of Adsorbed Proteins
onto Acrylic Materials and Its Implication in Secondary Cataract

Yi-Shiang Huang1,2, Virginie Bertrand1, Gabriel Mazzucchelli1, Dimitriya Bozukova3,
Christophe Pagnoulle3, Marie-Christine Durrieu2, Edwin De Pauw1, Marie-Claire De Pauw1
1

University of Liege, Liege, Belgium, 2University of Bordeaux 1, Bordeaux, France, 3PhysIOL, Liege, Belgium

Keywords:
Intraocular lens, Acrylic hydrophobic, Acrylic hydrophilic, Protein adsorption, Posterior Capsular Opacification
Novel aspects:
We illustrate the relationship between the absorbed proteins and the developement of secondary cataract. This finding will help
scientists to design a new intraocular lens with lower secondary cataract rate.
Abstract:
The intraocular lens（IOL）is a polymer implant designed to replace the natural lens after cataract surgery. When the implant isintroduced into the lens capsule, the polymer starts to interact with the aqueous humour and the exchange of molecules between
the solid and the liquid begins. The nature of exchange in water, ions, and biomolecules may result in several postoperativecomplications including glistening, calcification, and posterior capsular opacification. The posterior capsular opacification（PCO,
also called“Secondary Cataract“）is raised from the over-growth of residual lens epithelial cells. The first step of the over-growth
process of the cells is their adhesion to the deposited biomolecules, such as proteins involved in extra-cellular matrices.
The purpose of this study is to identify the principal proteins adsorbed onto the acrylic polymers by mass spectrometry. The
concept of adsorbome is to generate a list of adsorbed proteins to the hydrophilic and hydrophobic polymers, and then compare
the difference to the original component of aqueous humour in order to see the affinity of individual protein to each material. Twokinds of hydrophilic and two kinds of hydrophobic acrylic polymers were tested for their adsorbomes by treating them with anaqueous humour analogue and the major adsorbed proteins were identified by mass spectrometry. Interestingly, the hydrophilicacrylic polymer shows a relative lower protein adsorption rate but shows a higher incidence of secondary cataract. Thisphenomenon implies the adsorbed proteins play a crucial role in progress of secondary cataract.

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 6: Novel Approaches in Proteomics Analysis

PMo-124

13:30 － 14:40

Analysis of SOD in cell line HepG2 by LC-nanospray-MS for screening
natural antioxidants

Shih-Ying Dai, Kuo-Lung Ku

National Chiayi University, Chiayi City, Taiwan

Keywords:
LC-nanospray-MS, proteotypic peptide, absolutely quantity, screening antioxidant activity
Novel aspects:
A cheap, low dose and label-free absolutely quantitation by LC-nanospray-MS is established and tested for CuZn-SOD in HepG2.
Abstract:
Generally, artificial free radicals, DPPH or ABTS are employed in vitro to screening antioxidant activity in natural resources.
However, it is hard to infer directly the obtained free radical scavengers, neither the metabolites of the scavengers possessing
antioxidant activity in vivo. Therefore, that is why bio-guided screening approaches are still in use, although they are cost and highdose of testing sample needed. In the present study, we developed an efficient bio-guide assay by detecting CuZn-SOD in HepG 2
using LC-MS for screening antioxidants. The novel aspect of study is cheap, low dose and label-free absolutely quantitation . We
adopted trypsin to hydrolyze CuZn-SOD of HepG 2 and found good ion efficiency peptide sequences by LC-nanospray-MS. It issimply the recognition of the fact that not all of the peptides are equally likely to be detected by current mass spectrometrybased techniques. Therefore, we selected proteotypic peptide that is always detected and its analogue to synthesize. The
analogue peptide sequence has been replaced one amino acid to anticipate that have similar ion efficiency to proteotypic
peptide. These two synthesized peptides were used to acquire the absolutely quantity of CuZn-SOD in HepG2. And the CuZnSOD in HepG 2 was in different treated by resveratrol. The quantity of CuZn-SOD was evaluated by the induced resveratrol .
Nevertheless, if quantity of resveratrol adding in HepG 2 was too high, the CuZn-SOD containing in cell line HepG 2 would havedecreased. Furthermore, the technique should be applicable for screening widely natural antioxidants.

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 6: Novel Approaches in Proteomics Analysis

PMo-125

11:10 － 12:20

Comparing and validating protein expression in multiple experimental
conditions of antiviral immune response using clustering techniques onlabel-free LC-MS/MS data

Aivett Bilbao1,2, Ying Zhang1, Dario Bottinelli3, Bandar Alghanem1, Frederic Nikitin2, Markus Mueller2,
Frederique Lisacek2, Jeremy Luban3, Caterina Strambio De Castillia3, Emmanuel Varesio1,
Gerard Hopfgartner1

Life Sciences Mass Spectrometry, School of Pharmaceutical Sciences, University of Geneva, University of Lausanne,
Geneva, Switzerland, 2Proteome Informatics Group, Swiss Institute of Bioinformatics, Geneva, Switzerland, 3Departmentof Microbiology and Molecular Medicine, University of Geneva, Geneva, Switzerland

1

Keywords:
LC-MS/MS, bioinformatics, label-free quantification, data quality control, analytical proteomics
Novel aspects:
Development of an alternative data quality control method to compare and validate protein expression across multiple
experimental conditions
Abstract:
Classical vaccination approaches capable of controlling viruses have not worked for HIV-1. Several studies recently publishedindicate that mammalian cells have response mechanisms to HIV- 1 through activation of innate sensors. The central coordinatorsof innate and acquired immune responses are the dendritic cells（DCs）and little is known about how they can detect retroviruses
and help mounting an effective protective immunity against HIV-1.
A study applying a system biology approach capable of exhaustively exploring the protein landscape and the complexity of the
innate immune recognition pathways in the DCs represents an opportunity to unravel the mechanisms by which these cells
determine disease outcome and may provide important insights for the design of HIV therapies and vaccines.
Mass spectrometry has turned into the analytical technique widely used for data collection in proteomics research. At the sametime, bioinformatics supplies computational methods for protein identification and quantification in these large-scale experimental
data sets.
In this project, primary human DCs generated from peripheral blood monocytes of individual healthy donors（monocyte-derived
DCs or moDCs）have been analyzed using liquid chromatography combined with tandem mass spectrometry（LC-MS/MS）
instrumentation. These cells either elaborate a potent, innate, antiviral state that suppresses HIV- 1 infection to undetectablelevels or they are rendered completely permissive for HIV- 1 infection.
As an initial screening, we are applying the bottom-up shotgun MS/MS workflow： after cell lysis of the moDCs samples, protein
samples are digested and the label-free samples are analyzed using data-dependent（IDA）or data-independent（SWATH）
acquisition schemes on a QqTOF mass spectrometer（with a resolving power of 30,000）. Then, standard peptide fragment
fingerprinting（PFF）tools such as MASCOT and ProteinPilot are used for protein identification.
We are currently exploring approaches to compare and validate the protein expression level consistency across different donors
and across different replicates（within the same donor）, as well as the protein expression level variations as a result of viral
infection.
Preliminary results have shown some variability in the protein expression content among the multiple experimental conditions；however, to quantify and perform similarity analysis on these complex biological mixtures supporting the investigation of theantiviral response mechanisms is a difficult task.
To achieve this, we are developing a novel approach that consists in clustering the samples according to the different
experimental conditions. Each sample is represented by a fixed-length vector where each dimension corresponds to a protein（from the list of identified proteins）and the value for the corresponding dimension is an estimated quantification. Different
identification results and different label-free quantification strategies such as spectral count and MS 1 -based profiling（e.g.
generated by SuperHirn）will be investigated using several clustering algorithms and similarity measures. In addition, we plan toextend the analysis to peptides and study post-translational modification profiles.
We expect the results to indicate whether the protein/peptide quantification content properly characterizes these multiple
experimental conditions in order to： i）validate the protein expression level consistency intra and inter donors under the same
condition（i.e. grouped in the same cluster）, and ii）identify possible distinctive patterns in each of the different conditions（i.e.
samples under different conditions clearly separated in different clusters）.
The usage of such method as an alternative data quality control process is an important step in our project in order to reduce
artefacts induced by individual variability and reliably categorizes the list of differentially expressed candidate proteins andsignature peptides of the complex mixture under several basal and perturbed experimental contexts. Items of the output list will
be further validated and characterized to create and refine models describing the biological pathways under study.

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 6: Novel Approaches in Proteomics Analysis

PMo-126

13:30 － 14:40

Identification of a modified amino acid residue in the heme protein using
LC/MS/MS

Jun Watanabe1, Yuki Ando2, Keiko Matsumoto1, Junko Iida1, Shun Hirota2
1

Shimadzu Corporation, Kyoto, Japan, 2Nara Institute of Science and Technology, Nara, Japan

Keywords:
LC/MS/MS, peptide, triple quadrupore
Novel aspects:
Modified amino acid sequence identified using ultra fast LC/MS/MS scanning with multiple collision energy events.
Abstract:
An amino acid in a heme protein can be modified by reaction with hydrogen peroxide, resulting in loss of physiological activity ofthe protein. Additionally, it has been reported that the insertion of tryptophan near the heme in myoglobin can be oxidized by anorganic acid peroxide.
In this study, we found that a methionine residue is modified in a mutant of another heme protein. However, among the peptide
fragments obtained by the peptidase digestion of the modified protein, two peptides were present with the same molecularweight, containing methionine and constructed with the same set of amino acids. From this we identified the modified amino acidin the mutant protein using LC-MS/MS.
The mutant of the heme protein used in this study was a mixture of modified and non-modified proteins, with about 50% of thenative protein oxidized. After digesting the mutant of the heme protein with trypsin, the obtained peptide fragments wereanalysed by mass spectrometry. The sample was analyzed using a double-focusing mass spectrometer with electrosprayionization. Then LC-MS/MS analysis was performed using UHPLC（Nexera, Shimadzu Corporation, Japan）and a triple quadruple
mass spectrometer（LCMS-8030, Shimadzu Corporation, Japan）. Separation was achieved using an ODS column, Shim-pack XRODS II（150 x 2.0 mm, 2.2um）. The sample was eluted at 0.2 mL/min with a binary gradient system and analysed by MS/MS in
positive ion electrospray.
The tryptic digested sample of the heme protein was analyzed by ESI-MS. Peptide F 2 （sequence： IFIMK）and peptide F 8（sequence： MIFIK）at m/z 651（M+H）differed in sequence. Peak at m/z 667, which was 16u larger than the original peak is likely
to be due to the oxidized species of peptide F 2 or F8. MS/MS analysis of two products at m/z 326（M+ 2 H）corresponded to themass of the peptides F 2 or F8, and the peak at m/z 334（M+ 2 H）to the modified peptide. In order to analyze the sequence of thepeptide exhibiting these peaks, product ion scan（MS/MS）measurements were performed with three collision energies（CE=10,
20, 30V）and a high scan rate（15000u/sec）. The results of product ion scan data showed that the two peaks at m/z 326 were F 2
and F8, respectively, and that the peak of m/z 334 was F 2 （IFIMK）.

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 6: Novel Approaches in Proteomics Analysis

PMo-127

11:10 － 12:20

Population proteogenomics: qualitative and quantitative analysis of SNP
and protein markers for obesity from plasma proteome

Nam Young Hong1, Jin Nyoung Choi1, Sarah Yang2, Joohon Sung2, Kwang Pyo Kim1
1

Konkuk University, Seoul, Korea, 2Seoul National University, Seoul, Korea

Keywords:
single nucleotide polymorphism, SNP, multiple reaction monitoring（MRM）, obesity
Novel aspects:
We developed proteomics pipeline to analyze genetic variations in the proteome level analysis for population scale.
Abstract:
With the completion of the Human Genome Project, more than 3 billion bases in the human genome were fully sequenced.
Among the human genome, 99.9 % of bases are exactly the same from person to person. Approximately 3 million bases in thehuman genome are different, and these variations make people unique. Single nucleotide polymorphisms（SNPs）are the mostcommon type of these genetic variations among people and the major genetic basis of physiological or pathological variants forindividuals. There are two types of SNPs within coding region： synonymous and non-synonymous. The non-synonymous SNPs（nsSNPs）result in variation of amino acid sequences and often have significant impact on functions of the corresponding proteins
and also phenotypes. However, little attention has been paid to study SNPs at the proteome level in population scale so far.
Therefore, in this study, we present a new proteomic analysis pipeline based on high-confidence Uni-prot annotation and proteinsthat were analyzed by shotgun proteomics strategy. In this study, we applied a targeted proteomic approach, multiple reaction
monitoring（MRM）, in total 300 plasma samples to quantitatively analyze and find associations between nsSNP and obesity at theproteome level in population scale.

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 6: Novel Approaches in Proteomics Analysis

PMo-128

13:30 － 14:40

Optimization of chemical derivatization of nitro-peptides for fluorous
solid-phase extraction.

Ji Hye Hong, Kyung-Cho Cho, Jeong Won Kang, Kwang Pyo Kim
Konkuk University, Seoul, Korea

Keywords:
nitration, Post-translational modification, Mass spectrometry,
Novel aspects:
To enhance enrichment efficiency of nitro-peptides, we optimized the blocking step of free amino groups on the nitro-peptides.
Abstract:
Protein tyrosine nitration is nitric oxide（NO·）dependant post-translational modification（PTM）associated with a number of
diseases and biological aging. To correlate protein tyrosine nitration mechanistically with specific physiological and pathologicalconditions, it is important to identify protein target sequences and to quantify protein- 3 -NT residues on these proteins. Since
protein nitration is a low-abundant post-translational modification, development of an effective enrichment method is needed todetect nitrated peptides or proteins from the limited amount of patho-physiological samples. The developed enrichment method
includes blocking, reduction, and specific chemical tagging steps of the nitro-peptide prior to capturing the derivatives withfluorine carbon tag. However, blocking by acetylation resulted the reduction of mass signal. As the blocked peptides withacetylated lysine on C- terminal have positive charge at amine group, they are induced to be neutralized during ionization in ESIMS. Thus, the choice of acetic anhydride as an acetylating agent results in fatal reduction of peptide ion signals during ionizationin MS. To overcome the problem associated with signal reduction due to acetylation, in this study we introduced dimethylation
step to block amino groups on tryptic peptides keeping their positive charge after the blocking reaction.
At first, we synthesized two artificial peptides with only differed C-terminal sequence. One had C-terminal lysine, the other had
C-terminal arginine as a control. Then two peptides go through the same processes of blocking, acetylation and dimethylation,
followed by analysis on ESI-MS. Our results indicate that the peptide with C-terminal lysine showed significant reduction of masssignal when used acetylation for blocking, however, we could show the mass signal gain when we use dimethylation as a
blocking.
We conclude that the blocking step of tryptic digested peptides using dimethylation is more efficient than acetylation without lossof signal for ESI-MS analysis.

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 6: Novel Approaches in Proteomics Analysis

PMo-129

11:10 － 12:20

Quantification of biological drugs in plasma by liquid chromatographytamdem mass spectrometry.

Nozomu Kato1, Takesada Shimura2, Atsuko Takeuchi4, Hisahide Nishio3
1
3

Kobe University, Japan / Mitsubishi Tanabe Pharma Co., Osaka, Japan, 2Mitsubishi Tanabe Pharma Co., Osaka, Japan,
Kobe University, Kobe, Japan, 4Kobe pharmaceutical University, Kobe, Japan

Keywords:
Quantification, biological drugs, LC-MS/MS
Novel aspects:
Quantification of biological drugs in plasma by liquid chromatography-tamdem mass spectrometry was performed.
Abstract:［Objectives］
In many cases, enzyme-linked immunosorbent assay（ELISA）methods are developed to quantify the biological active proteins inplasma samples. However, in early stage of drug discovery, ELISA will not be available for candidate proteins because of not to beprepared yet the specific antibody for ELISA. Then the discussion on the relationship between PK and PD might be skipped,though it is very important to make go/no go-decision even in early stage. Our objective is to establish the method for quantifying
the biological drugs in plasma and take PK information in animals, without a specific drug antibody, using liquid chromatographytandem mass spectrometry（LC-MS/MS）.
［Methods］
To indicate the usefulness of LC-MS/MS method, three types of biological drugs on market , Palivizumab as a whole IgG（Abbott）,
Etanercept as a fusion protein of TNFalpha-Receptor and Fc fraction（Pfizer）, and Peginterferon as a PEGylated IFN-alpha- 2 b（MSD）, were examined. Firstly, a specific peptide, which was necessary as a probe peptide for each protein to be detectable bymass-spectrometry, was selected from a portion of protein sequences using available tools and data base on the web site
（Expazy, NCBI or Swiss-prot）considering some defined conditions. The each specific peptide and its peptide with stable isotope（IS）for proteins were synthesized. Next the standard samples were prepared for calibration curve and QC. A specific peptide
and IS were spiked in the tryptic digested plasma and samples were injected to LC-MS/MS（UPLC-Quattro Premier, Waters）toquantify the specific peptide. A validation（recovery, linearity of calibration curve, intra-day and inter-day assays）were performed.
Finally, a single dose PK study in animals was carried out to examine the usefulness of LC-MS/MS method against ELISA method.
［Results and Discussion］
The validation results were satisfied our criteria for Palivizumab. This validated method was adapted to the PK studies of
Palivizumab（rat and monkey）. In results, the PK parameters are good correlated good correlated to previous reported values
determined by ELISA. For Etanercept the recovery % from standard samples was relative low, therefore Etanercept was added toplasma before tryptic digestion and digested in order to correct the recovery %. This protocol could provide results with goodprecision. The validation of Peginterferon is still under consideration.

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 6: Novel Approaches in Proteomics Analysis

PMo-130

13:30 － 14:40

Label-Free Semi-Quantitative Analysis of Histone Modification Dynamics
using High-Resolution Mass Spectrometer

Kazuki Yamamoto, Yoko Chikaoka, Takeshi Kawamura, Takao Hamakubo, Tatsuhiko Kodama

Laboratory for System Biology and Medicine, Research Center for Advanced Science and Technology, The University ofTokyo, Tokyo, Japan

Keywords:
LC-MS/MS； Label-Free semi-Quantitation； Histone Modifications； ETD； Cell Cycle
Novel aspects:
Improvement in sensitivity and resolution of proteomic analysis for histone modification dynamics.
Abstract:
Purpose： Improve methodology and workflow of histone modification analysis to identify function-dependent combinatorial
histone codes.
Background： Histone modification has been an attractive target for MS. Especially, two types of histones, H 3 and H 4 have many
modification sites（amino acid residues）which can be methylated（mono-, di-, tri-）, acetylated, phosphorylated, and more. These
modifications can occur in any combination and produce thousands of possible patterns. Different modification pattern does notnecessarily mean different mass or chemical property. Rather, the difference is often extremely subtle, so the good separation andthe fast high-resolution mass measurement are essential. In addition, increasing interest in epigenetics demands more sensitive
analysis. Histones carry rich epigenetic information in the form of posttranslational modifications on their N-terminal tailsequences, playing crucial roles in dynamic cellular function. Their combinations of modifications constitute the“histone code“
which regulates epigenetic events.
Methods： Our approach is label-free semi-quantitative LC/MS/MS to investigate time-course changes in multiple modification
patterns. For H 4 analysis, histone was extracted from cell-cycle synchronized HeLa S 3 cells, digested with Asp-N, and analyzed
with nano LC-MS/MS（Orbitrap ELITE ETD）. We used Progenesis software（Nonlinear Dynamics）for quantitative analysis. Data
were then subjected to multivariate analyses.
Results： With about 1 μg of histone proteins, we determined over 50 patterns of combination of modifications. Notably, thephosphorylation of serine in H 4 （H 4 S 1 ph）was well-quantified, which has been otherwise poorly detected or quantified so far. In
the cell-cycle synchronization experiments, we observed cell cycle phase-dependent ups and downs of H 4 S 1 phosphorylationlevel with good reproducibility, as well as H 4 K20 methylation changes described previously.
Conclusion： We have developed an improved methodology and workflow to analyze histone modification dynamics.

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 6: Novel Approaches in Proteomics Analysis

PMo-131

11:10 － 12:20

Simultaneous quantification of CYP1A2, 2D6, and 3A4 isoform proteins
by liquid chromatography-tamdem mass spectrometry using a single
internal standard peptide.

Nozomu Kato2, Tetsuya Narita1, Yasuhiro Yamada2, Atsuko Takeuchi1, Hisahide Nishio2
1

Kobe Pharmaceutical University, Kobe, Japan, 2Kobe University, Kobe, Japan

Keywords:
quantification, cytochrome P450, LC-MS, single internal standard peptide
Novel aspects:
Simultaneous quantification of CYP 1 A2, 2 D6, and 3 A 4 isoform proteins by liquid chromatography-tamdem mass spectrometry
using a single internal standard peptide was performed.
Abstract:［Objectives］
Nowadays, quantification of enzyme proteins by liquid chromatography-tamdem mass spectrometry（LC-MS/MS）is adopted
widely in evaluation of drug-drug interaction caused by drug-metabolizing enzymes. Typically, in quantifying each cytochrome P450
（CYP）isoform protein, a stable isotope-labeled peptide specific to each CYP isoform protein is utilized as an internal standard（IS）. In this method, a stable isotope-labeled peptide has to be synthesized for each isoform to be measured, which constitutes
a drawback. Therefore, we established a simultaneous quantification method using a single IS peptide for major three CYP（CYP 1 A2, 2 D6, 3 A 4 ）isoform proteins, which are important in the incipient period of drug discovery.
［Methods］
Probe peptides of CYP 1 A2, 2 D6, and 3 A 4 isoforms were selected from analysis data of Matrix Assisted Laser Desorption/
Ionization Time-of-Flight mass spectrometry（MALDI-TOF MS）and literature information. A peptide that does not exist in vivo wasused as a single IS peptide. By using these peptides, we measured content of each CYP isoform protein in individual（Lot HG3,
HG6, HG23, HG43, HG56, HG66, HG89, BD Gentest）or pooled human liver（Mix gender, Pool of 15, Lot PR09004, Xenotech）andintestinal microsomes（Mixed sex human, Pool of 6, Lot UGU, BD Gentest）as well as recombinant human（cDNA-expressed）CYP
isoform proteins（CYP 1 A 2 Lot 456203, CYP 2 D 6 Lot 456217, CYP 3 A 4 Lot 456202 ： baculovirus-expressed microsome, BD Gentest
and CYP 1 A 2 Lot C 1 A 2 R007/A, CYP 2 D 6 Lot C 2 D 6 R013/A, CYP 3 A 4 Lot C 3 A 4 R010/A ：Escherichia coli expressed microsome,
Cypex）. We also measured content of CYP isoform proteins in cryopreserved human hepatocytes treated with rifampicin. These
quantitative results were compared with those measured with a stable isotope-labeled peptide IS specific to each CYP isoform
and validity of the measurement method that we established was verified.
［Results and discussion］
Contents of CYP 1 A2, 2 D6, and 3 A 4 isoform proteins in liver microsomes of each donor obtained with the measurement method
using the single IS peptide were almost identical to those results using the stable isotope-labeled peptide ISs for each CYP
isoform. Moreover, the measurement results in intestinal microsomes and recombinant human CYP microsomes with a different
matrix in which a CYP protein exists were almost the same. Furthermore, expression induction of CYP 3 A 4 isoform alone wasconfirmed when contents of each CYP isoform protein after rifampicin（a CYP 3 A typical inducer）treatment using the
cryopreserved human hepatocytes were quantified. Thus, the assay that we established is one of an effective tool in drug
discovery because contents of multiple CYP isoform proteins are measurable with the single IS protein.

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 6: Novel Approaches in Proteomics Analysis

PMo-132

13:30 － 14:40

Testing of LC-MS/MS identification criteria for ricin protein

Martin T Söderström, Paula Vanninen

VERIFIN, Department of Chemistry, University of Helsinki, Finland

Keywords:
ricin, identification criteria
Novel aspects:
Establishment and testing of well-defined criteria for target protein identification in an forensic-type analysis.
Abstract:
The Chemical Weapons Convention（CWC）, which entered into force in 1997, prohibits the development, production, acquisition,
stockpiling, retention, transfer or use of chemical weapons by Member States. The Organisation for the Prohibition of Chemical
Weapons（OPCW）has been established to implement the CWC. Compared to other treaties limiting weapons of mass
destruction, the CWC aims at complete elimination of this type of weapons. It also includes a verification aspect in the form ofroutine and possible challenge inspections as well as investigations of alleged use of chemical weapons.
Typical analytes covered by the CWC are small molecules, which can quite routinely be analysed by GC-EI/MS, LC-ESI/MS or LCAPCI/MS. Ricin is the only protein toxin included in the CWC. As a minimum, the identification by two different analyticaltechniques is required. These identification criteria are not directly applicable to a much more complex system as the ricin protein.
The Scientific Advisory Board（SAB）of the OPCW and its temporary working group has been discussing the establishment ofidentification criteria for ricin.
Ricin protein is found in seeds of Ricinus communis, the castor plant. It is a ribosome inactivating protein（RIP）, which consists of
A and B chains（30 kDa and 29 kDa without glycosylation）. The mass of the glycosylated protein is 63 kDa. The B chain is
responsible for allowing the toxin to enter cells while A chain is RIP active. The seeds also contains Ricinus communis agglutinin（RCA）which consists of two A and two B chains and has weight of 120 kDa. RCA is closely related to ricin and these two proteins
contain common sequence fragments.
Typically LC-MS/MS identifications of proteins are based on database searching. The analysis is done from an enzymaticallydigested sample. In these analyses, the product ion spectra are measured using either clearly defined or data-dependentmeasurements. The data, full raw data or pre-processed data, are searched in protein databases. Finally, the resulting peptide
matches are correlated with possible proteins present in the samples. The reliability of result is increased with increasing
sequence coverage.
The identification of ricin for the purposes of the CWC has several requirements： 1 ）the presence of both A and B chains have tobe verified, 2 ）it has to be verified that the chains are connected and 3 ）the ricin protein is RIP active. The traditional
identification method described above could fulfil the two first requirements. In our studies, we have chosen a more direct way
also able to fulfil these two requirements for the identification, which aims for high sensitivity. The third requirement can besatisfied only with activity assays, which are outside the scope of this paper.
The starting point for our work has been the analysis of the sequences of both ricin and RCA. The aim has been to find tryptic
peptides, which can be used to differentiate ricin and RCA as well as be as characteristic to ricin as possible. The analysis of ricin
samples has been carried out by a triple quadruple LC-ESI/MS/MS instrument using both product ion scan and selected reaction
monitoring（SRM）. The basic identification criteria used for the evaluation of this data include comparison retention time
information as well spectral or SRM data between sample and reference. For protein identification, the number and properties ofthe peptides as well as the relative intensities seleted to represent the protein play an important role. The detection limits depend
greatly on the selected criteria.

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 6: Novel Approaches in Proteomics Analysis

PMo-133

11:10 － 12:20

The Precision of Heavy-Light Peptide Ratios measured by MALDI-TOF
Mass Spectrometry and its Application to LC-Free SISCAPA Protein
Quantification

Detlev Suckau1, Leigh Anderson2, Morteza Razavi2, Terry Pearson2, Rainer Paape1
1

Bruker Daltonik GmbH, 2Andersson Forschung

Keywords:
peptide, quantitation, MALDI, SISCAPA
Novel aspects:
Peptide quantitation using stable isotope standards and MALDI-TOF MS yields accurate results with CVs in the 1 - 2 % range fromplasma.
Abstract:
The most critical step in translating candidate protein markers into clinical diagnostics, the so-called verification step, requires
accurate protein and peptide quantitation in large sample sets（usually plasma or serum）. Nano-LC and multiple reaction
monitoring（MRM）mass spectrometry have been methods of choice providing precise relative quantitation of proteotypicsignature peptides representing proteins of interest in clinical samples. However, the use of nanoLC inhibits use of this approachin clinical settings. SISCAPA（Stable Isotope Standards and Capture by Anti-Peptide Antibodies）are introduced and combinedwith MALDI-TOF MS as an LC-free method for high throughput protein quantitation. MALDI-TOF is characterized as an extremely
simple and precise assay methodology that may be applicable for measurement of diagnostic proteins in clinical laboratoryenvironments.
Six pairs of synthetic proteotypic peptides were used in 2 forms： unlabeled（L）and labeled（H）in the C-terminal K or R residue.
Peptides were proteotypic for human proteins such as thyroglobulin, PCI, HER 2 /neu, HE-4, amongst others. Peptides were usedfrom 10 mM stocks to prepare mixtures at varying L：H ratios across 11-point calibration series spanning ranges up to 2047 fold.
Dilutions were prepared in 96 well plates using a liquid-handling robot（Bravo, Agilent）and spotted on prespotted AnchorChip
MALDI plastic targets（Bruker Daltonik）in quadruplicate. The SISCAPA assays utilized Invitrogen´s protein G dynabeads, rabbit
monoclonal antibodies selected for high peptide affinity and human plasma with titrated levels of spiked peptides. MALDI-MS
spectra were acquired on an autoflex speed LRF MALDI-TOF in positive ion mode.
The precision of peptide quantitation by MALDI-TOF MS was explored using 6 pairs of proteotypic peptides（L）and samesequence stable isotope labeled synthetic internal standards（H）. These were combined in dilution curves spanning up to 2,047fold ratios. Coefficients of variation（CV）of L：H peak area ratios were examined across 4 replicate MALDI spots per sample..
Averaged across 11 points of a 100-fold dilution curve and over all 6 peptides, the overall CV was 2.5% at 11 fmol total（light +heavy）of each peptide applied per spot. The average CV of measurements at near-equivalence（the center of the dilution curve）
for the six peptides was 1.0%. Response curves across the 100-fold range could be closely modeled by a power law fit giving R²
values > 0.998 for all peptides.
The MALDI-TOF MS method was used to determine the endogenous level of a proteotypic peptide（EDQYHYLLDR）of humanprotein C inhibitor（PCI）in a plasma digest after enrichment by capture on a high affinity anti-peptide antibody, a technique called
Stable Isotope Standards and Capture by Anti-Peptide Antibodies（SISCAPA）. The level of PCI was determined to be 770 ng/mL
with a replicate measurement CV of 1.5% and a >14,000-fold target enrichment via SISCAPA-MALDI-TOF. These results indicate that
MALDI-TOF technology can provide precise quantitation of high-to medium abundance peptide biomarkers over a 100-folddynamic range when ratioed to same-sequence labeled internal standards and enriched to near purity by specific antibody
capture. The robustness and throughput of MALDI-TOF in comparison to conventional nano-LC-MS technology could enable
currently impractical large-scale verification studies of protein biomarkers.

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 6: Novel Approaches in Proteomics Analysis

PMo-134

13:30 － 14:40

Production of Standards for Quantitative Proteomics andPhosphoproteomics with Focus on ICP-MS and ESI-MS

Wolf D Lehmann

German Cancer Research Center (DKFZ)

Keywords:
quantitative proteomics, peptide standards, protein standards, phosphorus, selenium,
Novel aspects:
Production of peptide/protein standards calibrated via ICP-MS and phosphorus or selenium detection and their transformation
into phosphorus-free or selenium-free peptide/protein standards under preservation of their accurate quantification.
Abstract:
Quantitative data are of outstanding importance in the life sciences. Regular quantifications result in concentration data.
Quantitative proteomics started with relative quantifications, designed for measurement of concentration ratios. In general,
relative quantification methods are connected with differential analyte labeling, so analytes from samples to be compared can be
distinguished when analyzed as mixture. Later, methods for regular quantification（“absolute quantification“）followed, which
deliver concentration data. For this purpose, the method of choice is to use a standard which is both labeled and calibrated. For
further development of the field of quantitative proteomics, there is urgent demand for accurately quantified and certified peptide
and protein standards. Techniques capable for generation of such standards are described with focus on inductively-coupledplasma ionization mass spectrometry（ICP-MS）and electrospray ionization mass spectrometry（ESI-MS）as quantification methods.
For quantification of phosphopeptides by ICP-MS, phosphorus is the element of choice.（PASTA peptides［ 1 ］）. Using
quantitative dephosphorylation controlled by ESI-MS or LC-ESI-MS, quantified phosphopeptide standard solutions can beconverted into peptide standard solutions, with preservation of the quantification accuracy. In this way it is possible to produce
phosphorus-quantified but phosphorus-free stable isotope labeled peptide standards. Mixing of calibrated peptide andphosphopeptide solutions results in ratio standards, which serve e.g. as standard for determination of a site-specificphosphorylation status［ 2 ］. Ratio standards work without quantification, so that the error of the adjusted ratio is only connectedwith a volumetric mixing error.
For specific quantification of intact proteins by ICP-MS, selenium is the element of choice. This concept is based on the
production of selenomethionine（SeMet）labeled proteins by cell-free protein synthesis. Recently, stable isotope（ 13C, 15N）and
SeMet labeled proteins were produced by cell-free synthesis and quantified by ICP-MS and selenium detection（RISQ,［ 3 ］）.
Another possibility is to introduce these two types of label into protein analogs expressed separately. The SeMet-containing
protein is quantified by ICP-MS and then used as standard for quantification of its stable isotope labeled analog. In this way it ispossible to produce SeMet-quantified but SeMet-free stable isotope labeled protein standards. The necessary extra quantification
step can be performed with a relative standard deviation around 1%［ 4 ］. Examples for protein quantifications and theircalculated accuracies are given and discussed.
References［ 1 ］Zinn N, Hahn B, Pipkorn R, Schwarzer D, Lehmann WD. Phosphorus-based absolutely quantified standard peptides forquantitative proteomics. J Proteome Res 2009, 8, 4870-4875.
［ 2 ］Hahn B, Boehm ME. Raia V, Zinn N, Möller P, Klingmüller U, Lehmann WD. One-source peptide/phosphopeptide standards
for accurate phosphorylation degree determination.
Proteomics 2011, 11, 490-494.
［ 3 ］Zinn N, Winter D, Lehmann WD. Recombinant isotope labeled and selenium quantified proteins for absolute protein
quantification. Anal Chem 2010, 82, 2334-2340.
［ 4 ］Konopka A, Boehm ME, Rohmer M, Baeumlisberger D, Lehmann  WD. Improving the precision of quantitative bottom-up
proteomics based on stable isotope labeled proteins. Anal Bioanal Chem 2012, in press.

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 6: Novel Approaches in Proteomics Analysis

PMo-135

11:10 － 12:20

Exploring the dynamic range of label-free mass spectrometricquantification in complex samples

Stephanie Kaspar, Wolfgang Jabs, Markus Lubeck, Carsten Baessmann
Bruker Daltonik GmbH, Bremen, Germany

Keywords:
Label-free proteomics, dynamic range on UHR-TOF, quantitation in low fmol range
Novel aspects:
The in-depth determination of the dynamic range of label-free proteomic studies in complex mixtures using a Q-TOF instrument.
Abstract:
Introduction:
The proteome is characterized by its high complexity and wide range of analyte concentrations which are constantly in flux due todevelopmental and stress-related processes. Thus, discovery proteomics approaches are required which are completely unbiased
and cover the complete proteome.
Accurate quantification of differently expressed proteins is still challenging and relies heavily on a stable analytical platform. Dueto advances in instrumentation it is possible to perform quantification without the need for labeling based on the comparison ofion intensities delivering quantitative information for a theoretically unlimited number of samples.
In this study we will investigate the quantitative performance of label-free approaches with a focus on dynamic range.
Methods:
Tryptic peptides were separated on a nano UHPLC system（U3000 RSLCnano, Dionex）using a 120min gradient and data for labelfree quantification were acquired with an Ultrahigh Resolution（UHR）Q-TOF system（maXis impact, Bruker Daltonics）equippedwith a Captive Spray ionization MS source. A proteomic standard of 48 proteins spanning a concentration range of 6 decades intotal（UPS- 2 standard, Sigma）was used to determine the dynamic range for label-free quantification. This standard was spiked at
two different concentrations（250 fmol to .5 amol and 500 fmol to 5 amol）into a highly complex E.coli background（500ng）
mimicking the complexity typically found in biological samples. The label-free quantitation workflow was based on signal
intensities from LC-MS runs for quantification and data from LC-MS/MS runs were used for identification information.
Results:
The determination of the dynamic range of label-free quantification was performed for a standard protein mixture spiked at 2
different concentrations into a constant E.coli background. The experimental setup covered concentration ranges from 500fmol
down to 2.5amol. Separation of tryptic peptides on a nano LC system coupled to UHR-TOF detection showed excellent retention
time and signal intensity stability for five replicates per concentration, both being a prerequisite for label-free quantification
studies. Quantitative information was obtained from MS signal intensities. Signals originating from the same peptide, e.g. different
charge stages and isotopes were combined and used for quantification. Results show almost no variation for E.coli background,
whereas regulation ratios were determined for the standard peptides spiked into the complex background. In total 27 proteinsfrom the UPS 2 mixture were detected as significantly regulated（p<0.05）. Very accurate quantification was obtained down to thelow fmol range. Results also show the label-free approach to be capable of providing the correct quantification of peptides in theamol range. The high dynamic range of this approach allows in-depth studies of quantitative changes in biological systems.
Therefore the method will be applied to determine the effect of sodium butyrate treatment of Chinese hamster ovary cells.

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 6: Novel Approaches in Proteomics Analysis

PMo-136

13:30 － 14:40

Multiple Products Monitoring Using Q-Exactive Mass Spectrometer(Q-MpM)

Je-Hyun Baek, Eugene C Yi

Molecular Medicine and Biopharmaceutical Sciences, Seoul National University, Seoul, Korea

Keywords:
accurate target quantification, multiple products monitoring, Q-Exactive
Novel aspects:
Combination of MpM with Q Exactive MS（producing fast and accurate MS 2 spectra）is very compatible and promising for
accurate target quantification with high dynamic range in high complex sample.
Abstract:
Mass spectrometry-based proteomics has greatly emerged as useful technology for biomarker discovery and target verification.
Target verification from biological and clinical samples is still challenging upon high complexity and high dynamic ranges. Since an
MS 2 -intensity based quantification shows higher sensitivity and specificity, selected reaction monitoring（SRM）and multiple
reaction monitoring（MRM）has been widely used for target quantification on triple-quardrupole mass spectrometry. However, wepreviously reported a robust target peptide quantification method： multiple products monitoring（MpM）from full MS 2 scans
produced by ion trap mass spectrometer（Journal of Proteome Research, 2009, 8, 3625-3632）. A new type of orbitrap analyzer withquadrupole mass filter（Q-Exactive）was recently developed. Q-Exactive mass spectrometer enables fast multiplexed single ion
monitoring following full range of highly accurate MS 2 scans（12 scans/sec with R=17500）. We applied MpM approach to
Q-Exactive mass spectrometer platform. Moreover, we tested our MpM method（quantitative accuracy of target peptide
quantification）applying Q-Exactive multiplexed single ion monitoring with standard light and heavy peptides showing highdynamic range. Combination of MpM approach with Q-Exactive（Q-MpM）is very promising for accurate target quantification withhigh dynamic range in high complex sample.

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 6: Novel Approaches in Proteomics Analysis

PMo-137

11:10 － 12:20

In-Source Decay for the high-throughput sequencing of animal toxins

Loic Quinton1, Michel Degueldre1, Nicolas Gilles2,3, Pierre Escoubas3, Edwin De Pauw1
1
3

Mass Spec Laboratory - University of Liège - BELGIUM, 2CEA-iBiTEC-S-SIMOPRO- Gif-sur-Yvette - FRANCE,
VenomeTech- Valbonne- FRANCE

Keywords:
venomics, toxins, ISD, MALDI
Novel aspects:
High-throughput sequencing by ISD
Abstract:
Animalvenoms are complex chemical cocktails, comprising a wide range of biologically active reticulated peptides that target withhigh selectivity and efficacy avariety of membrane receptors such as ion channels and G-Protein Coupled Receptors. Based on
various disulfide-linked scaffolds, they represent anenormous structural and pharmacological diversity. Assuming the fact that eachof the 170,000 venomous species reported can produce in their venom more than 250 bioactive, the global animal venom resource
can be seen as a collection of more than 40,000,000 bioactive peptides and proteins. These toxins are continuously selected andhighly refined by the evolution process, up to the point where every molecule is endowed with pharmacological properties thatare highly valuable in the context of human use and drug development.
A FP 7 European Project called VENOMICS（http：//venomics.eu/）was designed and started in last November. This project
combine resources, skills and knowledge of the eight partners that represent an efficient mix of academic institutions（CEA（France）, University of Liège（Belgium）, Université de la Méditerranée（France））and SMEs（VenomeTech（France）, Sistemas
Genómicos（Spain）, NZYTech（Portugal）,Zealand Pharma（Denmark）, Vitamib（France））. The goal of this project is toconstruct a toxin bank of 10,000 sequences generating from the study of 200 selected venomous species. This bank will be
exploited to discover new peptides that can be used as human drugs. This ambitious project imposes high-throughput strategies
combining transcriptomic and proteomic sequencings, chemical and biochemical productions, and biological activity screenings.
High-throughput proteomics of crude venoms represent one of the major bottlenecks of this project. Part or total peptidic
sequences of toxins are hardly needed to allow the other partners to start their own tasks.
This work presents the strategy designed to generate high throughput accurate peptide sequences from crude venoms. The
sequencing strategy rests on the chromatographic purification of venoms hyphenated with MALDI-In-Source Decay. ISD has
already been demonstrated efficient for toxin sequencing［ 1 ］, and especially when using 1,5-DAN as reducing matrix［ 2 ］. ISD
yields sequences covering more than 70% of the sequence by series of singly charged c-type ions even for high molecular mass
toxins（> 6500 Da）. ISD spectra are simpler than classical MS/MS spectra and automation of spectra interpretation, difficult withother fragmentation techniques（CID, ETD）, is possible from ISD spectra. ISD requires however simple peptides mixtures that canbe obtained using LC fractions collection on the MALDI place（LC-MALDI）. In addition ’in-house’software performed automatedsequence analysis even when up to three peptides are within the same MALDI spot.［ 3 ］
Another part of our work is based on the use of Ion mobility（IMS）employed to control the quality of peptides produced byrecombinant technology, with a special focus for the correct pairing of cysteins in disulfide bridges.
［ 1 ］Quinton,L., Demeure, K., Dobson, R., et al., New method for characterizing highly disulfide-bridged peptides in complex
mixtures： Application to toxin identification from crude venoms, 2007, J. Proteome Res., 6 （ 8 ）,3216-3223.
［ 2 ］Demeure, K., Quinton, L., Gabelica, V., et al., Rational selection of the optimum MALDI matrix for top-down proteomics byinsource decay, 2007, Anal.Chem, 79（22）, 8678-8685.
［ 3 ］Zimmerman, T., Debois, D., Mazzucchelli, G., et al., An analytical pipeline for MALDI in-source decay mass spectrometry
imaging, 2011, Anal.Chem., 83（15）, 6090-6097.

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 6: Novel Approaches in Proteomics Analysis

PMo-138

13:30 － 14:40

Screening and Quantitative Analysis of Specific Proteins in ClinicalSamples by LC-ESI-MS/MS

Rieko Goto1, Yasushi Nakamura2, Shohei Shioyama1, Yukiko Nishida1, Tomonori Takami1, Tokio Sanke2
1

JCL Bioassay Corporation, Hyogo, Japan, 2Wakayama Medical University, Wakayama, Japan

Keywords:
Proteomics, Protein, LC-MS/MS, LTQ FT, QTRAP 5500
Novel aspects:
Specific biomarker proteins to disease were identified with combination of screening and quantitative analysis by LC-MS/MS using
small amounts of clinical samples.
Abstract:<Introduction>
To explore biomarkers for the identification specific diseases（e.g. cancer）, it’s important to target the proper biomarker protein
candidates and evaluate their expression including low level expression proteins from clinical samples, directly. Using clinicalsamples obtained from patients is one of the most effective ways to evaluate disease specific proteins because the identified
proteins information can be linked to the disease state. One of the ways to evaluate if that the targeted proteins are disease
specific, is immunoassay including ELISA or ECL. However, immunoassay requires an unique antibody and determines only one
target. Therefore, as previous stage for immunoassay, it is beneficial to screen for and verify disease specific proteins from clinicalsamples by LC-MS/MS.
<Objective>
The objective of this study is to establish rapid and effective screening methods to identify positive biomarker protein candidates
from the huge number of proteins in clinical samples and quantitative verification methods of the targeted proteins.
<Methods>
Non-metastasis tissues of breast cancer（subjects 6 ）and the metastasis tissues of breast cancer（subjects 19）were used fromclinical samples. The cytoplasm was separated from each tissue specimen by centrifugation. The purified samples for each subject
were alkylated with iodoacetamide and digested with trypsin. For the first screening process, the tryptic digested samples（ 4 µg
total protein）were analyzed with 2 DLC-LTQ FT. For the second screening process, the digested samples（0.4 µg total protein）
were semi-quantitatively analyzed by multiple reaction monitoring（MRM）mode without internal standard using a QTRAP 5500,
and the highest level peptides were selected as potential biomarker proteins. Finally, the tryptic digested samples（ 3 µg total
protein）were quantitatively analyzed by MRM with stable isotope-labeled peptides as internal standards. From the results,
expression of the targeted proteins was verified and evaluated the specificity to the metastasis group.
<Results>
During the first screening, 59 proteins were selected for analysis by 2 D-LC-LTQ FT. The second screening, by semi-quantitative
MRM analysis, found four proteins that contained higher levels of certain peptides and were selected as biomarker specificproteins candidates. Finally during MRM analysis using stable isotope peptides as internal standards, two proteins were identified
as biomarker specific proteins to metastasis of breast cancer. Furthermore, the most disease specific protein was also positively
confirmed by quantitative RT-PCR.

<Conclusion>
Positive biomarker proteins specific to metastasis of breast cancer were selected efficiently from huge number of proteins at alow cost using a small number of clinical samples using LTQ FT. The selected proteins were identified and confirmed to be
specific to metastasis of breast cancer at low cost using semi-quantitative and quantitative LC-MS/MS. Therefore, the combinationof the screening methods and the quantitative verification methods is an effective way to identify the disease specific proteinsusing clinical samples.

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 6: Novel Approaches in Proteomics Analysis

PMo-139

11:10 － 12:20

Analysis of modified amyloid beta peptide by Mass Spectrometry

Akutsu Hiroaki, Tsumura Naomi, Funakoshi Hiroshi, Nakamura Masao
Asahikawa Medical University, Asahikawa Japan

Keywords:
Amyloid beta peptide, Tyr10, Met35, Asp1, Halogen
Novel aspects:
Here we present the different reaction of Amyloid beta with hypochlorous acid and hypoiodous acid.
Abstract:
Introduction
Accumulation of the amyloid beta peptide（Aβ ）in the form of senile plaques（SPs）is a pathological hallmark of Alzheimer’s
disease（AD）. Although the progression of AD is linked to inflammatory and oxidative processes, the biochemical mechanism
underlying AD is uncertain. SPs consist of Aβ 1 -42 and Aβ 1 -41, and contain high concentrations of iron, copper and zinc. Recent
findings show that myeloperoxidase（MPO）and microglia areco-localized with SPs. In addition to phagocytosis, microglia secrete
proteases that degrade Aβ. In addition, microglia are activated by Aβin vivo and generate H 2 O 2 .The interaction of Aβ withmicroglia is reported to stimulate the inflammatory process and may contribute to neuronal damage. We have reported thereaction of full length Aβ（Aβ 1 -42, Aβ 1 -40）and Aβ25-35 with hypochlorous acid 1). The results indicate that Met35 in Aβ rapidly
reacts with hypochlorous acid. In this study, we focused on the reaction of various Aβ containing no Met with MPO-derived
oxidant, hypohalous acid and compared with the result of full length Aβ.
MethodHypochlorous acid（HOCl）and hypoiodous acid（HOI）were formed by reaction of MPO in the present of H2O2 and KCl or KI.
Reaction mixture containing 40μM Aβ 1 -12 was treated with MPO system. Reactions were carried out in 20mM phosphate buffer（pH 6 ）. Mass spectra were observed by LC/MS/MS at indicated time after reaction was started. A NanoFrontier eLD LC/MS/MS
system（Hitachi High-Tech）was used to obtain Mass spectra with MonoCap C18 Fast-flow（0.05 x 150 mm, GL Sciences）. The
mobile phase consisted of solvent A（ 2 % acetonitrile with 0.1% formic acid）and B（98% acetonitrile with 0.1% formic acid）. The
acetonitrile gradient consisted of 15 to 40% at the flow rate of 200nL/min.
Results and Discussion
The mass spectrum of native Aβ 1 -12 showed a signal at m/z 712（［M+ 2 H］2+）and 475（［M+ 3 H］3+）, respectively. When the Aβ
1 -12 was treated with HOCl, the spectra gave a 17Da（double charge ion）increase at m/z 729（retention time 14.1min and 15.0min）
and 22Da（double charge ion）loss at m/z 690（retention time 14.6min）. CID analysis revealed that the signal of m/z 729 is 35Da
increase at b-ion and a-ion respectively（retention time 15.0min）and b10+34 ion（retention time 14.1min）. A peak at retention
time 14.1min corresponds to the chlorinated Aβ 1 -12 at Tyr10. A peak at 15.0min corresponds to the chlorinated one at terminal
amine of Asp1. The signal of m/z 690 is 44Da loss at b-ion and a-ion, respectively. These results depict the decarboxylation of
Asp 1 2). When the Aβ 1 -12 was treated with HOI, the spectra gave a 63Da（double charge ion）, 42Da（triple charge ion）at m/z775, 517（retention time 8.2min）and 126Da（double charge ion）, 84Da（triple charge ion）at m/z 838, 559（retention time 9.9min）increase of m/z 712, respectively. CID analysis confirmed that the signal of m/z 775 is b10+126 ion and a10+126 ion. The results
indicate that the one iodine was added to Tyr10 and the signal of m/z 838 is a10+252 ion indicates that the two iodines wereadded to Tyr10.
There is a difference between the reaction mechanism of HOI and HOCl with Aβ 1 -12. Chlorination of Asp 1 was followed by thedecarboxylation of Asp 1 through the reaction of Aβ 1 -12 with HOCl. Subsequently, chlorine transfer from Asp 1 to Tyr10 was
observed. In contrast, iodination of Tyr10 was readily observed without formation of Asp 1 iodination. We also report the result ofAβ25-35 with HOCl for the comparison.
References
1 ）M.Nakamura et al., Free Radical Research. 44,734-741（2010）2 ）H.Akutsu et al., 59th MSSJ Annual Conference, 2 P-22（2011）

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 6: Novel Approaches in Proteomics Analysis

PMo-140

13:30 － 14:40

The trial of lower background in using a nanoelectrospray (nano ES)
interfaceToshie Takahashi1, Takashi Usui2
1

University of Tokyo, Tokyo, Japan, 2Nikkyo Technos, Co., Ltd. Tokyo, Japan

Keywords:
nanoelectrospray LC/MS/MS, Proteomics, peptide
Novel aspects:
We have a prototype made by appending a filter to the cover of nanES interface. Using filter attached caver, we were able toreduce the low background.
Abstract:
Recently, nanoelectrospray LC/MS/MS was used for in the field of biological samples analysis should not be without. We analyzed
a femtomole level biological sample, it became a problem to get a good results was that the low background.
Usually, nanoelectrospray（nanoES）interface was often attached to use in the open air space. Therefore, the influence of theestablishment environment of the device was often taken. We tried to put the normal cover on the nanoES interface that we had
been obtained to reduce the low background. Next, we had fabricated a cover which was wearing a filter to a part of the cover.
We confirmed to be lower than background measurement was performed filter cover attached to the nanoES interface that wasused only nomal cover.
It thinks that it becomes more possible by a background having fallen down for the measurement of the quantity without a low
concentration biological sample.

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 6: Novel Approaches in Proteomics Analysis

PMo-141

11:10 － 12:20

Fully automated chip-based nanoelectrospray combined with electrontransfer dissociation for high throughput top-down proteomics

CORINA FLANGEA1,2, CRISTINA MOSOARCA1,3, MARILENA MANEA4, CATALIN SCHIOPU1,5,
EUGEN SISU5,6, ALINA D ZAMFIR1,2

1
Aurel Vlaicu University, Arad, Romania, 2National Institute for Research and Development in Electrochemistry andCondensed Matter, Timisoara, Romania, 3West University Timisoara, Romania, 4Zukunftskolleg, University of Konstanz,
Germany, 5Victor Babes University of Medicine and Pharmacy, Timisoara, Romania, 6Chemistry Institute of RomanianAcademy, Timisoara, Romania

Keywords:
NanoMate, ETD/CID, top-down analysis, proteomics
Novel aspects:
NanoMate robot was for the first time combined with electron transfer dissociation and introduced in proteomics for peptide andbioconjugate analysis as well as for top-down protein fragmentation.
Abstract:
Collision-induced dissociation（CID）, previously the only available fragmentation technique on ion traps exhibits some limitation,
among which, the low throughput and impossibility to characterize in details posttranslational modifications in terms of site（s）
and structure. The recently implemented electron transfer dissociation（ETD）performed on advanced and versatile mass
spectrometers is regarded as an important step with real perspectives for top-down proteomics. The last few years havewitnessed the breakthrough of microfluidics for electrospray（ESI）, in which our group was intensively involved［1,2］, and theirsuperior performance in terms of ionization efficiency, spray stability, analysis reproducibility, sensitivity and pace. Applied
together, ETD and CID may significantly increase the sequence coverage and provide added confidence to peptide/protein
identification［3,4］. In the present work, chip-based nanoESI performed on a NanoMate robot was combined for the first timewith ETD and ETD/CID on a high capacity ion trap（HCT）MS to yield an analytical platform on which peptide sequencing andhigh throughput top-down protein analysis is feasible.
Mass spectrometry experiments were performed on a High Capacity Ion Trap Ultra mass spectrometer incorporating an ETD
module with fluoranthene as anionic reagent. Fully automated chip-based nanoelectrospray was conducted on a NanoMate robot
incorporating ESI 400 Chip technology.
NanoMate-ETDapproach was optimized and tested first for high throughput sequencing of a simple peptide. As the analyte thestandard substance P neuropeptide containing 11 amino acids（MW 1347.63 Da）was used. This advanced protocol developed for
Substance P was further applied to a complex functionalized synthetic peptide： gonadotropin-releasing hormone（GnRH）-III
linked via amide bond to Daunorubicin（Dau）-GFLG,（MW 2257.32 Da）. NanoMate-ETD of the triply protonated form of GnRH-III（Dau-GFLG）produced sequencing of the peptide backbone and N-C alpha cleavage of the NH-CO linkage between the peptide
and Dau moiety, with the preservation of the labile glycosidic bond. The［M+ 2 H+］2+ product ion corresponding to Dau at m/z
257.53 enhanced the further characterization of Dau moiety by a subsequent CID experiment using the alternate ETD/CID mode
available on the HCT MS instrument. Furthermore, with the present setup, the first top-down protein analysis by ETD could bedemonstrated on a medium-size protein. As a model substrate, horse apomyoglobin（MW 16.95 kDa）was chosen. In thisexperiment, of major importance is the molecule multiple protonation under chip-nanoESI conditions； this process evidenced by
the signals corresponding to high charge states, ranging from 14+ to 21+, is a fundamental prerequisite of a successful top-down
fragmentation by ETD. For the experiment using［M+16H+］16+ion detected at m/z 1060.32 as the precursor, the fragmentation by
ETD MS 2 could be successfully accomplished within only 30 s and with a sample consumption of only 12 fmols. ETD mass
spectrum summed over the scans acquired for 30 s yielded a number of c- and z-type of product ions corresponding to 80 %coverage of apomyglobin sequence.
The obtained results on simple and functionalized peptides as well as on a medium size intact protein allow the possibility toaccomplish peptide sequencing by ETD and top-down protein fragmentation in a high throughput regime. A significant reductionof the time and sample consumption required for reliable peptide/protein identification was also noticed. In view of NanoMateETD potential, extension to large proteins and proteins bearing posttranslational modifications are certainly planned and foreseenin the near future.
［ 1 ］C. Flangea, A. Serb, E. Sisu, A.D. Zamfir, Biochim. Biophys. Acta. 20111811, 513-535.
［ 2 ］C. Flangea, E. Sisu, D.G. Seidler, A.D. Zamfir, Anal.Biochem. 2012420, 155-162.
［ 3 ］N. P. Sargaeva, C. Lin, P. B. O’Connor, J. Am. Soc. Mass Spectrom. 2011, 22, 480-491.
［ 4 ］H. Hahne, B. Kuster,J. Am. Soc. Mass Spectrom. 2011,22, 931-942.

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 6: Novel Approaches in Proteomics Analysis

PMo-142

13:30 － 14:40

Analysis of nitroproteome in S.cerevisiae using chemical approach

Jeong Won Kang1, Na Young Lee2, Kyung-Cho Cho1, Sang-Hyun Park2, Kwang Pyo Kim1
1

Konkuk University, Seoul, Korea, 2Seoul National University, Seoul, Korea

Keywords:
Protein Tyrosine Nitration（PTN）, Nitroproteome, mass spectrometry, signal transduction
Novel aspects:
Proteomic scale study was performed to elucidate physiological functions of protein tyrosine nitration in mating signaltransduction of S.cerevisiae.
Abstract:
Protein tyrosine nitration（PTN）is a post-translational modification occurring under the action of series of nitrating agents
generated under oxidative stress. It results in the addition of a nitro group（NO2）in ortho position to the phenolic hydroxyl groupof tyrosine residue. The analysis of 3 -nitrotyrosine are very important in biologically and clinically because PTN is affects in many
pathological events such as various cancers, diabetes, neurodegenerative and age-related disorders. However, the functional andbiochemical analysis of PTN has been hampered by the low-abundance of nitrated species and the lack of efficient enrichment
methods. We described here a chemical approach to enrich nitrated peptides or proteins by incorporating specific tagging groups
in the peptides through simple chemical transformations followed by mass spectrometric analysis, and also we apply this method
to S. cerevisiae. The study of S.cerevisiae nitration is very important because S.cerevisiae is a most simple eukaryote. Therefore,
we can indicate how nitration affects in vivo signal transduction or protein-protein interaction through the study of nitroproteome
in S.cerevisiae. In this study, we try to identification of nitroproteins from S.cerevisiae, and detection of biochemical functions of
nitroproteins on mating pathway from several over-expressed proteins involved mating signaling. Finally, we try to find motif ofnitropeptides, and structural analysis.

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 6: Novel Approaches in Proteomics Analysis

PMo-143

11:10 － 12:20

Mass spectrometric and bioaffinity MS investigations of proteins involved
in metabolic and age related diseases

Marius I Iurascu1, Claudia Cozma1, Michael Gross2, David Clemmer3, Michael Przybylski1

1
University of Konstanz, Konstanz, Germany, 2Washington University in St. Louise, MO, USA, 3Indiana University,
Bloomington, IN, USA

Keywords:
Affinity-MS, IMS, Fabry, Alzheimer’s, oligomers
Novel aspects:
Mass spectrometry, ion mobility spectroscopy and bioaffinity MS aplications on proteins involved in metabolic and age relateddiseases
Abstract:
New mass spectrometric（MS）methods have proven lately an increasing range of applications within the life science domain,from the rare diseases in newborns, like Fabry, to high level occurrence, age related disorders, like Alzheimer’s. Fabry is alysosomal storage disease（LSD）characterized by the deficiency of the enzyme alpha galactosidase A（a-GLA A）which leads tothe accumulation of globotriaosylceramide in different organs, causing their failure. To understand the molecular working of the
enzyme the affinity mass spectrometry was chosen and a recombinant human alpha-galactosidase A was used. A mousemonoclonal antibody to human alpha-galactosidase A was immobilized on a sepharose column and its affinity to the a-GLA A wasinvestigated by in-gel tryptic digestion. For the epitope identification an epitope-excision experiment was performed. The a-GLA A
was immobilized on the antibody column and proteolytically digested with trypsin. After the wash of the supernatant, the epitopewas eluted with 0.1% TFA in milliQ and identified by mass spectrometry. Alzheimer’s disease（AD）is a progressive
neurodegenerative disorder which affects an increasingly proportion of the elderly population. The hallmark of the disease is theaccumulation of intercellular high molecular weight aggregates in the form of amyloid plaques and fibrils, although, the key
neurotoxic moieties are lately suggested to be the Amyloid-beta（Aß）oligomers. Aß is a 39-43 aa peptide that is formed
proteolytically from the large ß-amyloid transmembrane precursor protein（APP）and is highly prone to spontaneously aggregate.
For the Aß-oligomers studies by（i）ion mobility spectroscopy（IMS）and（ii）mass spectrometric proteome analysis, Aß（ 1 -40）
was synthesized using solid phase peptide synthesis（SPPS）, Fmoc chemistry, and the crude peptide purified by RP-HPLC［ 1 ］.
The peptide was characterized by electrospray（ESI）ion trap mass spectrometry and by high resolution MALDI FT-ICR MS. Aftersynthesis and purification, the Aß（ 1 -40）peptide was incubated for five days at 37 °C in（i）phosphate buffer saline（PBS）, pH 7.5［ 2 ］, and（ii）10 mM ammoniumacetate buffer, at various pHs, in order to yield oligomers. After the incubation, the aggregateswere further characterized by TrisTricin Polyacrylamide Gel Electrophoresis, IMS and MS. The gel electrophoresis analysis
ascertained the successful oligomerization of the Aß（ 1 -40）peptide, evidentiating the formation of dimers, trimers, tertramers, andlarge, high molecular weight aggregates with masses over 200 kDa. Further characterization of the oligomers was made by MALDITOF mass spectrometric measurements. The MS spectra confirmed the Aß（ 1 -40）oligomers formation, showing evidence of
dimers, trimers and tetramers, in accordance with the gel electrophoreris assay. The ion mobility measurements were of mostimportance, evidentiating two distinct species with different cross-section areas, and the methionine oxidation at position 36.
These new mass spectrometric techniques proved to be of key importance in the elucidation and characterization of a wide rangeof diseases. This project was supported by the EU-US grant MS-Life（Integrating performance mass spectrometry tools withapplications in life science）.
［ 1 ］Iurascu M.I., Cozma C., Tomczyk N., Rontree J.,Desor M., Drescher M., Przybylski M., Anal Bioanal Chem2009,8：2509-19［ 2 ］Harry LeVine et al., Biochemistry 2005,44：15937-43.

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 6: Novel Approaches in Proteomics Analysis

PMo-144

13:30 － 14:40

Higher resolution improves top-down protein ID results on an Orbitrap
mass analyzer for large (>40 kDa) proteins

Daisuke Higo1, David Horn2, Rosa Viner2, Seema Sharma2, Eduard Denisov3, Alexander Makarov3

1
Thermo fisher scientific, Yokohama, Japan, 2Thermo Fisher Scientific, San Jose, CA, 3Thermo Fisher Scientific (Bremen)GmbH, Bremen, GERMANY

Keywords:
Top-down, Protein ID, Orbitrap, ETD, HCD
Novel aspects:
For large intact proteins, the highest possible resolution produces the most confident top-down intact protein identifications.
Abstract:
Introduction
In Fourier transform-based mass spectrometers, there is a trade-off between the acquisition time for a single scan and theresolution of that scan. For bottom-up proteomics experiments, scientists often choose a resolution that is sufficient to resolve theisotopes of the fragments while keeping scan speed as high as possible. Here we study the effect of instrument resolution on theconfidence in MS/MS identification of large, intact proteins and it was found that maximizing the resolution rather than maximizing
throughput produces the most confident results.
Methods
Bovine serum albumin, enolase, and carbonic anhydrase were obtained from Sigma. The proteins were dissolved in water/acetonitrile at a concentration of 7.5, 10, and 3 pmol/uL, respectively. The proteins were direct infused at 3 uL/min into an
Orbitrap Elite system and electron-transfer dissociation（ETD）and high energy C-trap dissociation（HCD）MS/MS spectra wereacquired for 1 - 2 minutes at 60,000, 120,000, and 240,000 resolution with 5 microscans per spectrum. A single spectrum wasaveraged across the full elution profile and Xtract analysis was performed to produce zero-charge masses. The resulting data weresearched by ProSightPC and the E-values for the match against the target sequence were calculated.
Abstract
For enolase, three direct infusion experiments were acquired at the three different resolutions mentioned above for ETD of the
44+ charge state of enolase at m/z 1086. For the 60000 resolution experiment, 18 spectra were acquired in the 50 seconds of direct
infusion. The subsequent averaged spectrum was matched to the enolase sequence, producing an expectation value of 1.6e-67
matching 49 c ions and 21 matching z ions. For the 120000 resolution experiment, 14 spectra were acquired over 51 seconds and theresulting match produced an expectation value of 1.67e-84 and 58 matching c ions and 33 matching z ions. Finally, the 240000
resolution experiment produced 10 spectra over the 52 seconds of direct infusion and the match produced an expectation value of
5.6e-99 with 55 matching c and 38 matching z ions. Thus, as the instrument resolution was increased, the true fragment matches
increased accordingly while the matches to the background signal were reduced, resulting in improved sequence coverage aswell as improved confidence.
The higher resolution also enables extension of the range for top down analyses to higher mass proteins. We were able toproduce resolved isotope distribution of the 44+ of bovine serum albumin and identify both the HCD and ETD spectra from 1.3
minutes each of direct infusion with highly confident E-values（3.87e-42 for ETD, and 6.27e-19 for HCD）. This hints at the potential
for identification of such large proteins on a chromatographic timescale.
We will also present results from LC/MS runs of more complex intact protein mixtures at different resolutions.

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 6: Novel Approaches in Proteomics Analysis

PMo-145

11:10 － 12:20

HR/AM Targeted Peptide Quantification on Q Exactive: A Unique
Combination of High Selectivity, High Sensitivity and High Throughput

Yi Zhang2,3, Makoto Takahata1, Zhiqi Hao2,3, Markus Kellmann2,3, Andreas Huhmer2,31

Thermo Fisher Scientific K.K., Japan, 2Thermo Scientific, San Jose, CA, 3Thermo Scientific, Bremen, Germany

Keywords:
Orbitrap, Peptide Quan, Targeted Approach
Novel aspects:
High sensitivity, high selectivity and high throughput flexible HR/AM targeted quantification on a benchtop Orbitrap mass
spectrometer
Abstract:
Introduction
Quantitative proteomics enables the identification of large number of protein candidates, which display biologically interesting
dynamics on a global scale in the early discovery phase. Targeted MS approach, in particular selected reaction monitoring（SRM）,
has become the preferred platform for quantitatively analyzing tens to hundreds of peptide candidates across large number ofsamples, either for understanding of signaling regulation or for verification and selection of potential clinical biomarkers. In thisstudy, the Q Exactive, a true high resolution and accurate mass（HR/AM）mass spectrometer, was evaluated for targeted protein
quantification. The dynamic range and LOD/LOQ of two HR/AM methods, multiplexed targeted selected ion monitoring（msxtSIM）and targeted HCD（tHCD）, were investigated.
Methods
Different amounts of 11 heavy isotope labeled yeast peptides from 10 amol to 100 fmol were spiked into either 10ng or 500ng of Ecoli whole cell digest. The heavy peptides were analyzed with two HR/AM targeted methods, msx tSIM and tHCD, on the Q
ExactiveTM. The targets were quantified at precursor level in msx tSIM method. Up to 4 targets were monitored in a single scanwith a resolution of 140,000（m/z 200）. The targets were quantified at fragment level in tHCD method. Full HCD spectra wereacquired at 12Hz with a resolution of 17,500. The data were automatically processed using Pinpoint with 5 ppm mass tolerance toobtain peak areas, linear dynamic range and LOD/LOQ for each method.
Abstract
For msx tSIM method, the resolution of 140,000 allows baseline separation of two species as close as 30ppm. The high resolution
and high mass accuracy（< 5 ppm）ensures confident identification of target ion in complex matrix. The measured isotope
distribution is < 5 % from theoretical distribution. Advanced signal processing doubles the resolution for the same transient time,
which results in a scan speed of 3 Hz at 140,000 resolution. Two unique features, parallel filling and detection and spectrum
multiplexing, further increase total scan speed and throughput. More than eight scans with a resolution of 140,000 were acquired
across 12 sec LC peaks even at 10 amol level. A LOD of 10amol and an excellent linear dynamic range of 4 orders were obtained
for all heavy peptide targets in a medium complex background of 10ng of E coli whole cell digest. The LOD varied from 50 amol to1000 amol in a strong complex background of 500ng of E coli whole celldigest. For tHCD method, the high scan speed of 12Hz at aresolution of 17,500 allows acquisition of more than 8 scans across 12 sec LC peaks even at 10 amol level. The high mass accuracyof < 5 ppm and high resolution ensures unmatched confident identification of fragment ions compared to triple quadrupole based
SRM method. A LOD of 10amol and an excellent linear dynamic range of 4 orders were obtained for all heavy peptide targets in a
medium complex background of 10ng of E coli whole cell digest. The LOD varied from 10amol to 100 amol in a strong complex
background of 500ng of E coli whole cell digest.

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 7: New Ionization Methods and Related Topics for the Next Generation

PMo-146

13:30 － 14:40

Development of an Ion Source for the Analysis of Cuticular Hydrocabonsfrom Living Insects and Steps toward Comprehensive Hydrocarbon
Analysis

Alexander Pirkl1, Ann-Christin Buelter1, Joanne Y Yew2,3, Klaus Dreisewerd1

University of Muenster, Muenster, Germany, 2Temasek Life Sciences Laboratory, Singapore, Singapore, 3Department of
Biological Sciences, National University of Singapore, Singapore

1

Keywords:
ambient, cuticular hydrocarbons, drosophila, UV-LDI
Novel aspects:
An ion source design and sample preparation approaches are presented that could provide a comprehensive spatially-resolved
MS analysis of cuticular hydrocarbons from living insects.
Abstract:
Introduction
Insects and other arthropods express species-specific blends of cuticular hydrocarbons（CHCs）on their surface that serve as
pheromones and play an important role in behavior / 1 /. Recently, we demonstrated that using ultraviolet laser desorption/
ionization orthogonal time-of-flight mass spectrometry（UV-LDI-o-TOF-MS）enables the spatially-resolved analysis of CHCs fromwhole Drosophila melanogaster / 2 / and other insects / 3 /. However, one major limitation of this method is that it is typicallylethal to the animals. Moreover, so far, alkanes cannot be detected. In a second study, we showed that ion profiles can also be
generated from living insects at ambient pressure（AP）solely under the influence of a strong electrical field / 4 /. However, theresulting spectra are extremely complex, presumably due to oxidation reactions.
Methods
In order to address these limitations, we have developed an AP-ion source that provides better control over the ionization
conditions（e.g., by applying a protective gas environment and/or adjustable pressure）and the ion transfer into the mass analyzer（QStar-Pulsar, AB-Sciex）. For construction of the source, the particle discriminator approach by Schneider et al. / 5 / wasadopted. Capillaries of different diameters and lengths were tested to establish variable flow conditions. Using a coil heater（GCheat）, capillaries can be heated up to 400 °C. A Nd：YAG-laser（lambda=355nm）can be utilized for UV-LDI. We also tested
several strategies for the direct UV-LDI-MS analysis of alkanes and the characterization of double bond positions. Preliminary
experiments were performed on intact male and female Drosophila melanogaster.
Preliminary Data
In the poster, details on the design of the ion source and the vacuum interface will be presented. Preliminary experiments
demonstrated functionality of the source, including capillary heating and reproduction of our previous MS data obtained by thefield-based ion generation（FBIG）method / 4 /. We will show first results on the effect of instrumental parameters（gas pressure,gas type, electrical field strength, capillary design and temperature）on the generation of CHC profiles using FBIG and UV-LDI
mass spectrometry at pressures close to 1 bar. To increase the spatial resolution of the“FBIG“ method, we will present resultsfrom two approaches in which miniaturized extraction capillaries were employed to analyze CHCs from intact insects both withand without activation by a well-focused laser beam.
We will also present data demonstrating that（i）UV-LDI-MS analysis of alkanes is possible after application of silver colloids and（ii）the use of ozonolysis allows partial characterization directly from insect cuticles of the double bond positions of unsaturatedhydrocarbons.
/ 1 / Howard, R. W.； Blomquist, G. J. Ecological, behavioral, and biochemical aspects of insect hydrocarbons. Annu. Rev. Entomol.
2005, 50, 371–393.
/ 2 / Yew, J. Y., Dreisewerd, K.； Luftmann, H.； Müthing, H.； Pohlentz, G.； Kravitz, E. A. A new malesex pheromone and novel cuticular cues for chemical communication in Drosophila. Curr. Biol.
2009, 19, 1245–1254.
/ 3 / Yew. J. Y.； Soltwisch, J.； Pirkl, A.； Dreisewerd, K. Direct laser desorption ionization of endogenous and exogenous
compounds from insect cuticles： practical and methodological aspects. J. Am. Soc. Mass Spectrom. 2011, 22, 1273-1284.
/ 4 / Pirkl, A.； Dreisewerd, K.； Yew, J. Y.； König, S., Field-based ion generation from microscale emitters on natural and artificial
objects for atmospheric pressure mass spectrometry. Anal. Bioanal. Chem. 2010, 97, 3311–3316.
/ 5 / Schneider, B. B.； Lock, C.； Covey, T. R. AP and vacuum MALDI on a QqLIT instrument. J. Am. Soc. Mass Spectrom. 2004, 16,
176-182.

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 7: New Ionization Methods and Related Topics for the Next Generation

PMo-147

11:10 － 12:20

Oligonucleotide analysis by nanoparticle-assisted laser desorption/
ionization mass spectrometry

Shu TAIRA1, Daisaku KANEKO1, Yasuko KONISHI-KAWAMURA-2, Yuko ICHIYANAGI3
1

JAPAN Advanced Institute of Science and Technology, 2Ishikawa Prefectural University, 3Yokohama National University

Keywords:
nanoparticle-assisted laser desorption/ionization, oligonucleotide, ionization
Novel aspects:
Reference： S. Taira et al. Analyst vol. 137, 2006-2010（2012）,（Cover Article）
Abstract:
We analyzed oligonucleotides by nanoparticle-assisted laser desorption/ionization（nano-PALDI）mass spectrometry（MS）. To
this end, we prepared several kinds of nanoparticles（Cr-, Fe-, Mn-, Co-based）and optimized the nano-PALDI MS method toanalyze the oligonucleotides. Iron oxide nanoparticles with diammonium hydrogen citrate were found to serve as an effective
ionization-assisting reagent in MS. The mass spectra showed both［M-H］- and［M+xMe2+-H］-（Me： transition metal）peaks. The
number of metal-adducted ion signals depended on the length of the oligonucleotide. This phenomenon was only observed using
bivalent metal core nanoparticles, not with any other valency metal core nanoparticles. Our pilot study demonstrated that
ironoxide nanoparticles could easily ionize samples such as chemical drugs and peptides as well as oligonucleotides without theaid of an oligonucleotide-specific chemical matrix（e.g., 3 -hydroxypicolinic acid）used in conventional MS methods. These results
suggested that iron-based nanoparticles may serve as the assisting material of ionization for genes and other biomolecules.

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 7: New Ionization Methods and Related Topics for the Next Generation

PMo-148

13:30 － 14:40

Temperature Dependence of Proton Transfer and Conformation Change
for Ubiquitin Ions

Minami Kawashima, Ayako Sudo, Takashige Mori, Yuto Ohshima, Keishi Machida, Kanako Yokoyama,
Kazuki Yamashita, Shinji Nonose
Yokohama City University, Yokohama, Japan

Keywords:
temperature, conformation, proton transfer, ubiquitin
Novel aspects:
Temperature dependence of proton transfer for isolated multiply-charged ubiquitin ions with gaseous molecules was investigated.
Dramatic change was observed for distribution of product ions and absolute reaction rate.
Abstract:
Temperature dependence ofproton transfer for isolated multiply-charged ubiquitin ions with gaseous molecules of large proton
affinity（PA）was investigated with mass spectrometry. A home-made tandem mass spectrometer with electrospray ionization（ESI）was used for measurements. Multiply-charged ubiquitin ions were produced by ESI of a dilute solution of ubiquitin inmethanol-water mixture including acetic acid. The ions produced by ESI are admitted into the vacuum chamber through stainlesscapillary. The charge-selected ions emerging from a quadrupole mass spectrometer are admitted into a collision cell with octapole
ion trap. The collision cell is filled with He including gaseous molecules of large PA. We choose 1 -propylamine, 1 -butylamine,
1 -pentylamine, tert-butylamine, or pyridine as target molecules. Temperature dependence of reaction rate and branching
fractions for proton transfer from multiply-charged ions to the target molecules was measured, by changing temperature ofcollision cell. The parent and product ions are mass-analyzed by a time-of-flight mass spectrometer equipped with reflectron.
Proton transfer from ubiquitin ions to the target molecules was occurred by collisions in the cell. Absolute reaction rate for protontransfer was estimated with intensity of ions in the mass spectra. The reaction rate increased with increasing number of thecharges（z）. It also increases with increase of PA for the target molecule. By changing temperature of collision cell in region from
280 to 470 K, temperature dependence of reaction rate and branching fractions for proton transfer from ions to target molecules
was measured. For ions of z = 6 and 7, dramatic change was observed for distribution of product ions and reaction rate. For z=6,
reaction rate increases with decrease of temperature from 450 K to 380 K, whereas it decreases with decrease of temperature from370 K to 300 K. For z=7, the reaction rate increases rapidly with decrease of temperature from 320 K to 290 K, whereas it decreases
rapidly with decrease of temperature from 290 K to 280 K. Change of reaction rates would correlate with conformation of ubiquitin
ions, which originates in self-solvation of the proton by hydrophilic residues in polypeptide chains, delocalization of charges with
self-solvation, and Coulomb interaction between charges.

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 7: New Ionization Methods and Related Topics for the Next Generation

PMo-149

11:10 － 12:20

Development of a Dual Pressure Operating Hollow Cathode Glow
Discharge Ion Source for Detection of Explosives and Explosive-related
Compounds

Md Ahsan Habib, Lee Chuin Chen, Satoshi Ninomiya, Kenzo Hiraoka
University of Yamanashi, Kofu, Japan

Keywords:
Glow discharge, Explosives, Adduct ions
Novel aspects:
Hollow cathode glow discharge ion source has been proved to be a simple, rapid and robust ionization technique for trace level
detection with high selectivity and specificity of multiclass explosives
Abstract:［Introduction］： Detection of explosives at trace levels is imperative for security purposes because of increasing worldwide
terrorist threats at public places. Recently, we have developed a dual pressure operating hollow cathode glow discharge ion
source for the analysis of explosives and explosive-related compounds. The ion source was designed in such a way that theplasma can be generated alternatively at high pressure（~1900 Pa）and at low pressure（~140 Pa）regions. The plasma contains asufficient amount of reactant ions and electrons, and thus the gaseous analyte molecules were efficiently ionized when theypassed through the plasma.
［Experimental]： Explosive samples RDX, PETN, TNT, TNB, HMTD, and TATP were purchased as either 0.1 or 1 mg/L acetonitrile
solutions. DNT isomers were purchased as solid. Acetonitrile was used for dilution.
Briefly, the glow discharge ion source consists of two metallic tubes of different i.d.（e.g., 2 mm and 5 mm）, and connected withan insulator and an aperture. For sample introduction, a metallic capillary of i.d. ~0.2 mm and 16 mm in length was connected withthe tube of i.d. 2 mm. The ion source was coupled with a linear ion trap-MS（Thermo-LTQ）. Mass spectra were collected underautomatic gain control with maximum injection time of 10 ms. The targeted species were identified by MS/MS mode. About 1 - 2µL aliquot from each solution deposited on a glass slide was placed in front of the sample inlet at a distance of 0.5-1.0 cm. The
desorbed sample heated between 180-250 °C was introduced into the ion source.
［Results and Discussion］： All the nitro-compounds gave negative product ions under both high pressure plasma（HPP）and low
pressure plasma（LPP）conditions, whereas peroxide-based explosives e.g., HMTD and TATP produced only positive ions as either
protonated or cluster ions under HPP condition. No product ions were detected for these compounds under LPP condition.
Nitroaromatic molecules（M）formed mostly M- as a product ion under LPP condition at pg level, but, under HPP condition, thecompounds formed either M- or（M-H）-and also minor ions［e.g.,（M-NO）-,（M-OH）-,（M-H+O）-］. The observation of adduct ion of
the deprotonated nitroaromatic compounds with O,（M-H+O）-, is our first finding. Moreover, under HPP condition, TNB produced（~15%）
and（M-H+O）-（~20%）ions, whereas, M-（100%）ion was produced by TNB when it was（M-H）-（100%）, M-（80%）,（M-NO）
introduced with TNT. All DNT isomers produced only M-（100%）under LPP condition. Under HPP condition, however, 2,4-DNT and
2,6-DNT formed（M-H）- ,（M-NO）- and（M-H+O）- , and 2,3-DNT formed M -（100 %）and（M-H）-（60 %）. The dissimilarity in ion
formation by the nitroaromatic compounds can be explained by taking the following factors into an account：（i）acidic characterof ring and side chain hydrogen,（ii）electron affinity of the nitro-aromatic compounds, and（iii）profusion of reagent ions in HPP
region.
For nitro-ester explosives（e.g., PETN, RDX）under HPP condition, PETN produced adduct ion,（M+NO3）-, whereas RDX formed
adduct ions,（M+NO2）-and（M+NO3）-. Formation of only the（M+NO3）-adduct ion for PETN suggests the strong bond formation
for this cluster ion. Under LPP condition, RDX mostly formed（M-NO2-HNO2）-and（M-NO2）- , whereas PETN formed（M-NO2）-.

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 7: New Ionization Methods and Related Topics for the Next Generation

PMo-150

13:30 － 14:40

A Novel Source Design for the Analysis of both Polar and Non-Polar
Species

Steve M Smith1, Hilary Major1, Steve Bajic1, Anthony Bristow2, Andrew Ray2
1

Waters Corporation, Manchester, UK, 2AstraZeneca, Macclesfield, UK

Keywords:
novel ionization, non-polars
Novel aspects:
A new API source design capable of enhanced sensitivity analysis of both polar and non-polar species.
Abstract:
Introduction
Since the early commercialisation of API sources, analysts have utilized a combination of ESI, APCI and APPI sources to meet thechallenge of ionizing multi-component samples of widely ranging polarities. This approach is hampered by the requirement ofdifferent optimal chemistries and the time-consuming need to alternate between source techniques. More recently, multimodesource designs have strived to deliver the ultimate goals of broad polarity range applicability, equivalent analyte response andenhanced sensitivity over existing ionization methods. Here, we describe a high-sensitivity multimode API source where ionization
is derived from the interaction of a high velocity droplet stream from a grounded probe with a closely coupled high voltage
electrode.
Methods
The modified source components were incorporated into a Waters Xevo TQD tandem quadrupole mass spectrometer fitted withan ESI/APCI source housing and electrospray probe. Samples were introduced either by infusion using the system’s fluidics or bycoupling an ACQUITY UPLC for LC/MS experiments. An electrode voltage of 1 kV was used throughout and other source
parameters, such as cone voltage, source temperature and desolvation gas flow rate and temperature, were adjusted according
to the flow rate and samples under investigation. This“multi-mode“ source has the advantage of ionizing compounds with a wide
range of polarities in a single chromatographic run without having to switch ionization modes.
Comparison experiments were carried out using the same instrumentation and standard ESI/APCI and APPI sources.

Preliminary Data
This novel source has been found to be more sensitive than electrospray（typically 3 ）for the majority of polar species
investigated such as peptides, pesticides and pharmaceuticals.
The source has also been used to analyse a range of non-polar compounds more commonly analyzed by APCI and APPI and has
shown an improvement in sensitivity over these techniques. The results obtained for vitamins and poly-aromatic hydrocarbons（PAHs）show the principal ions generated are M+. radical cations and［M-H］+ ions by hydride abstraction while steroids generate［M+H］+ ions.
Data will be presented demonstrating the range of compounds which can be analysed by this source and the gain in sensitivity
achieved.

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 7: New Ionization Methods and Related Topics for the Next Generation

PMo-151

11:10 － 12:20

Sheath- flow PESI for Dry Samples

Md Obaidur Rahman, Yasuo Shida, Mridul Kanti Mandal, Satoshi Ninomiya, Lee Chuin Chen,
Kenzo Hiraoka
University of Yamanashi,Kofu,Japan

Keywords:
Electrospray, PESI, Sheath-Flow PESI
Novel aspects:
Probe electrospray ionization（PESI）associated with sheath-liquid flow is newly developed. This technique can be applicable toany samples regardless of their wetness.
Abstract:
Introduction：
Probe electrospray ionization（PESI）uses a solid needle or a wire as a sampling probe and an ESI emitter instead of a capillary.
PESI can be applicable to various biological samples without any special sample pretreatment. It was also found that PESI was
superior to nanoESI for samples with high concentration of salts, urea, and detergents. One drawback of PESI is that it is notapplicable to dry samples. In our laboratory, PESI coupled with a coaxial capillary, namely, sheath-flow PESI, was newly developed.
By supplying solvent through the capillary, this technique was found to be applicable to any samples regardless of their wetness.
Experimental：
An acupuncture needle（0.12 mm o.d.）was inserted into the fine glass or plastic capillary with i.d. of 0.3 mm. The acupunctureneedle was protruded from the tip of the capillary by 0.2-0.3 mm in length. The solvent such as methanol was flowed through thecapillary with flow rate of 1 microL/min（one micro litre/min）or less. When the PESI probe slightly touches the sample surface,
the solvent flowing out of the capillary makes the sample wet with the solvent and sample components were extracted to thesolvent liquid. After the sample extraction, the PESI needle was moved to the highest position. By the application of high voltage
of about 2.5- 3 kV to the PESI probe, the extracted samples were electrosprayed. Mass spectra were measured by a time-of-flightmass spectrometer（JEOL, AccuTOF）.
Results and Discussion
Because the solvent was continuously supplied to the tip of the acupuncture needle, this method can be applicable to dry andalso wet samples. Some examples are given below.
［Tablets］： Sheath-flow PESI was applied to several tablets, such as loratadine, atenolol, etizolam, triazolam, etc. The protonatedmolecule ions were observed as major ions for all the tablets examined.
［Human finger］： By touching the volunteer’s finger by the probe, intense signals originating from lipids and sometimes detergentswere detected. Dry saliva and urine also gave strong ion signals.
［Bank notes］： A US dollar bill was scanned/analyzed from the central to the terminal positions. In the central part of the bill,
little signals were observed. However, the signal of protonated cocaine was clearly observed at the edge of the bill.
［Plants］： Characteristic mass spectra could be obtained from plants such as vegetables, fruits etc. When a greenish potato was
examined, a strong signal of toxic solanine was detected on the surface, while no such signal was detected in the central part ofthe sample. The insecticides and fertilizers in plants could also be detected by this technique.
［Illicit drugs］： After the methanol droplet containing 100 pg of methamphetamine or morphine was deposited on the finger, thedried area（ 3 mm in diameter）was examined. Strong signals for both compounds could be detected.
The sheath-flow PESI can be applicable to any samples regardless of wetness. By using meter-long flexible capillary, it may be
used as a very fine endoscope. After the sampling with least invasion, the PESI mass spectra can be obtained by sheath-flow PESI.

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 7: New Ionization Methods and Related Topics for the Next Generation

PMo-152

13:30 － 14:40

An Electron Impact Ion Source for a Miniature Mass spectrometeryide zhao, meiru guo, liang wang, yuhua xiao, taiguo li

Science and Technology on Vacuum & cryogenics Technology and Physics Laboratory, Lanzhou Institute of Physics,
Lanzhou, China

Keywords:
Electron Impact； SIMION； optimal design
Novel aspects:
Thought optimal design by SIMION, The performance of the EI ion source combined with miniature magnetron sector mass
spectrometer was tested.
Abstract:
Miniature mass spectrometer, one of the most powerful and versatile methods of real time and in suit chemical analysis, is widely
utilized in harsh environment, such as space exploration, active volcanoes, underwater environment, and the battlefield. A
miniature magnetic sector mass spectrometer has been developed that consists of an electron impact（EI）ion source, a magnetic
mass analyzer, two continuous dynode multiplier detectors, and associated electronics circuits.
In this paper, the design, simulation and construction of the EI ion source is described, which consists of a small chamber
attached to a rigid mounting plate through which an electron beam is produced by thermal emission from tungsten filament. A
pair of collimation magnets mounted to two walls of the chamber which creates the field for the alignment of the electron beam.
Ions produced in the source are drawn out by a high voltage and collimated into a beam by a series of electrostatic lens system
which consists of repeller, exit slit of ionization chamber, extraction electrodes, focusing electrodes, main slit and alpha slit.
Charged particle electrodynamic modeling（SIMION 8.0）was employed to simulate the ion source electrostatic lens system. The
beam emittance and beam width as a function of the lens system voltage ratio for single charged ion trajectories have been
investigated. Influence of the atomic masses of the different elements has been studied on both the beam emittance and beamwidth.
Simulation results were used to verify and optimize the performance of the ion source before and during its fabrication. The
performance of the EI ion source combined with miniature magnetron sector mass spectrometer was tested.

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 7: New Ionization Methods and Related Topics for the Next Generation

PMo-153

11:10 － 12:20

Development of High Pressure Ion Sources with Operating Pressure Higher
Than Atmospheric Pressure

Lee Chuin Chen, Matiur M Rahman, Mridul Kanti Mandal, Kenzo Hiraoka
University of Yamanashi, Kofu, Japan

Keywords:
High pressure ion source, electrospray
Novel aspects:
Development of high pressure ion source with operating pressure high than one atmospheric pressure using ionization techniquessuch as ESI, CI and FD.
Abstract:
Early attempts to couple ESI ion source with mass spectrometry were conducted by placing the ion source in high vacuum. Due toinsufficient bath gas and desolvation, vacuum ESI suffered from poor sensitivity and could only handle limited types of solvents.
John Fenn et al. revolutionized the ESI method by bringing the ion source to the atmospheric pressure ambient, and the bath gas
provided sufficient thermal energy to the charged droplets for the vaporization of solvent without freezing them. Nowadays,
standard ESI ion source is operated under an atmospheric pressure and the mass spectrometer, which is designed to work with
ESI and other atmospheric pressure ionization（API）source, is referred as API mass spectrometer. To date, nearly all ion sources（e.g. MALDI, ESI, APCI, EI, CI, FI/FD, etc.）are operated either under high vacuum or one atmospheric pressure.
Super-atmospheric pressure electrospray ionization with operating pressure > 5 atm has recently been demonstrated in our lab［1,2］. By raising the operating air pressure of the ion source, we have verified that the threshold for the corona and arc discharge
increases but the threshold for the onset of electrospray remained unchanged. This is an interesting advantage because it allows
a stable electrospray ionization to be performed without the occurrence of disturbing corona and arc discharge even for purewater solution which is of high surface tension.
In this presentation, we will report some of our recent progress on the development of high pressure ion source with ionization
techniques other than electrospray. Some of our efforts have been put on the ionization of gaseous compounds because thenumber of gas phase interaction is increased with the increase of pressure. Also, by sufficiently quenching the electrical discharge
by increasing the ion source pressure, some of our preliminary results have showed that it is even possible to perform stable nonvacuum field desorption（FD）which is until now has to be performed under ultra high vacuum.

Reference［ 1 ］L.C. Chen, M. K. Mandal, K. Hiraoka, J. Am. Soc. Mass Spectrom. 22, 539-544（2011）［ 2 ］L. C. Chen, M. K. Mandal, K. Hiraoka, J. Am. Soc. Mass Spectrom. 22, 2108-2114（2011）

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 7: New Ionization Methods and Related Topics for the Next Generation

PMo-154

13:30 － 14:40

Conformation Change for Insulin and Insulin Chain B Ions; Temperature
Dependence of Proton Transfer

Ayako Sudo, Keishi Machida, Kanako Yokoyama, Minami Kawashima, Takashige Mori, Yuto Ohshima,
Kazuki Yamashita, Shinji Nonose
Yokohama City University

Keywords:
temperature dependence, proton transfer, insulin
Novel aspects:
Temperature dependence of proton transfer for insulin and insulin chain B ions with gaseous molecules was investigated.
Dramatic change was observed for distribution of product ions and absolute reaction rate.
Abstract:
Temperature dependence ofproton transfer for isolated multiply-charged insulin and insulin chain B ions with gaseous moleculesof large proton affinity（PA）was investigated with mass spectrometry. A home-made tandem mass spectrometer with electrosprayionization（ESI）was used for measurements. Multiply-charged insulin and insulin chain B ions were produced by ESI of a dilute
solution in methanol-water mixture including acetic acid. The charge-selected ions emerging from a home-made quadrupole mass
spectrometer are admitted into a collision cell with octapole ion trap. The collision cell is filled with He including gaseous
molecules of large PA. We choose 1 -propylamine, 1 -butylamine, 1 -pentylamine, tert-butylamine, or pyridine as target molecules.
Temperature dependence of reaction rate and branching fractions for proton transfer from multiply-charged ions to the target
molecules was measured, by changing temperature of collision cell. The parent and product ions are mass-analyzed by a time-offlight mass spectrometer equipped with reflectron. Reduction of disulfide bonds in insulin to produce insulin chain B ion was
carried out with 1,4-dithiothreitol（DTT）. A small amount of DTT was added to insulin aqueous solution（-0.1 mM）in distilledwater, such that the final DTT concentration was approximately 1 mM. After a period of over 20 minutes at 350 K, mass spectra ofinsulin chain B ions were obtained.
Proton transfer from insulin and insulin chain B ions to the target molecules was occurred by collisions in the cell. Absolute
reaction rate for proton transfer was estimated with intensity of ions in the mass spectra. The reaction rate increased withincreasing number of the charges（z）. It also increases with increase of PA for the target molecule. By changing temperature ofcollision cell in region from 290 to 470 K, temperature dependence of reaction rate and branching fractions for proton transfer fromions to target molecules was measured. For z = 4 of insulin ion reacted with pyridine, dramatic change was observed for
distribution of product ions and reaction rate. The reaction rate decreases with decrease of temperature from 450 K to 330 K, andit increases with decrease of temperature from 330 K to 290 K. For z= 3 of insulin chain B ion reacted with pyridine, reaction rateincreases rapidly with decrease of temperature from 350 K to 290 K, whereas the reaction rate with 1 -butylamine, 1 -pentylamineor tert-butylamine decreases gradually with decrease of temperature from 450 K to 280 K. Proton transfer would proceed by twostep mechanism； complex formation of ions with target molecules, and release of protonated target molecular ion. These
reaction rates would correlate with conformation change of insulin and insulin chain B ions with change of temperature. The
conformation change would originate in self-solvation of the proton by hydrophilic residues in polypeptide chains.

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 7: New Ionization Methods and Related Topics for the Next Generation

PMo-155

11:10 － 12:20

Development of high sensitive low vacuum dielectric barrier discharge
ionization method

Kazushige Nishimura1, Shun Kumano1, Masuyuki Sugiyama1, Masuyoshi Yamada1, Hidetoshi Morokuma2,
Yuichiro Hashimoto1
1

Hitachi Ltd., Tokyo, Japan, 2Hitachi High-Technologies Corp.,Ibaraki-ken, Japan

Keywords:
Dielectric_barrier_discharge
Novel aspects:
Development of high sensitive low vacuum dielectric barrier discharge ionization method
Abstract:
Introduction
Recently, ionization methods using dielectric barrier discharge（DBD）have become of great interest［1, 2 ］. In this study, wedeveloped a novel high sensitive DBD ionization method, which is called a low vacuum DBD ionization method. In thisconfiguration, samples directly pass through the inside of the DBD plasma where charge density is high. Because the ion source isplaced in a vacuum, the conductance between the ion source and the mass spectrometer is large, which leads to highintroduction efficiency. High ionization efficiency and high introduction efficiency result in high sensitivity.
MethodThe low vacuum DBD ion source consists of a glass tube, a tube-shaped discharge electrode, and a ring electrode. The groundeddischarge electrode was inserted into the glass tube, and the ring electrode was attached around the glass tube. The AC voltageof 2 kV was applied to the ring electrode. The DBD plasma was generated between the end of the discharge electrode and thering electrode.
The instrument consists of the low vacuum DBD ion source and an ion trap. Vaporized samples were discontinuously introduced
from ambient air into the ion source and passed through the inside of the plasma directly where gas molecules were converted toions. The ions produced were introduced into the ion trap and were mass analyzed.
Results
The mass spectra of methyl salicylate were measured by using a low vacuum DBD ion source. The ambient air introduced withthe samples gas was used as a discharge gas. Water cluster ions（（H 2 O）nH+）n = 1, 2, 3 ）and［M+H］+ ions of methyl
salicylate were observed as dominant ions. This result shows that the low vacuum DBD ion source is a soft ionization method.
To optimize the pressure of the region where ionization occurred, the intensity of the［M+H］+ ions of methyl salicylate wasmeasured as a function of ion source pressure. The intensity of the［M+H］+ ions became maximum when the pressure of theregion where ionization occurred ranged from 1000 to 3000 Pa.
The intensity of the［M+H］+ ions of methyl salicylate observed by using low vacuum DBD ion sources was 30 times higher thanthat observed by using conventional atmospheric pressure chemical ionization（APCI）ion source when we set the flow rate to be
the same for both configurations.
At the conference, we will report experimental results of other samples by using a low vacuum DBD ion source.
［ 1 ］Lee Chuin Chen, Zhan, Yu, Hiroko Furuya, Yutaka Hashimoto, Kenichi Takekawa, Hiroaki Suzuki, Osamu Ariyada, Kenzo
Hiraoka, J. Mass. Spectrom, 2010, 45, 861.
［ 2 ］Juan F. García-Reyes, Fabio Mazzoti, Jason D. Harper, Nicholas A. Charipar, Sheran Oradu, Zheng Ouyang , Giovanni
Sindona, R. Graham Cooks, Rapid. Common. Mass Spectrom. 2009, 23, 3057.

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 7: New Ionization Methods and Related Topics for the Next Generation

PMo-156

13:30 － 14:40

High ion yields of carbohydrates from frozen solution by UV-MALDI

Chi-Wei Liang, Po-Jul Chang, Yu-Jiun Lin, Yuan T Lee, Chi-Kung Ni
Academia Sinica, Taipei, Taiwan

Keywords:
MALDI, carbohydrate, frozen solution,
Novel aspects:
High ion yiels and low fragmentation from frozen solution by UV-MALDI
Abstract:
Aqueous acetonitrile solution containing oligosaccharide（maltopentaose）and matrix（2, 5 -dihyrroxybenzoic acid）was frozen at
100 K for ultraviolet matrix-assisted laser desorption ionization（UV-MALDI）mass analysis. Compared to the conventional UVMALDI of dried analyte and matrix mixture（after evaporation of solvent）, frozen solution generates more oligosaccharide ionsand less fragments from post-source decay. It is long-lasting and the analyte distribution is more homogeneous. The ion generation
efficiency is 20-30 times larger than conventional dried mixture. The percentages of fragmentation due to post-source decay forfrozen samples are almost zero（< 2 %）, compared to 17% and 40% for dried samples at low and high laser fluences, respectively.
The applications to polysaccharides also demonstrate that the ion intensity from frozen solution is 20 times larger thanconventional method.

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 7: New Ionization Methods and Related Topics for the Next Generation

PMo-157

11:10 － 12:20

ATMOSPHERIC PRESSURE PHOTOIONIZATION-MASS SPECTROMETRY OF
ESTROGENIC COMPOUNDS

Oscar Nunez1, Paolo Lucci2, Encarnacion Moyano1, Maria Teresa Galceran11

University of Barcelona, Barcelona, Spain, 2Pontificia Universidad Javeriana, Bogota, Colombia

Keywords:
APPI, UHPLC, Mass Spectrometry, Estrogenic Compounds,
Novel aspects:
Study of the effect of different dopants in the APPI ionization of estrogenic compounds
Abstract:
Among endocrine-disrupting chemicals（EDCs）, natural and synthetic estrogens are considered the most potent estrogenic
compounds. EDCs are widely distributed over the aquatic environment and due to their ecotoxic effects natural and synthetic
estrogens are of special relevance even at very low concentrations. Estrogenic compounds are usually excreted into the aquatic
environment through human and animal urine and the use of estrogens in medicine or in veterinary have caused their presence inaquatic ecosystems［ 1 ］. Liquid chromatography coupled with tandem mass spectrometry using both electrospray ionization（ESI）
and atmospheric pressure chemical ionization（APCI）is the technique of choice for the LC-MS analysis of these compounds［2,3］.
However, estrogens are non polar compounds and are usually hard to ionize using classical LC-MS sources mainly ESI.
Atmospheric pressure photoionization（APPI）which can ionize both polar and non polar molecules with high sensitivity can beconsidered an alternative and has began to be used for the analysis of these compounds［ 4 ］. An additional advantage of using
APPI is that ionization can be enhanced adding dopant substances and in some cases the choice of an adequate dopant is as
much important as the choice of the source of ionization itself［ 5 ］.
The aim of this study was to compare the behavior of three ionization sources（ESI, APCI and APPI）on analyzing eight estrogenic
compounds（dienestrol, diethylstilbestrol, estrone, 17β-estradiol, 17α-estradiol, mestranol, 17α-ethinylestradiol and estriol）. LCMS was carried out using an Accela liquid chromatograph system coupled to a triple quadrupole mass spectrometer TSQ
Quantum Ultra AM（ThermoFisher Scientific）. To evaluate APPI performance six dopant solvents（toluene, acetone, ethylacetate,anisole, clorobenzene, tetrahydrofuran）as well as some dopant mixtures（toluene-anisole and tolune-chlorobenzene）were used
and their sensitivity and their capability to ionize estrogens was compared. Negative ESI mode only provided the deprotonatedmolecules as base peak and low responses while in positive APCI the dehydrated fragment ion was found for the majority of the
estrogenic compounds and better signals were obtained. As regards APPI in general most of the dopants provided the positive
radical ions as the most abundant peaks. However, when using anisole or the mixture toluene-anisole as dopants the protonatedion［M+H］+ resulted favored against the positive radical ions. For all the estrogenic compounds the highest signal responses were
found with chlorobenzene and the mixture toluene-chlorobenzene except for dienestrol that gave higher signals using anisole,although signal-to-noise ratio worsened due to dopant solvent ionization. The detectability of the selected estrogens by APPI
using chlorobenzene was found to be more effective than that of APCI and ESI, and was then selected for the analysis of
estrogenic compounds.
A fast and high-efficient chromatographic separation of all estrogenic compounds was achieved in less than 4 min with an
Ascentis Express Phenyl-Hexyl HPLC column（150 mm x 2.1 mm i.d., 2.7 µm）and gradient elution using water and acetonitrile asmobile phases. Good method performance was observed in terms of LODs（ppt level）, linearity and run-to-run precision（RSD <4 %）. The method was applied to the analysis of wastewater samples from a Hospital after off-line SPE using HLB cartridges, andestrone and estriol were detected and quantified at low ppb levels.
［ 1 ］. M. Auriol, Y. Filali-Meknassi, R.D. Tyagi, C.D. Adams, R.Y. Surampalli, Process Biochem. 41（2006）525-539.
［ 2 ］P. Labadie, E M. Hill, J Chromatogr A. 1141（2007）174-81.
［ 3 ］P. Lucci, O. Nunez, M.T. Galceran, J. Chromatogr A. 1218（2011）4828-4833.
［ 4 ］L.Viglino, K. Aboufaldl, M. Prevost, S. Sauve, Talanta, 76（2008）1088-1096［ 5 ］. L. Hollosi, T. Wenzl, J Chromatogr A. 1218（2011）23-31.
This work has been supported by CARBOSORB project［（FP 7 Marie Curie Industry-Academia Partnerships and Pathways（IAPP）］

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 7: New Ionization Methods and Related Topics for the Next Generation

PMo-158

13:30 － 14:40

Application of Electrospray Droplet Impact / SIMS to synthetic polymers

Rio Takaishi, Yuji Sakai, Satoshi Ninomiya, Kenzo Hiraoka

Clean Energy Research Center, University of Yamanashi, Yamanashi, Japan

Keywords:
Electrospray Droplet Impact； SIMS； Cluster ion； synthetic polymers
Novel aspects:
EDI is capable of atomic- and molecular level etching without leavingthe damage after irradiation. It would be a valuable
technique for thenext-generation surface science.
Abstract:
The electrospray droplet impact secondary ion mass spectrometry（EDI/SIMS）has been developed as cluster SIMS［ 1 ］. The
typical primary droplets are roughly represented as［（H2O）90,000 + 100H］100+（mass of 1.6×106 u）. The kinetic energies of charged
water droplets are 10 6 eV with the velocity of ~12 km/s. EDI/SIMS has the atomic/molecular level etching ability with highionization efficiencies. Furthermore, degradation products do not accumulate on sample surfaces（i.e. surface-cleaning effect）［ 1 ］.
EDI/SIMS has been applied to many inorganic and organic samples. For synthetic polymers, EDI mass spectra were found toreflect the backbone structures for the polymers［ 2 ］［ 3 ］. In addition, no selective etching［ 2 ］［ 3 ］was observed for all
samples examined, such as PS, PI, PET, PMMA, etc.
Synthetic polymers are the target for the active investigation in semiconductor field, for example, package of semiconductor,
flexible printed circuit, flexible display etc. Analysis of thin-film synthetic polymer used in the semiconductor field is of significant
importance. In this paper, the application of EDI to thin and bulk samples of various synthetic polymers will be presented. The
comparison of etching rates for organic and inorganic materials will also be given.
Reference［ 1 ］K. Hiraoka, D. Asakawa, S. Fujimaki, A. Takamizawa, K. Mori, Eur. Phys. J. D38, 225（2006）［ 2 ］K. Hiraoka, Y. Sakai, Y. Iijima, D. Asakawa, K. Mori, Appl. Surf. Sci. 255, 8947（2009）［ 3 ］K. Hiraoka, Y. Sakai and Y. Iijima ： J. Vac. Sci. Technol. A28, 510

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 7: New Ionization Methods and Related Topics for the Next Generation

PMo-159

11:10 － 12:20

Development of high pressure (>1 atm) probe electrospray ionization
mass spectrometry

Md Matiur Rahman, Lee Chuin Chen, Kenzo Hiraoka
University of Yamanashi, Kofu, Japan

Keywords:
PESI, HP-PESI, gas breakdown,
Novel aspects:
HP-PESI can suppress the occurrence of the gas breakdown. It is readily applicable to aqueous sample solutions in both positive
and negative mode of operations.
Abstract:
Probe electrospray ionization（PESI）has been developed in our laboratory［ 1 - 4 ］. It can be applicable to wet real-world samples
without any sample pretreatment. Because it uses a fine metal needle, the gas breakdown is apt to take place for aqueous
sample solutions, especially in the negative-mode of operation. In order to circumvent this problem, a high-pressure PESI（HP-PESI）
was developed.
The PESI ion source chamber was pressurized up to 7 bars with dry air supplied from an air compressor. To couple the highpressure ion source directly to the mass spectrometer, the default ion transfer tube of the ion trap mass spectrometer（Thermo,
Velos）was replaced with a custom made one with 0.25mm in id and 1 /16“ in od. The desolvation chamber was made ofaluminum alloy that has two view ports made of polycarbonate for the optical microscopic observation.
HP-PESI was found to have several advantages. First it can provide denser heat bath gas for the desolvation of the charged
droplets. Second, the gaseous breakdown was almost totally suppressed in both positive- and negative-mode of operations.
These features are particularly useful for the analysis of peptides and proteins in aqueous solutions.
A comparative study of PESI and HP-PESI was performed. HP-PESI was found to be superior to PESI for the aqueous sample
solutions in the negative-mode of operation because negative-mode PESI measurements were sometimes disturbed by theoccurrence of corona discharge at the tip of the needle. HP-PESI would be an attractive tool particularly for the analysis of native
proteins because it can handle the aqueous solutions that contain no organic solvents.
References：
1. Hiraoka, K. et al. Rapid Commun. Mass Spectrom, 21, 3139（2007）.
2. Chen, L. C. et al. J. Phys. Chem. B 112, 11164（2008）.
3. Yu, Z. et. al. Rapid Commun. Mass Spectrom. 24, 1507（2010）.
4. Chen, L. C. et al. J. Mass Spectrom. 44, 1469（2009）.

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 7: New Ionization Methods and Related Topics for the Next Generation

PMo-160

13:30 － 14:40

Development of an Atmospheric Pressure Laser Ionization Techniqueusing a Novel 6 um-band Mid-Infrared Tunable Laser and Liquid Water
Matrix

Ryuji Hiraguchi, Hisanao Hazama, Kunio Awazu
Osaka University, Osaka, Japan

Keywords:
Mid-infrared tunable laser, Atmospheric pressure ionization, Water, Matrix-free, O-H bending vibration
Novel aspects:
We ionized peptide in aqueous solution under atmospheric pressure by irradiation of mid-infrared laser with a wavelength around6 um and peak intensity was strongest at the wavelength 5.95 um.
Abstract:
Purpose: The purpose of this study is to develop a new laser ionization method using absorption corresponding to O-H bending
vibration of water and to evaluate the wavelength dependence of the ion signal intensity. Ionization of peptides in aqueous
solution by using a mid-infrared laser with the wavelength of 3 μm corresponding to the O-H stretching vibration under theatmospheric pressure has been demonstrated1) 2). On the other hand, the 6 μm-band mid-infrared has advantages compared tothe 3 μm-band as follows：
Various molecular vibration modes such as O-H bending vibration, C=O and C=N stretching vibration have characteristic
absorption in the 6 μm-band. Thus, not only water but also various solvents and substances can be used as matrix. In addition,
the change in the absorption peak caused by the temperature rise for the O-H stretching vibration at the wavelength of 2.94μm.
However, the atmospheric pressure ionization using 6 μm-band mid-infrared laser has never been reported bacause it has beendifficult to obtain a high power tunable laser in the 6 μm-band. We have investigated using a 6 μm-band mid-infrared tunable
laser which has recently been developed.
Materials and Methods: A mid-infraraed tunable pulesed laser using difference-frequency generation（DFG）was used. The midinfrared DFG laser has a tunable wavelength range of 5.5-10.0μm, a repetation rate of 10 Hz, and a pulse width of 5 ns. The
tunable range of this laser includes the absorption peak of water corresponding to the O-H bending vabration at the wavelengh of
6.07μm.
--------In this research, atmospheric pressure laser ionization ion source was constructed and assembled on the commercial ion trap
mass spectrometer（LCQ Classic, Thermo Finnigan）. A heated capillary of the ion trap mass spectrometer was extended with aself-produced extension capillary, and the liquid sample on the stainless plate in front of the extension capillary was irradiatedwith DFG laser. Angiotensin（1045.54 Da, SIGMA-ALDRICH）was dissolved in 0.1% solution of trifluoroacetic acid（TFA）in water andadjusted to a concentration of 10 pmol/μL. A drop of this sample solution（ 1 μL）was dropped on the sample plate and laser
irradiation and acquiring mass spectrum was started within 1 minute after dropped. Laser energy was 250μJ/pulse, and injection
time of ion trap was 300 ms and measured 30 mass spectra were averaged.
Results and Discussion: Mass spectra of protonated angiotensin II（［M+H］+ ）were observed around the wavelength 6.07μm
corresponding to O-H bending vibration mode. Ionization was not observed on the wavelength of 5.75μm or less. Wavelength
dependence of the peak intensity of angiotensin II was similar to the IR absorption spectrum of liquid water. This result indicatedthat liquid water functioned as matrix. However, peak intensity at the wavelength of 5.95μm was stronger than at the wavelengtharound 6.07μm. As a reason for this experimental result, it is supposed that the pulse energy of 250μJ is too high at the
wavelength around 6.08μm. In this method, aromatic matrix such as
α-cyano- 4 -hydroxycinnamic acid （CHCA） and
2,5-dihydroxybenzoic acid（DHB）were not mixed into sample solution. Liquid water as solvent was utilized as also matrix.
Therefore, this technique showed promise for the application as an interface connecting liquid chromatography（LC）and mass
spectrometer. In the future work, laser irradiation condition will be optimized and online connection between LC and mass
spectrometer by this ionization technique will be investigated.
References
1 ）V. V. Laiko et al., J. Am. Soc. Mass Spectrom., 13, 354-361,（2002）
2 ）E. Rapp et al., Anal. Chem., 81, 443-452,（2009）

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 7: New Ionization Methods and Related Topics for the Next Generation

PMo-161

11:10 － 12:20

Influence of source parameters on the detection and the fragmentation ofaroma compounds using PTR-SRI-ToF MS

Xaviera Pennanec1,2,3, Etienne Semon1,2,3, Jean-Luc Le Quere1,2,3

INRA, UMR1324 Centre des Sciences du Gout et de l’Alimentation, Dijon, France, 2CNRS, UMR6265 Centre desSciences du Gout et de l’Alimentation, Dijon, France, 3Universite de Bourgogne, UMR Centre des Sciences du Gout etde l’Alimentation, Dijon, France
1

Keywords:
PTR-SRI-MS ； Flavouring Process ； Flavour Release ； Switchable Reagent Ion ； Real-Time Aroma Perception
Novel aspects:
Using Proton Transfer Reaction-Switchable Reagent Ion-Mass Spectrometry to measure headspace of aroma（in-vitro）and flavour
release in the expired air（in-vivo）to understand aroma perception and improve flavouring processes
Abstract:
Acceptability or rejection of foods by consumers highly depends on their aroma quality. Food aroma is a complex mixture ofseveral volatile compounds in a wide range of concentration and of chemical structure. During consumption, aroma release fromfoods is a complex sequential process involving interactions of flavour molecules with the matrix, the saliva and the chewing
activity. The gas phase is transferred to the nasal cavity where it has to reach the sensory receptors to be perceived. The gaseousatmospheres in the air above foodstuff（in-vitro conditions）and created during food consumption in the oral cavity（in-vivo
conditions）may be significantly different. Studying these two aspects is essential to investigate flavour release before and during
eating and ultimately to understand sensory perception. Proton Transfer Reaction mass spectrometry（PTR-MS）is a very efficienttechnique to continuously monitor volatile organic compounds. It can be advantageously used to analyse the headspace of anaroma solution（in-vitro conditions）but also air in the nasal cavity during eating（nosespace）, taking into account the dynamics
of aroma release induced in-vivo.
Proton Transfer Reaction mass spectrometry（PTR-MS）is a specific chemical ionization using H3O+ as precursor ion. H2O is firstly
ionized in the ion source to create H3 O + by electron impact. Then, reagent ions react with neutral molecules into a drift tube
mainly leading to the production of［M+H］+ protonated molecules. The reaction of proton transfer is only possible for analytemolecules with a proton affinity（PA）superior to the one of water（PA water = 165.1 kcal.mol -1 ； IP water = 12.62 eV）. The PA
consideration limits ionization opportunity. Thus a Switchable Reagent Ions（SRI）system on the PTR-MS source（Ionicon Analytik,
Innsbruck）enables to switch to other precursor ions i.e. O2 +or NO+. Hence, charge transfer ionization is possible for moleculeswith a lower ionization potential than the one of O2（PAO2 = 100.6 kcal.mol-1 ； IPO2 = 12.06 eV）. The use of NO+ mainly leads todehydrogenated cations［M-H］+ （PA NO = 125.7 kcal.mol -1 ； IP NO = 9.26 eV）. In addition to the SRI system, detection andfragmentation of analytes can be highly modified by varying drift tube parameters and more particularly the E/N ratio（E： electricfield strength in the drift tube； N： buffer gas number density in the drift tube）. The E/N ratio is interlocked with the physicochemical nature of the analytes and then to the mass spectra pattern through fragmentation and needs to be adapted to theapplications.
The aim of this work was to analyze the impact of the precursor ions produced in the SRI source combined with several E/N ratios
to optimize the analyses of aroma compounds. Analyses were conducted on the headspace of twelve different aroma compounds
belonging to various chemical classes, chosen for their frequent use in flavouring. Comparison of the signal response was followed
according to the reagent ion selected and 15 E/N ratios, focusing on the intensity, the sensitivity, the resolution and thefragmentation pattern of the analytes under every experimental condition. The results highlighted that both reagent ion andoptimal E/N parameter were closely linked to the chemical structure of the ionized molecules and that experimental parameters
have to be adapted to the applications, i.e. more or less fragmentation needed. When optimal parameters are determined, in-vivo
experiments can be realized.
These first in-vitro and in-vivo results show the massive potential of the PTR-SRI-MS technique to study flavour release in order tounderstand aroma perception and improve flavouring processes in food industry.

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 7: New Ionization Methods and Related Topics for the Next Generation

PMo-162

13:30 － 14:40

Expansion of an ionization technique in nonpolar solvents with cation
tetrakis(3,5-bis(trifluoromethyl)-phenyl)borate

Atsushi Takamizawa1,2, Naoto Niizuma2, Takashi Korenaga2
1

Japan Science and Technology Agency (JST), Saitama, Japan, 2Tokyo Metropolitan University, Tokyo, Japan

Keywords:
nonpolar solvent, ionization method, cationization reagent, electrolyte, nebulizer
Novel aspects:
An ionization method, developed in our laboratory, in nonpolar solvents with using cation tetrakis（3,5-bis（trifluoromethyl）-phenyl）borate（cation-TFPB）was improved by using a biological characteristic and interfacial chemical reactions.
Abstract:
Generally selection of ionization method changes by molecular species or experimental condition. The most efficient technique forone molecule is not useful for other molecules in many cases. In weakly polar molecules, it is difficult to observe a large part ofthese molecules by conventional methods. We have studied a novel method to enable to ionize a lot of weakly polar molecules
softly. This method has two important features using nonpolar solvent and cationization reagent（electrolyte）. Recently, it was
found that electrolytes containing tetrakis（3,5-bis（trifluoromethyl）phenyl）borate anion（TFPB - ）were useful as cationization
reagents to lipophilic analytes in nonpolar solvents. The TFPB anion possesses two characteristic properties, a large lipophilicity
and a small surfacecharge density, that result from the anion’s size； this makes the anion readily soluble in nonpolar solvents witha small donor number（DN）, such as dichloromethane and toluene. In most cases, cation or anion hardly exists independently innonpolar solvent. Above cationzation reagents（Li+,Na+ and Ag+ TFPB- ）, however, can slightly dissociate； unstable dissociatedcations easily can react with analyte molecules and form ion pair with TFPB-. This reactivity depends on chemistry between an
analyte molecule and a cation； the improvement of reaction efficiencies closely connects with increase of measurement
sensitivity. In this research, we discuss some methods for the improvement of reaction efficiency concerning cation-analyte
reaction in nonpolar solvents.
It is expected to enable ionization of only molecules containing a certain functional group by the suitable selection of cation
species. To achieve this selective ionization, we paid attention to a molecular selectivity in biological systems. The selectivity invivo is precise, and can be available for selective ionization. First, we focused on 8,13-Bis（ethyl）-3,7,12,17-tetramethyl-21H,23Hprphine-2,18-dipropionic acid iron（III）chloride Mesohemin（Fe（III）Mesoporphyrin IX chloride）, to synthesize electrolyte
containing a TFPB-. This molecule resembles heme closely and may combine to oxygen atom selectively. In fact, our experimental
results showed that Fe（III）Mesoporphyrin -TFPB had strong Lewis acidity for molecules with hydroxyl functional group（-OH）.
A liquid-liquid interface and a liquid surface are different from the bulk due to unbalanced forces exerted on particles in thisregion. We built a new sprayer with triple tube to use these conditions. A conventional sprayer is double tube and can flow asample solution and nitrogen（N2）. In a new triple tube, other one solution or air can be flowed； a liquid-liquid or a bubble-liquidinterface can be formed in the inside of the sprayer. With our ionization method using the conventional sprayer, cationizedmultimeres are produced in many cases. With the triple tube, a cationized monomer is mainly observed. These result indicate thatthe ionization process of analyte molecules in triple tube is different from the case of using the conventional sprayer.
Experimental
Cationization reagents were synthesized from sodium tetrakis（3,5-bis（trifluoromethyl）phenyl）borate（NaTFPB, purchased from
Dojindo）and salts containing objective cations by a solvent extraction method. Sample solutions were injected by syringe pumpand nebulized by a sprayer（in-house built）to form fine droplets. The sprayer was positioned parallel to the axis of a samplingorifice at a distance of about 1 mm from the orifice.

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 7: New Ionization Methods and Related Topics for the Next Generation

PMo-163

11:10 － 12:20

Gas chromatographic separation and accurate mass determination by low
temperature plasma ionization API-QTOF-MS

Asger W Noergaard, Vivi Kofoed-Soerensen, Peder Wolkoff, Per A Clausen
National Research Centre for the Working Environment, Denmark

Keywords:
Low temperature plasma； Gas chromatography； GC interface； Adduct ion formation；
Novel aspects:
A sensitive and versatile interface between a GC and an API-QTOF-MS based on low temperature plasma ionization facilitating
mass measurements with accuracies of 5 mDa.
Abstract:
Introduction
Low temperature plasma（LTP）is a recent ambient ionization technique where cold plasma（~30 °C）is utilized for direct mass
spectrometric analysis of liquid and solid samples, and compounds in the gas phase. The plasma is generated inside a small glass
tube by an alternating high voltage applied to an outer ring electrode, a grounded center electrode and a low flow（< 0.3 l/min）of discharge gas（He, Ar, N2 or air）. LTP is based on dielectric barrier discharges that are produced when a high voltage applied
to electrodes with dielectric insulators causes a series of micro-discharges due to electrical breakdown of discharge gas between
the electrodes. This series of micro-discharges induces a cascade of reactions that form a non-equilibrium low temperatureplasma rich in metastable neutrals as well as ionized species. When He is used as discharge gas, He 2 + is the dominant positive ion
in the discharge region inside the glass tube. As the plasma enters the open atmosphere, N2 + and subsequently water clusters（H2O）nH+ are formed by charge transfer reactions. Thus, LTP ionization in the positive mode usually yields protonated molecules,
however adduct ions such as［M+OH］+,［M+NO］+ and［M+NO2］+ may also be formed to some extents.
Since it’s launching in 2008 LTP has been used for a wide range of applications, e.g. coating products, explosives, pharmaceuticals
and olive oil. Compounds of interest are liberated from surfaces by thermal processes, thus LTP works best for compounds with avapour pressure. Compounds that elutes from a gas chromatograph（GC）should therefore be ideal for ionization by LTP. In order
to exploit the facilities for accurate mass measurements offered by an atmospheric pressure ionization quadrupole time-of-flightmass spectrometer in combination with a GC, LTP was used as an interfacing ionization technique.
Experimental
An LTP probe was mounted in a modified APCI source and carefully aligned with a GC-column in front of the inlet orifice. A heatedtransfer line was utilized in order to keep the column temperature at 225 °C from the GC to the ion source. Sample volumes of 1
ml were introduced via split-less injection onto a 30 m Agilent VF- 5 column with an inner diameter of 0.25 mm. The mass
spectrometer, a Bruker micrOTOF-Q, was operated in the positive mode and spectra were recorded at rate of 1 Hz. A massaccuracy of 5 mDa was obtained by lock mass calibration using phthalic anhydride（m/z 149.0223）as calibrant.
Preliminary results
Aromatic compounds, terpenes and terpenols in a test solution（dichloromethane）were successfully baseline separated. The GC
peaks had widths of 7 -14 s and the limit of detection was about 0.5 ng for most compounds. Protonated molecules［M+H］+ was
observed as the most abundant ion for all compounds. However, radical cations M +  · and adduct ions such as［M+OH］+ ,［M+O 2 H］+ ,［M+NO］+ and［M+NO 2 ］+ were also observed as abundant ions for several compounds. The number and type ofobserved ions differed with the analyte. For instance, a-pinene showed［M+H］+,［M+OH］+,［M+NO］+ and［M+NO2］+, while xylene
showed M +  ·,［M+H］+,［M+OH］+ and［M+O 2H］+. In general, the compound groups yielded different adduct ion patterns. These
differences in adduct ion formation might be useful for the identification of functional groups in analyte molecules.

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 7: New Ionization Methods and Related Topics for the Next Generation

PMo-164

13:30 － 14:40

Fast, High Throughput LC-MS/MS Method for ImmunosuppressantsAnalysis Using Dual Probe Electrospray

Lisa Cousins, Heather Gamble, Hui Qiao, Frenny Ruparelia, George Scott, Sha Joshua Ye
IONICS Mass Spectrometry, Toronto, Canada

Keywords:
Immunosuppressants； dual source； mass spectrometer
Novel aspects:
Demonstration of a highly sensitive dual electrospray ion source method that optimizes throughput for clinical settings
Abstract:
Objective
Immunosuppressant panels are now commonly used by clinical mass spectrometrists for patient management, for example toensure succesful organ transplants. A fast, sensitive, and accurate LC-MS/MS quantitation method is presented using a novel,
highly sensitive dual probe electrospray ion source for analysis of Tacrolimus, Sirolimus, Everolimus and Cyclosporin A using
Cyclosporin D and Ascomycin as internal standards.
Methods
Whole blood spiked samples were cleaned up by mixing one volume of serum with two volumes 0.1 M ZnSO 4 precipitation
solution containing the internal standards（Cyclosporin D and Ascomycin）. Three level QCs were purchased from UTAK（Valencia,
CA）. The mixture was vortexed for one minute followed by centrifugation for 15 min. The supernatant was transferred to a clean
vial for quantitation by two-dimension LC-MS/MS. 20 µL of supernatant were loaded on a Poros R 1 /20 pretreatment column（30x2.1mm）for on-line washing with water for a half minute at a liquid flow rate of 3 mL/min, then eluted by an Imtakt Cadenza
CD-C18HT analytical column（50x2.0mm, 3 µm）with 98% methanol in 10mM NH 4 OAc and 0.1% acetic acid for 1.5 min at 0.6 mL/
min. The signal is detected by an IONICS 3 Q 100 Series dual probe ion source triple quadrupole coupled to two Shimadzu
Prominence UPLC systems（each LC is equipped with a set of pretreament and analytical columns）. The total LC cycle time for
an injection is 2 min and the sample analysis time is 1 min with alternating injections from each LC. All the solvents used in thismethod are HPLC grade.
Results
This method covers a concentration range of three orders of magnitude from 0.2 to 200 ng/mL for Tacrolimus, Sirolimus, Everolimus
and 2 to 2000 ng/mL for Cyclosporin A, while maintaining good linearity（R 2 = 0.998）. The intraday and interday CVs for threelevels QCs were all < 9 % and < 11%, respectively. No interference or cross contamination was observed.
Conclusion
A sensitive, reliable and accurate LC-MS/MS method was developed and validated for quantification of Tacrolimus, Sirolimus,
Everolimus and Cyclosporin A in whole blood. The use of novel dual electrospray source multiplexing technology allows a sample
analysis time of only one minute, which doubles the throughput. This LC-MS/MS method requires simple sample preparation andis well-suited for routine therapeutic drug monitoring of immunosuppressive drugs.

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 7: New Ionization Methods and Related Topics for the Next Generation

PMo-165

11:10 － 12:20

Evaluation of Pt-deposited Porous Alumina Target Plate with MALDI
Spiral-TOFMS

Takafumi Sato1, Kanae Teramoto1, Yoshihisa Ueda1, Tomohiko Tamura2, Moriyuki Hamada2,
Ken-Ichiro Suzuki2, Yoshinao Wada3, Takashi Yanagishita4, Hideki Masuda4,5

1
JEOL ltd., Tokyo, Japan, 2NITE Biological Resource Center, Kisarazu, Japan, 3Osaka Medical Center and ResearchInstitute for Maternal and Child Health, Osaka, Japan, 4Tokyo Metropolitan University, Tokyo, Japan, 5Kanagawa
Academy of Science and Technology, Sagamihara, Japan

Keywords:
SALDI, Matrix free, MALDI, Spiral-TOFMS, Mycolic acid
Novel aspects:
Pt-deposited Porous alumina plate as a target plate of SALDI MS was developed. Combination of Pt-deposited porous alumina
target plate and MALDI Spiral-TOFMS is allowing easy identification of mycolic acids.
Abstract:
Introduction
MALDI-MS came into wider use for biological samples. However, low molecular weight compounds including lipids are difficult tobe analyzed by MALDI-TOFMS, because many intense peaks derived from chemical matrix appear in the low mass region.
We have developed a surface-assisted laser desorption/ionization（SALDI）target plate made of Pt-deposited porous alumina
surface. The pore dimensions were 100-200 nm in diameter and ca. 500 nm in depth and Pt thickness is less than 50nm. As porousalumina target plate does not require matrix, the resulting mass spectra are clean in the low mass region, and sample preparation
is easier than that for MALDI.
In this study, mycolic acid methyl esters were analyzed by Pt-deposited porous alumina target plate for evaluation of its suitability
for the SALDI platform.
Experimental
Mycolic acids were extracted from actinomycetes（Rhodococcus erythropolis NBRC100887, R. erythropolis NBRC13914, R. equi
NBRC101255T, Gordonia amarae NBRC15530T）and they were methylesterified. Each sample was dissolved in tetrahydrofuran（THF）, and sodium iodide（NaI）, a cationizing reagent, was dissolved in THF at 1 mg/ml. For sample preparation, the NaI
solution was deposited on a porous alumina target plate, and then overlaid with sample solution on the plate. In case of
conventional MALDI measurement, 2,5-dihydroxybenzoic acid as a matrix compound was overlaid with sample and NaI solutions
on a stainless target plate.
MALDI Spiral-TOFMS（JMS-S3000： JEOL）equipped with Nd：YLF laser（UV： 349nm）was used for SALDI MS and MALDI MS. The
effective flight length of Spiral-TOFMS is ca. 17m, and over 60,000 of resolving power（m/Δm： FWHM）is obtained at m/z 2,500.
Acquired mass spectra were analyzed by Polymerix（Sierra Analytics, Modesto, CA）software and a commercially available spread
sheet software.
Results
In both of SALDI and MALDI mass spectra, the sodium-adduct ions of mycolic acid methyl esters were observed in a m/z 400-900
region where five different species of mycolic acid molecules were identified. Both mass spectra were similar each other withrespect to the relative intensities of the mycolic acid ions. However, the peaks derived from the matrix compound were notobserved in the SALDI mass spectrum, allowing easy identification and assignment of the ions especially in the low mass region
compared with MALDI mass spectrum. These results indicate that the Pt-deposited porous alumina target plate is useful as asample target of SALDI MS.
Acknowledgement
The work was partly funded by a grant from the Institute for Fermentation, Osaka（IFO）.

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 7: New Ionization Methods and Related Topics for the Next Generation

PMo-166

13:30 － 14:40

New Version of Desorption Corona Beam Ionization Source andApplications

Yupeng Cheng, Junsheng Zhang, Joseph Ting, Wenjian Sun
Shimadzu Research Laboratory (Shanghai) Co., Ltd, Shanghai, China

Keywords:
DCBI； ionization source； direct analysis
Novel aspects:
DCBI source is capable of direct analysis without sample pretreatment.
Abstract:
A Desorption Corona Beam Ionization（DCBI）source has been reported previously with its capability of providing an alternative
direct analysis method mainly for analytes with weak polarity and low mass. In the current version of our DCBI source, a numberof new features were introduced while maintaining helium as discharge gas for forming the visual corona beam which serves
desorbing and ionizing samples from surface. A new set of DCBI source was developed and it contains a newly designed DCBI
probe and its control module. The thin wall stainless steel tubing used for gas heating is effective for fast temperature variationand the desorption temperature the probe can reach is beyond 375 o C at its maximum flow rate of 2 L/min. Due to the highdesorption temperature, molecules with mass up to 1544 dalton have been detected successfully. For implementing highthroughput and reproducible analysis, automated sampler as well as manual sampler has been developed. Some features of the
DCBI source including thermal stability of the probe（+/- 5 &#8451；, maximum 375&#8451；）, sensitivity（hexyl cinnamaldehyde
1 pg, S/N= 6 ： 1 at m/z 217, scan mode）, reproducibility（RSD= 8 %（n= 4 ））and dynamic range（ 5 orders）have been obtained.
With this improved version, the DCBI source was further tested in the fields of food/drug safety and environmental monitoring.
Results will be shown for the analysis of compounds in complex matrices such as herbal medicine, blood and urine. Data for
combining this DCBI source with some newly developed sample preparation techniques（such as Frontal Elution Paper
Chromatography and Micropipette Tip-based Micro-extraction）will also be presented for the purpose of furthering reducing theeffect of matrices.

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 7: New Ionization Methods and Related Topics for the Next Generation

PMo-167

11:10 － 12:20

Development of TLC/MS by LI-API and WAr-APPI (Photoionization in
Argon Gas Contained Water) Method

yoshiyuki mochida, gennki kobayashi, futoshi matumoto
Kanagawa Univercity, Yokohama, Japan

Keywords:
Thin Layer Chromatography, TLC/MS, Atmospheric Pressure Ionization, Photoionization, Penning Ionization
Novel aspects:
A photoionization method with the UV lamp which we developed was performed in argon gas soaked with water. and, New TLC/
MS using this method and LI method were developed.
Abstract:
A WAr-APPI method, ionization with low-pressure mercury lamp which installed in flow of moist argon gas, has been found by me.
This method is one of a photoionization different from penning ionization of direct analysis in real time（DART）. A penningionization（LI-API）method of the liquid developed by Tuchiya which does not contain water in gas was also considered. As asimple separation method and a detection for sample, thin layer chromatography（TLC）is widely used like column chromatographeven now. In mass spectrometry of TLC, TLC/MS using desorption electrospray ionization（DESI）, DART, and matrix-assisted laser
desorption/ionization（MALDI）as a detecting element will be in use. They, WAr-APPI and the LI-API, were discussed for ion sourceof the TLC/mass spectrometry. Additionally, we developed desorption method of sample which developed on thin layer（TL）
plate and the method of ionizing efficiently. They were carried out by the following methods. TL plate was intercalated in the
portion which processed to concave state a tip of the tungsten pipe. And, hot argon gas that controlled temperature was sentfrom lower side of pipe. A vaporized sample detached from TL plate was ionized by different two ionization methods used underatmospheric pressure. In the case of a put sample on sample holder, in ionizing of peroxides sensitive with heat by LP-API,
although it is the ionizing method excellent in the usual sample, No protonated molecule（［M+H］+ ）of their sample could beobserved. However, it was solved by using an addition reagent. On the other hand, by the WAr-APPI method, the［M+H］+ ofperoxide such as t-butyl peroxy laurate appeared without decomposition. The reason why the peroxide sensitive with heat does
not disintegrate will be that water acted as a decomposition suppressant. To this verification, the effect of the added water inargon gas and the appearance state of the fragment ion which occurs with heat was investigated. With the increase in water, thefragment ion of peroxide generated with heat disappeared, and no［M+H］+ was observed in case of dry argon gas. Therefore, it
seemed that this method was good to detect ion of the peroxide. As for helium gas, the effect was smaller than that of argon gas.
No nitrogen gas was observed. At TLC, or desorption from TL plate of sample, the ionization was performed to both LI-API and
WAr-APPI. Both solid methyl stearate and liquid methyl laurate at the room temperature which used as a primary standard sample
presented the signal of the beautiful［M+H］+. The limit of detection of methyl stearate by LI-API method was 6 ng which isenough to actually use. This fact shows that desorption of sample and ionization were fully performed. A new method ofdesorption of sample on TL plate was a method which is heated from the two side of the surface of the plate, unlike the reflective
method on the plate of DART or desorption electrosplay ionization（DESI）. Therefore, it is thought that sample detached withthis method from TL plate efficiently. Furthermore, as dimethyl sulfoxide（DMSO）solvent is difficult for MALDI/MS, WAr-APPI also
examined. A［M+H］+ of the synthesized sample was clearly observed without the influence of the solvent. As for LI-API, theinfluence of various solvents such as DMSO, tetramethylsilane）（TMS）, and tetrahydrofuran（THF）was investigated similarly. The
analytes were used the synthesized sample with the functional group（such as hydroxy, oxo, double bond, and bromide）which
may be affected by heat. We were satisfied with the obtained result. In addition, Ni complex has also been measured on a parwith fast atom bombardment（FAB）used for metal complex.

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 7: New Ionization Methods and Related Topics for the Next Generation

PMo-168

13:30 － 14:40

Charged-droplet beam source using vacuum electrospray of an ionicliquid for secondary ion mass spectrometry (SIMS)

Yukio Fujiwara, Naoaki Saito, Hidehiko Nonaka, Shingo Ichimura
AIST, Tsukuba, Japan

Keywords:
vacuum electrospray, charged droplet, ionic liquid, ion beam, cluster, SIMS
Novel aspects:
Obtained results are important to develop a primary ion beam source for SIMS, which is capable of producing massive ions at
higher current density with a smaller beam spot size.
Abstract:
Secondary ion mass spectrometry（SIMS）is a powerful technique for characterizing the surface and interface compositions of
inorganic and organic materials. Polyatomic（or cluster）primary ion beams can greatly increase yields of higher-molecular-weight
secondary ions, enabling molecular imaging of organic and biomolecules.［ 1 ］For these reasons, cluster ions, such as Au3+, C60+,（H2O）90,000+100H］100+, have been used in recent years.［ 1 - 4 ］
Ir（CO）
4
7 +, Ar n + and charged droplets［
For further improvement in SIMS analysis, it is required to develop a new cluster ion source capable of producing massive ions at
higher current density with a smaller beam spot size. From the viewpoint of generating such massive ions, vacuum electrospray ofionic liquids is expected to have a great potential； for instance, it probably improves beam-transport efficiency, thereby increasing
current density as well as beam current［ 5 - 7 ］.
Ionic liquids［C+A-］represent room-temperature molten salts consisting of a cation［C+］and an anion［A-］. Since they have anegligible vapor pressure and a high ionic conductivity, it is possible to electrospray them in a vacuum. A wide variety of ionicliquids has been synthesized commercially in recent years. Among these, we tested a quaternary ammonium ionic liquid, N,Ndiethyl-N-methyl-N-（ 2 -methoxyethyl）ammonium bis（trifluoromethanesulfonyl）amide（DEME-TFSA）.［ 8 ］
To develop a vacuum-electrospray beam source for SIMS, we investigated beam characteristics of charged particles
electrosprayed in vacuum in negative-ion mode as well as positive-ion mode.［9,10］In terms of practical use as a primary ion
beam source in SIMS, several factors have to be taken into account； e.g.,（i）beam current,（ii）efficiency of beam generation, and（iii）distributions of mass and charge of emitted droplets. For these reasons, we have investigated such factors under various
experimental conditions［ 8 -11］. Obtained results indicate that vacuum electrospray of ionic liquids is applicable to a massivecluster beam source for SIMS.
［ 1 ］N. Winograd, Anal. Chem. 77, 143A（2005）.
［ 2 ］Y. Fujiwara et al., J. Appl. Phys. 100, 043305（2006）.
［ 3 ］S. Ninomiya et al., Nucl. Instr. and Meth. B, 256 493（2007）.
［ 4 ］K. Hiraoka et al., Eur. Phys. J. D., 38, 225（2006）.
［ 5 ］Y. Fujiwara et al., Proc. 7 th Int. Symp. Atomic Level Charact. for New Materials and Devices, 2009, p471.
［ 6 ］Y. Fujiwara et al., Jpn. J. Appl. Phys. 48（2009）126005.
［ 7 ］Y. Fujiwara et al., Vacuum 84（2010）p544.
［ 8 ］Y. Fujiwara et al., Nucl. Instr. and Meth. B, 268（2010）1938.
［ 9 ］Y. Fujiwara et al., Chem. Phys. Lett., 501（2011）335.
［10］Y. Fujiwara et a., Jpn. J. Appl. Phys., 51（2012）036701.
［11］Y. Fujiwara et al., J. Appl. Phys., 111（2012）064901.

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 7: New Ionization Methods and Related Topics for the Next Generation

PMo-169

11:10 － 12:20

High Throughput Molecular Weight Confirmation of Pharmaceutical
Compounds Using DART MS Analysis with Ultra-fast Polarity Switching

Teruhisa Shiota1, Hirotaka Honda1, Jun Watanabe2, Kazuo Mukaibatake2, Junko Iida2, Tsubasa Ibushi3,
Manabu Ueda2
1

AMR, Inc., Tokyo, Japan, 2Shimadzu Corporation, Kyoto, Japan, 3Shimadzu Techno Research, Kyoto, Japan

Keywords:
Ultra-fast Polarity Switching, Direct Analysis in Real Time, Pharmaceutical Compounds, DMSO
Novel aspects:
A high throughput analysis method for pharmaceutical compounds dissolved in non-LC/MS compatible solvent utilizing the ultrafast polarity switching was developed.
Abstract:
Introduction
DART, a direct atmospheric pressure ionization source, is known to have little or no negative effect from the solvent used todissolve the sample. Even with problematic solvents for LC/MS operation such as non-volatile solvents or solvents containing salt,
the target compounds can be instantly identified using DART-MS without any sample preparation. High-throughput MW
confirmation of synthesized compounds is difficult to achieve using time of flight mass spectrometers, which have been a common
choice to interface DART, due to its limitation in switching between positive and negative ion modes.
By combining an automated DART ion source with a quadrupole mass spectrometer with ultra-fast polarity switching capability, 11different pharmaceutical compounds with various polarities were successfully determined in approximately 10 sec/sample.
MethodDART-MS analyses of 10ppm pharmaceutical compounds dissolved in 100 % DMSO were carried out. The DART-SVP ion source（IonSense, MA, USA）was coupled to the single quadrupole LCMS-2020（Shimadzu, Kyoto, Japan）, and ID CUBE（IonSense）, the
new type of DART, was also coupled to the said mass spectrometer. Introduction of samples for DART-SVP was automated byusing the X-Z scanner, which is capable of automating up to 96 samples/run. Ultra-fast polarity switching was utilized on the mass
spectrometer to collect full scan data. LCMS-2020 can achieve the polarity switching time of 15msec and the scanning speed of up
to 15000u/sec, therefore the loop time can be set at less than 1 second despite the relatively large scanning range of 100-1000u.
Preliminary DataFirst, the new ID CUBE ion source was tested and successfully interfaced to LCMS-2020. Mass spectrum was identified for both thepositive standard sample of 100ppm quinine and the negative standard sample of 100ppm methylparaben. 5 uL of samples werespotted on a specialized sampling card and inserted into the ID CUBE source. Vaporization of sample is more rapid with ID CUBE
than it is with DART since the sample is directly heated with electric current running through the metal mesh on which the sample
is applied, while the DART heats the sample with heated gas. Next, the DART gas（helium）heater temperature was raised to 350C
and commercially available pharmaceutical compounds such as Atenolol, Warfarin, Yohimbine, Cilostazol, Nifedipine, Diazepam,
Nitrendipine, and Diphenhydramine were applied on a metal mesh of the X-Z tool, which has the same size and configuration as
the 96 well microtiter plate, to automatically introduce them into the DART ionization gas. Relatively large DMSO peak was
observed in positive mode scan, but the target compounds were successfully identified as well. Positive ion spectrum wasobserved for Atenolol, Diazepam and Diphenhydramine. Negative ion spectrum was observed for Nifedipine and Nitrendipine.
Both positive and negative ion spectra were observed for Warfarin, Yohimbine and Cilostazol. DART-MS with the ultra-fast polarity
switching and ultra-fast scanning demonstrated its ability to perform high throughput analysis of pharmaceutical compounds
dissolved in high concentration DMSO at approximately 10sec/sample.

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 7: New Ionization Methods and Related Topics for the Next Generation

PMo-170

13:30 － 14:40

Componential Analysis of Pepper of Various Origins Using DART-MS Using
Ultra-fast Polarity Switching

FUMIHIKO USUI1, Teruhisa Shiota1, Manabu Ueda2, Kazuo Mukaibatake2, Junko Iida2, Tsubasa Ibushi3,
Yasuhiko Bando1, Jun Watanabe2
1

AMR INCORPORATED Tokyo,Japan, 2Shimadzu Corporation, Kyoto, Japan, 3Shimadzu Techno Research, Kyoto, Japan

Keywords:
Flavor, Origin, Multivariable Analysis, Ultra-fast Polarity Switching, Direct Analysis in Real Time
Novel aspects:
Rapid DART analysis of food samples using ultra-fast polarity switching to conduct multivariable analysis to distinguish their origins.
Abstract:
Introduction
DART（Direct Analysis in Real Time）, a direct atmospheric pressure ionization source, is capable of analyzing food samples withlittle or no sample preparation. Time of flight mass spectrometers, which have been a common choice to interface DART, were
not suitable in carrying out simultaneous detection of compounds with different polarities from the same sample due to its
limitation in switching between positive and negative ion modes in terms of time required to do so,.
Analysis of different parts of pepper（exodermis, endodermis and seeds）along with olive oil and soy source of different types andorigins were carried out using the DART combined with a mass spectrometer with ultra-fast polarity switching capability to
conduct multivariable analysis.
MethodCommercially available peppers from different origins were introduced to the DART gas using tweezers. Glass capillary was used
to dip and transfer olive oil to the ionization area, and soy source was spotted on a metal mesh to scan with the DART beam. TheDART（IonSense, MA, USA）was interfaced onto the single quadrupole mass spectrometer LCMS-2020（Shimadzu, Kyoto Japan）.
Ultra-fast polarity switching was utilized on the mass spectrometer to collect full scan data. LCMS-2020 can achieve the polarity
switching time of 15msec and the scanning speed of up to 15000u/sec, therefore the loop time can be set at less than 1 second
despite the relatively large scanning range of 100-1000u.
Preliminary Data
Capsaicin（C18H27NO3, MW 305）, which is responsible for the spicy flavor of pepper, is usually most abundant in placenta.
Commercially available peppers from Korea, China and several areas in Japan were cut and separated to each part of theexodermis, endodermis and the seed and each sample was analyzed by DART-MS. The signal indicating capsaicin was detected inboth polarities； at m/z 306 in positive ion mode, m/z 304 in negative mode. After careful optimization of analytical conditions using
different DART gas heater temperatures at 200C, 350C and 500C, it was determined that 350C was the suitable temperature setting
to analyze capsaicin. In most of the samples, the signal of capsaicin was most intense in seeds, which implicates that there wereplacental fragments on the surface of the seeds, which, even in small quantity, was giving out the intense signal for capsaicin.
Next, soy sources and fish sources from different origins were analyzed. Low molecular weight spectra were observed in different
profiles depending on the sample ingredients, indicating the different balances in amino acid contents.
For olive oil and grape seeds oil samples, high molecular weight spectra were more dominant in both polarity at temperature
setting of 500C than 200C and 350C, suggesting the possibility to analyze the differences in triglycerides contents.
Multivariable analyses were conducted using these spectral data to distinguish different food samples from different origins and/or deferent ingredients. This indicates that DART-MS can be a powerful tool for origin determination/authentication of agricultural
produce and processed goods.

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 7: New Ionization Methods and Related Topics for the Next Generation

PMo-171

11:10 － 12:20

Gas Chromatography/Ambient Ionization Mass Spectrometry (GC/AMS)
for the Analysis of Long Chain n-Alkanes in Crude Oil

Jentaie Shiea1, Min-Zong Huang1, Yi-Tzu Cho2
1

RIKEN ASI, Wako, Japan, 2Yuh-Ing Junior College of Health Care & Management, Kaohsiung, Taiwan

Keywords:
Ambient Ionization Mass Spectrometry；Gas Chromatography；Alkanes；Crude Oil
Novel aspects:
Gas chromatography/ambient ionization mass spectrometry was demonstrated to analyze long chain n-alkanes in crude oil.
Abstract:
Gas chromatography（GC）is a well established analytical tool commonly used to separate organic mixtures. The analytes eluted
from GC were ionized by electron impact ionization, chemical ionization, electrospray ionization（ESI）, or atmospheric pressurechemical ionization（APCI）（also referred as corona discharging mechanisms）. In this study, we combined gas chromatography
with ambient mass spectrometry（AMS）using a plasma based-APCI as the ionization source and interface. The interface of GCAMS consists of a heated metal tube inserted in a stainless steel chamber and a plasma based-APCI ion source. The ion source isa glass tube covered by a small stainless steel tube as the counter electrode and a ring electrode. As helium stream flows throughthe APCI source, plasma is generated between the two electrodes.
For a typical analysis, samples were injected in gas chromatography for separation. The separated analytes were carried throughthe heated stainless steel tube at high temperature, and flowed into the plasma based-APCI source. The analytes were ionized byinteracting with the helium plasma species, and subsequently detected by a Q-TOF mass analyzer. For example, a mixture of long
chain n-alkanes（n-C 8 to n-C 34 ）and fatty acid methyl esters（FAMEs）standards were prepared and used to examine theperformance of the GC/AMS. As it was expected, protonated molecular ions（M+H）+ were detected for all FAMEs. Since themolecules of long chain n-alkanes contain no functional groups to receive proton during ion-molecule reactions, no protonatedmolecular ions was detected. Instead, nitrogen adduct ions［（M+N-H）+］were detected for all n-alkanes.

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 7: New Ionization Methods and Related Topics for the Next Generation

PMo-172

13:30 － 14:40

A Combined DSMC - CFD Approach for the Numerical Simulation of an
Under-expanded Jet

Athanasios Zacharos2, Ioannis Nikolos3, Dimitris Papanastasiou2, Roger Giles1, Andrew Entwistle1,
Emmanuel Raptakis2

1
Shimadzu Research Laboratory (EUROPE) LTD, 2Fasmatech, Athens, GR, 3Department of Production Engineering &Management, TUC, Chania, Crete, GR

Keywords:
DSMC, Under-expanded Jet, CFD
Novel aspects:
A new method for calculating the free jet expansion region with greater precision has been developed. A combined approach
using DSMC and CFD is employed.
Abstract:
A combined Direct Simulation Monte Carlo（DSMC）Computational Fluid Dynamics（CFD）approach is adopted to calculate thefree jet developed through a long capillary inlet at the fore vacuum of a mass spectrometer. The transition from the continuum tothe rarefied flow regime requires different solvers to be employed in order to describe the free jet with greater precision. The
simulation domain is divided into two regions, the continuum regime where the CFD method can be applied successfully and themolecular flow regime where the DSMC method is well known to provide more physically accurate results. The combined solution
is compared with the one obtained by applying the Navier-Stokes equations across the entire domain.
The CFD solver is first applied across the entire domain, which includes the atmospheric pressure region connected to the forevacuum region through a 100 mm long 0.5 mm ID capillary. Nitrogen pressure in the vacuum region is set to 20 mbar. The DSMC
solver is applied across the last 2 mm of the capillary and throughout the low pressure region. The DSMC boundary conditions at
the inlet of the domain are determined by the CFD calculations. The DSMC code has been modified to accept the parabolic
velocity profile of the gas flow near the capillary exit. Salome TM open-source software is used for the generation of the
unstructured mesh and computations are performed in OpenFOAMTM. The DSMC simulations are performed on the grid computer
system of the Technical University of Crete, comprising 44 HP Proliant BL465c server blades with two AMD Opteron Model 2218
processors each.
The CFD solution for the nitrogen free jet expansion at 20 mbar shows the formation of a barrel shock, the presence of a Machdisk formed a few mm downstream the capillary outlet followed by the silent zone. Oscillations of the axial velocity observed
further downstream are attributed to shock waves of decreasing strength. The onset of turbulence at the jet breaking point is also
clearly observed including the region where the flow becomes subsonic. The region where CFD is expected to provide resultswith greater accuracy is the flow across the capillary where the drop in pressure is accompanied by a significant acceleration of
the gas toward the outlet. No slip conditions are imposed across the length of the capillary. In contrast, the DSMC solver showsthat slip velocity conditions develop near the outlet despite the short distance allowed for the gas to accelerate near the capillaryoutlet. The two solutions are compared in terms of the structure of the barrel shock, the Mach disk, the axial velocity oscillations
and radial velocity profiles. The turbulence and subsonic regions developed deep inside the vacuum region where the DSMC
solution is expected to give more accurate results are also investigated in greater detail. The discrepancies and limitation of the
DSMC and CFD methods with respect to their applicability on the free jet theme and with particular interest on the design ofmass spectrometers are highlighted.

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 7: New Ionization Methods and Related Topics for the Next Generation

PMo-173

11:10 － 12:20

Proposal of the New Method for 3DAP Analysis of Biological Molecules
Utilizing Photocatalyst and Mesoporous Materials

Masato Morita, Mahito Shimizu, Yuya Hanaoka, Masanori Owari
The University of Tokyo, Tokyo, Japan

Keywords:
3 DAP, Photocatalyst, Biological molecules, Mesoporous, Laser
Novel aspects:
The application of photocatalyst to the field evaporation for analyzing covalent materials in the 3 DAP.
Abstract:
The three dimensional atom probe（ 3 DAP）is one of the most unique three dimensional imaging technique which has high spatial
resolution and high detection sensitivity. The basic principle of the atom probe is based on field evaporation of surface atoms in
the needle whose apex is typically 100 nm in radius. These atoms are chemically mass indentified by time-of-flight. Three
dimensional position of each atom is calculated from the coordinates on the position sensitive detector and the sequence ofdetection. From such information, the three dimensional structure of the sample can be observed with atomic scale. However, theanalysis of covalent materials such as organic and bio molecules has not succeeded. If the 3 DAP analysis of organic material ispossible, many contributions to various fields are expected. In particular, the analysis of biological specimen may dramaticallyadvance researches of medicine and pharmacy.
During the 3 DAP analysis, a high voltage is applied to the sample. This results in a very strong electric field more than 10 V/nm at
the apex of the sample allowing ions to field-evaporate from the surface. The strong electric field induces mechanical forces（electric stress）at the sample, which eventually lead to the rupture of the sample. Therefore, a certain amount of conductivity
and mechanical strength are needed for the sample of 3 DAP analysis. From the beginning, 3 DAP specializes to the analysis ofhigh electric conductivity materials such as alloy. In recent years, by application of the femto second pulsed laser, 3 DAP has many
good contributions in the analysis of semiconductor-based electronics. However, in the case of analysis of covalent materials,
there are some difficulties： fragment ions which have various sizes are generated at field evaporation, sample fabrication byfocused ion beam（FIB）is very difficult, a rupture of sample by high electric stress frequency occurs, and so on. The fieldevaporation of carbon atom requires very high electric field（103 V/nm）compared to metallic atom. Therefore, a higher voltage isapplied to the organic sample and a higher electric stress causes the rupture of sample earlier than field evaporation. On theother hand, the fragment ions complicate the mass spectrum and worsen the spatial resolution. To resolve these problems,application of the short wavelength pulsed laser for the 3 DAP was proposed. However, the short wave length pulsed laser is very
expensive and causes the low output power. Since the photon energy assists the ionization, the adjustment of wavelength isneeded depending on the materials. Moreover, alignment between the sample apex and laser irradiation position is very difficult
with invisible wavelength.
In this study, we proposed the new ionization process for 3 DAP analysis of organic materials utilizing the photocatalyst. Thismethod enables to make organic materials ionize with lower voltage and the conventional wavelength laser. The adjustment ofwavelength is not required depending on the materials. It is expected that the extent of fragmentation is controlled and the mass
spectrum are not complicated. In this study, we evaluated the effect on the mass spectrum in the case of the new ionization
process utilizing the photocatalyst. Another problem to be resolved is the sample fabrication. The 3 DAP sample was preparedutilizing mesoporous materials. The mesoporous material was designed with photocatalyst material and an organic material was
enclosed in pore. Since a frame is an inorganic material, the needlelike sample fabrication by FIB is possible. Moreover, the sample
of covalent material is supplemented with a certain amount of conductivity and mechanical strength.

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 7: New Ionization Methods and Related Topics for the Next Generation

PMo-174

13:30 － 14:40

High Sensitivity Spray Ionization Approaches at AP and Vacuum with
Matrices or Solution With or Without Voltage or a Laser

Charles N McEwen1, Sarah Trimpin2
1

University of the Sciences, 2Wayne State University

Keywords:
Spray ionization, inlet ionization, MALDI, electrospray ionization, mechanism
Novel aspects:
Several new ionization approaches are presented that produce multiply charged ions from solution or a solid matrix, fromatmospheric pressure or vacuum, with or without a laser or voltage.
Abstract:
In mass spectrometry ’spray ionization’, means electrospray ionization（ESI）, but in nature charged droplets or particles arecommon, being produced from an ocean spray or even as a spray of charged ice particles from a freezing water droplet.
Producing ions from sprayed solutions for mass spectrometry has been accomplished using ESI, sonic spray ionization,
thermospray ionization, and more recently we introduced solvent assisted inlet ionization. There are also techniques such as fast
atom bombardment which may produce ions by ’microspray’ ionization . The commonality of these methods, operating at AP or
vacuum, are that the ions observed using mass spectrometry are produced from charged droplets similar to ESI, but without
applying a high voltage. We will demonstrate a highly sensitive solution spray ionization approach that uses a combination of
methods in which the ionization selectivity（or suppression）can be switched to favor different classes of compounds.
’Spray ionization’ is also applicable to ionization from solid matrices. Recently, laserspray ionization, a method operationally nearly
identical to AP-MALDI, was shown to produce multiply charged ESI-like ions from solid matrices commonly used in MALDI.
However, the laser was not necessary for ion production, and simply introducing the matrix analyte into a heated inlet tube linking
AP and the first vacuum region of a mass spectrometer produced nearly identical ESI-like ions. This approach was extended to
produce multiply charged ions using a vacuum MALDI source. We will provide data supporting the conclusion that solid matrices,
acting as solutions, produce ions by a form of spray ionization. Again, neither a laser nor a voltage is required, and we
demonstrate this by ionizing proteins directly from ice. The data provided by these experiments show that photochemical
ionization is not necessary for producing ions from proteins as large as BSA under MALDI conditions.
We will present new highly sensitive ionization methods, producing ions from solution or solid matrix introduced to the mass
analyzer from atmospheric pressure or directly into the vacuum, with or without use of a laser or voltage for ion production. The
mechanistic implications of these studies for the ionization of nonvolatile compounds will be related to a variety of ionization
approaches used in mass spectrometry.
Pagnotti VS, Chubatyi ND, McEwen CN： Solvent Assisted Inlet Ionization: An Ultrasensitive New Liquid Introduction Ionization
Method for Mass Spectrometry. Anal Chem 2011, 83：3981-3985.
Trimpin S, Inutan ED, Herath TN, McEwen CN： Laserspray Ionization - A New AP-MALDI Method for Producing Highly Charged
Gas-Phase Ions of Peptides and Proteins Directly from Solid Solutions.Mol Cell Proteomics 2010, 9：362-367.
McEwen CN, Pagnotti VS, Inutan ED, Trimpin S： A New Paradigm in Ionization: Multiply Charged Ion Formation from a Solid Matrix
without a Laser or Voltage.AnalChem 2010, 82：9164-9168.
Trimpin S, Ren Y, Wang B, Lietz CB, Richards AL, Marshall AG, Inutan ED： Extending the Laserspray Ionization Concept to Produce
Highly Charged Ions at High Vacuum on a Time-of-Flight Mass Analyzer. Anal Chem 2011, 83：5469-5475.

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 7: New Ionization Methods and Related Topics for the Next Generation

PMo-175

11:10 － 12:20

Fundamental Aspects of Solvent free Atmospheric Pressure Chemical
Ionization and Laser Diode Thermal Desorption Ionization Source (LDTD)

Pierre Picard1, Real E Paquin2
1

Phytronix Technologies Inc, Quebec, QC, Canada, 2Universite Laval, Quebec, QC, Canada

Keywords:
Desorption, Thermal； Ionization, Atmospheric Pressure Chemical； Mass Spectrometry
Novel aspects:
New insights in thermal desorption process involved in LDTD-APCI source are introduced in terms of enthalpy of volatilization,
thermodynamics and solvent less APCI.
Abstract:
Introduction
The Laser Diode Thermal Desorption（LDTD）ionization source allows the analysis by mass spectrometry of samples at
atmospheric pressure, minimizing preparation steps. The LDTD uses a Laser Diode in the transmission geometry to produce a
rapid thermal desorption of the sample deposited in a dedicated 96 well holder followed by atmospheric pressure chemical
ionization. The purpose of this work is to present the measurements of the main characteristics of the source and the associatedthermodynamics behavior.
Methods
A LDTD source was modified to monitor the temperature on the 96 well sample holder during a real-time thermal desorption
experiment. Different combinations of thermal desorption profile and carrier gas flow have been used to monitor the sample
holder surface temperature produced by the laser diode radiation. The thermal desorption of sulfadiazine in function of thesurface temperature was measured while the influence of the carrier gas flow on the signal intensity was evaluated using
clomiphene. Thermodynamic theory is applied to corroborate the experimental data and electron microscopy is used tocharacterize the sample crystallization on the surface of the well. All the experimental work has been performed on a LDTD model
T-960 interfaced to a TSQ Quantum Ultra AM.
Preliminary results
The gas flow applied to carry the vaporized neutral sample into the APCI region for ionization influences the signal forclomiphene. Maximum signal is obtained with a carrier gas flow set between 2.0 and 3.5 L/min. At a lower or higher carrier gas
flow, the signal intensity decreases and the variability increases. The intensity of the signal as a function of temperature for
Sulfadiazine gives the behavior of the volatilization process, showing beginning of the process at 60°C up to a maximum starting at
95 °C and stable until 140 °C is attained. The influence of the temperature sensor on the measurement is evaluated to be 14 %Computed temperature for the incident power shows good agreement with measured values at 200 °C. Deposit dimension
measured with the electron microscope on the surface are in the nanometer range as expected. The theory of the crystallization
at the nanoscale can then be applied to explain the lower value of volatilization enthalpy observed on compounds that should
decompose before melting as in the bulk experiment. Characterization of the APCI by monitoring the water concentration andsolvent addition show proton affinity effect. Optimums are confirmed by the protonation of a fully saturated fatty acid. Flow ofavailable protons produced by the corona discharge should be in theory at 10 -10 Mol/sec. Binary mixture of compounds
corroborate showing 50% suppression of the signal at 3.8x10-10 Mol vaporized.

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 7: New Ionization Methods and Related Topics for the Next Generation

PMo-176

13:30 － 14:40

Photo-ionisation mass spectrometry for on-line monitoring: Technology
and real-time monitoring applications such as characterisation of thecoffee-roasting and cigarette-smoking process

Ralf Zimmermann1, Romy Hertz1, Matthias Bente2, Mohammad Saraji2, Thorsten Streibel1

Joint Mass Spectrometry Centre of University Rostock and Helmholtz Zentrum München, Oberschleissheim/Rostock,Germany, 2Photonion GmbH, Schwerin Germany

1

Keywords:
photo ionisation, on-line real-time measurement, coffee-roasting, cigratte smoking
Novel aspects:
New application of on-line photo ionisation-MS, Setup of optimised VUV-light sources, On-line-analysis of roasting-degree andflavour formation-mechanisms（coffee）as well as pyrolysis chemistry and formation-mechanisms of hazardous chemicals（cigarette）
Abstract:
In the last decade, several soft ionisation Mass Spectrometric（MS）techniques for on-line monitoring of complex gas mixtures
were developed. This included Chemical Ionisation techniques as well as photo ionisation（PI）based mass spectrometrictechnologies. Photo Ionisation mass spectrometry is particularly well suited to monitor the chemical signatures in combustion andpyrolysis processes. On the one hand, the laser based Resonance Enhanced Multi-Photon Ionisation technique（REMPI）allows theultra-sensitive and selective detection of aromatic substances in complex gases； on the other hand the Single Photon Ionisation
method（SPI, with VUV-photons）features an overview characterisation of all organic compounds. Several photo-ionisation mass
spectrometric application e.g. for on-line monitoring of puff-resolved tobacco smoke［ 1 ］, waste incineration flue gas or pyrolysis
processes have been reported. In an on-line analysis study of the coffee-roasting process it was demonstrated that a prediction of
the degree of roasting from the chemical signature of the roasting-off gas is feasible by laser ionisation mass spectrometry（SPI/
REMPI）［ 2 ］. Recently also a less sophisticated SPI mass spectrometric photo-ionisation method, based on a novel lamp-like VUVlight-source（electron beam pumped rare gas excimer light-source, EBEL）, has been developed［ 1 ］. The EBEL-SPI technology
allows fast real-time mass spectrometric analysis of organic mixtures. Depending on the used wavelength（i.e. type of rare gas
filling of the EBEL）all organic substances are accessible including alkanes, alkenes, naphthenes, aromatic compounds, carbonylcompounds or heterocyclic species. Detection limits in the low ppb concentration range are achieved at a time-resolution of someseconds. The novel EBEL-SPI technique up to now has been used in conjunction with different mass spectrometric technologies（Quadruple-（QMS）, Time-Of-Flight-（TOF-MS）and Ion Trap-mass spectrometers（ITMS））. The EBEL-SPI-ITMS approach has
advantages for direct analysis of complex mixtures such as coffee roasting gases as a direct confirmation of the recorded analyte
is possible by tandem-MS［ 3 ］. Several practical applications of the SPI-MS technology with EBEL-VUV-light-sources（and laser
based REMPI/SPI）are discussed. This includes the analysis of the coffee roasting process, the puff-resolved detection of chemicals
in tobacco smoke as well as field-applications for process-monitoring（on-site-analysis）.
For the coffee-roasting application in addition to the measurement of the bulk gases also a newly developed µ-probe samplingtechnique was applied. This approach allows the monitoring of the formed roasting gases directly inside individual coffee beans：
A hole of ~400 µm diameter was drilled in the green coffee bean and the 300µm diameter stainless steel tip of the µ-probe wasinserted in the hole, which was sealed thereafter with inorganic glue. Subsequently the individual bean was hot air-roasted at 200°C and the evolved gases from the inside of the investigated bean were directly sampled into the PI-TOFMS. During the roastingprocess, a variety of organic compounds are sensitively detectable. The on-line detected chemical signature comprises a variety ofcompounds and compound classes including phenols, sulphur-compounds, carboxylic-compounds, nitrogen-compounds, furanderivatives, fatty-acids and of course caffeine. Typical time-intensity profiles were observed and different compounds have also a
different precursor behaviour and react also differently on the rupture of the cellular structure of the coffee-bean during roasting（cracking, caused by the CO 2 formation due to decarboxylation of ferula acid）. Measurements of several individual bean
roasting-profiles from the Robusta and Arabica coffee varieties have been statistically analysed. Markers for roast-degree andvariety-type have been identified. The µ-probe also was applied to analyse the chemical compounds in the pyrolysis- andcombustion-zones within a cigarette during puffing.
Additionally on-line process analysis results at a biomass-to-liquid plant are discussed.
References：［ 1 ］Anal. Chem. 77（2005）4708［ 2 ］Anal. Chem. 76（2004）1386［ 3 ］Anal. Chem. 81（2009）4456

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 8: Collision Dynamics and Spectroscopy Using Ion Storage Rings and Traps

PMo-177

11:10 － 12:20

Design of Electrodynamic Ion Funnel-Stacked Ring Trap

Liulin Deng, T-W Dominic Chan

The Chinese University of Hongkong, Hongkong

Keywords:
ion funnel, ion funnel trap, ion funnel-stacked ring trap
Novel aspects:
IF-SRT is consisted of ion funnel and stacked ring trap
Abstract:
Electrodynamic ion funnel（IF）is a device developed by Shaffer and co-workers1-3 for transmission of ions across the medium
pressure region（i.e. 1 30 torr）of typical atmospheric ionization sources. This device can transmit ions with wide spatial andenergy dispersions and focus them into a narrow ion beam. It was designed to replace the conventional skimmer electrode to
improve the ion transmission efficiency while maintaining the conductance limiting function. Electrodynamic ion funnel trap（IFT）
was proposed by Ibrahim and co-workers4 for accumulation of ions behind pre-IF and followed by post-IF which can extract andrefocus all trapped ions to down-stream region, high trapping efficiency can improve sensitivity of IMS and MS instruments. As partof the instrumental rejuvenation program, a 100-plate electrodynamic ion funnel was designed and fabricated in house to improve
the performance of the 4.7 Tesla FTMS instrument. Moreover, electrodynamic ion funnel-stacked ring trap（IF-SRT）we proposed
here has a different structure compared to IFT, it has two main sections： the first section is a IF which consists 66-plate to capture
and focuses ions with high transmission efficiency； the second section has 34 constant internal diameter（3.0mm）electrode rings,
effective potential in radial direction is generated by applying RF potentials with opposite phases in adjacent electrode rings,
different DC gradient potentials by adjusting a pulse voltage applied to the last electrode are utilized in axis direction to control
ions working in transmit（+ 5 V/cm）or trap（- 5 V/cm）mode. We have constructed IF-SRT model in SIMION 8.0 software andinvestigated ion transmission and trapping performance with different DC potentials of IF-SRT.
Reference:［ 1 ］ S. A. Shaffer； K. Tang； G. A. Anderson； D. C. Prior； H. R. Udseth； R. D. Smith Rapid Communications in Mass
Spectrometry1997, 11, 5.
［ 2 ］S. A. Shaffer； D. C. Prior； G. A. Anderson； H. R. Udseth； R. D. Smith Anal. Chem.1998, 70, 9.
［ 3 ］Y. Ibrahim； K. Tang； A. V. Tolmachev； A. A. Shvartsburg； R. D. Smith J Am Soc Mass Spectrom2006, 17, 7.
［ 4 ］Y. Ibrahim； M. E. Belov； A. V. Tolmachev； D. C. Prior； R. D. Smith Anal. Chem2007, 79, 7845.

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 8: Collision Dynamics and Spectroscopy Using Ion Storage Rings and Traps

PMo-178

13:30 － 14:40

Delayed electron emission and radiative cooling of stored molecular ionsin TMU electrostatic ion storage ringToshiyuki Azuma1,2, Motoshi Goto1,3, Jun Matsumoto3, Haruo Shiromaru3, Takeshi Furukawa2,
Takuya Majima2, Hajime Tanuma2, Tsuneto Kanai1, Erika Sunden4, Klavs Hansen4

RIKEN, Wako, Japan, 2Department of Physics, Tokyo Metropolitan University, Tokyo, Japan, 3Department of Chemistry,
Tokyo Metropolitan University, Tokyo, Japan, 4Department of Physics, University of Gothenburg, Gothenburg, Sweden

1

Keywords:
electrostatic ion storage ring, laser excitation, cooling process, slow dynamics
Novel aspects:
An electrostatic ion storage ring allows us to keep molecular ions in vacuum for a period up to a few minutes to study their slow
dynamics by combination of laser-excitation.
Abstract:
An electrostatic ion storage ring allows us to keep molecular ions circulating in vacuum for a long period up to a few minutes tostudy their slow dynamics. Since this type of electrostatic rings is free from restriction of the ion mass, large polyatomic molecularions can be stored.
By introducing a visible tunable laser, we investigated the evolutional change of the delayed electron emission from negative ionsdepending on the ion storage time, and succeeded in extracting the dynamics of the de-excitation process of the hot molecularions through delayed electron emission and IR radiation.
One example is determination of the cooling rate and the internal temperature of hot large polyatomic ions. The cooling rates of
hot C60 -   have been measured in the time region up to several tens of ms by observing the delayed electron detachment yield with
one-photon laser excitation. The absolute energy scale is established by the photon energy and the cooling time interval isderived from the non-exponential statistical decay of the ensemble of hot molecules.
Another typical example is a small molecular ion. One-photon laser induced decays have been measured for C5 - ions with smallheat capacity. Photon-induced decays were found to be again statistical and influenced by radiative cooling. We measured thephoto-enhanced decay at different storage times and photon energies. The electron detachment yield increased as the storage
time advances, and after the maximum, they reversely decreased. This characteristic behavior is well explained by the concept ofenergy window for electron detachment observation using this ion storage ring, and again allowed determination of the coolingrate. The average internal energy content varies as time to the power of 0.5, which is consistent with a T4 cooling power.
When the stored ion is a large molecule with a large heat capacity at moderately hot temperatures, these one-photon methods
are not applicable due to shortage of the necessary excess energy for electron emission. To heat such targets sufficiently, analternative scheme with sequential multi-photon laser excitation was also tried for zinc phthalocyanine anions, C32H16N8Zn-. We
extracted the information on the cooling behavior by observing the change in the fluence dependence of the laser induced
electron detachment yield.
Thus, by applying a variety of methods suitable for each target ion, we confirmed that the cooling behavior of the molecular ionsis well understood by the electrostatic ion storage ring and combined with a laser excitation.

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 8: Collision Dynamics and Spectroscopy Using Ion Storage Rings and Traps

PMo-179

11:10 － 12:20

Develapment of a cryogenic ion storage ring in RIKEN

yoshinori enomoto, yuji nakano, takuya masunaga, toshiyuki azuma
RIKEN, Wako, Japan

Keywords:
cryogenic ion storage ring
Novel aspects:
Our new ion storage ring realize completly cryogenic environment.
Abstract:
We have been developing a new electrostatic ion storage ring in RIKEN.
Compared with storage rings which use magnetic field to control ions, electrostatic ones can control ions regardless of their mass.
Thus heavy ions like biomolecule can be stored in the same way as light atomic ions.
First electrostatic ring, ELISA, was developed in 1997 at Aarhus University. Subsequently, similar rings were constructed several
places. However, these rings are operated in a room or liquid nitrogen temperature. Stored ions are heated by radiation fromelectrodes and walls of vacuum chamber. Vibrational or rotational states of them are excited and obtained spectra areconvolution of many states. In addition, storage time of ions is limited by collision with residual gas molecules. Although pressurein these rings is about 10-11 Torr, typical storage time is only 1 s. Thus it is difficult to observe slow or rare process.
As next generation storage rings to overcome these issues, cryogenic rings, whose environmental temperature is below 10 K, areunder development at several institutes in the world.
Features and design objectives of our ring is as follows.
（ 1 ）Ultimate environmental temperature is 5 K.
（ 2 ）Ultimate pressure in the ring is 10-14 Torr.
（ 3 ）Liquid He free cooling system.
At present, we finished detailed design of the ring, and it is under construction. We will show details and status of our new ring at
the conference.

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 9: Imaging II

PMo-180

13:30 － 14:40

Development of MALDI-MS-based imaging technique for visualizing thegreen tea polyphenol EGCG in mammalian tissues

Yoshinori Fujimura, Yoon Hee Kim, Takatoki Hagihara, Daichi Yukihira, Ayumi Yamaguchi, Miho Irie,
Daisuke Miura, Hiroyuki Wariishi, Mitsuru Shindo, Hirofumi Tachibana
Kyushu University, Fukuoka, Japan

Keywords:
MALDI-MS； Imaging； Polyphenol； EGCG； Mammalian-tissue
Novel aspects:
We succeeded for the first time in developing a novel MALDI-MSI technique for visualizing the green tea polyphenol EGCG on the
mammalian tissue section.
Abstract:
Introduction and objective： Epigallocatechin- 3 -O-gallate（EGCG）, the major active polyphenol in green tea（Camellia sinensis L.）,has been shown to possess various health promotion effects. Although the understanding of spatiotemporal distribution in tissues
is indispensable for elucidating the detailed mechanism of EGCG action, such information still remains unclear. Mass spectrometry
imaging（MSI）is a remarkable new technology that enables us to determine the distribution of endogenous or exogenous
molecules present in tissue sections by direct ionization and detection with matrix-assisted laser desorption ionization（MALDI）-MS 1 . Recently, we have developed the highly sensitive MSI technique for visualizing of spatiotemporal dynamics of the tissue
metabolome2. In this study, we attempted to develop a MALDI-MSI technique for visualizing two-dimensional distribution of EGCG
in tissue micro-regions.
Methods and results： We have recently reported that a MALDI-MS system with 9 -aminoacridine（ 9 -AA）achieved a great
improvement for the sensitivity of low-molecular-weight metabolite analysis that is advantageous for the simultaneous detectionof a variety of cellular and tissue metabolites2-4. Here we applied this technique for imaging the major green tea polyphenol EGCG
with higher sensitivity. First of all, more than 40 kinds of chemical compounds, including 2,5-dihydroxybenzoic acid（DHB）, α-cyano- 4 -hydroxycinnamic acid（CHCA）, sinapic acid（SA）, and 9 -AA, were examined for screening the potential matrix against
EGCG. Spots of mixed solutions of these chemicals and EGCG were measured by MALDI-TOF-MS in both positive and negative
ionization modes. An EGCG peak was not observed by the major matrix（DHB, CHCA, SA, and 9 -AA）. On the other hand, β-carboline derivatives, CHCA analogues, and naphthalene derivatives were able to detect EGCG with higher sensitivity（femtomole
order）in negative ionization mode. Almost all matrices capable of detecting polyphenols as previously reported had no ability todetect EGCG with the same sensitivity. To examine whether these chemicals can visualize EGCG on the tissue section, we were
undertaken to detect an image of EGCG spotted on the normal mouse liver section. Although MSI experiment revealed that apeak intensity of EGCG was greatly reduced as compared to the case of the screening test on a stainless MALDI sample plate,two-dimensional image of EGCG spot was obtained with signal-to-noise ratio of more than 100.
The principal findings： These results suggest that our selected chemicals act as a matrix against the green tea polyphenol EGCG
on both the sample plate and the tissue section. This finding is the first report showing that our proposed MALDI-MSI techniques
enable to visualize two-dimensional image of EGCG. Now, we are trying to detect EGCG in the liver section derived from EGCGadministrated mouse.
References：［ 1 ］J. Proteomics, in press, 2012；［ 2 ］Anal. Chem., 82, 9789, 2010；［ 3 ］Anal. Chem., 82, 4278, 2010；［ 4 ］Anal. Chem.,
82, 498, 2010
Acknowledgements： This work was supported in part by Grants for project research（Development of fundamental technology
for analysis and evaluation of functional agricultural products and functional foods（to H.T.）, and Grants-in-Aid for Scientific
Research（S）（Grant 22228002）（to H.T.）. We would like to specially thank Shimadzu Corporation.（Kyoto, Japan）for their fullcooperations.

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 9: Imaging II

PMo-181

11:10 － 12:20

Development of micro-focusing electrospray droplet impact gun for SIMS
analysis

YUJI SAKAI, RIO TAKAISHI, SATOSHI NINOMIYA, KENZO HIRAOKA
University of Yamanashi, Kofe, Japan

Keywords:
electrospray, electrospray droplet impact（EDI）, SIMS
Novel aspects:
A newly designed ambient electrospray droplet impact（EDI）gun could be focused to about 40 micron with 8 kV acceleration
voltage with the beam current of 0.5nA.
Abstract:
A noble ionization method, the electrospray droplet impact（EDI）, has been developed for cluster SIMS.［ 1 ］The projectiles
used in EDI are extremely large multiple charged water cluster ions with masses of several 106 u. The EDI gun was installed in an
orthogonal time of flight mass spectrometer（TOF-MS）（JEOL, Accu-TOF, Japan）. The organic, inorganic and semiconductors
materials were examined by EDI/SIMS. It was found that the surfaces etched by EDI suffered from neither degradation nor
preferential sputtering of particular elements. EDI was found to be capable of molecular level etching without leaving damage
after the irradiation.［ 2 ］,［ 3 ］
The charged water droplets were formed in ambient by electrospraying 0.01 M trifluoroacetic acid aqueous solution. The droplets
were sampled through an orifice with a 400-μm in diameter into the vacuum chamber, transported into a quadrupole ion guide,
and accelerated by 10 kV after exiting the ion guide. The electrospray droplets accelerated and focused impact a solid sample.
The diameter of the droplet beam for the original apparatus was approximately 3 mm in diameter, as determined by moving thesample stage. The beam focusing is mandatory for the micro area analysis. In this paper, a newly designed objective lens system
which consists of Einzel objective lens, deflector and angle aperture will be presented. The beam size was measured by observing
images of secondary electrons induced by EDI irradiation. The beam diameter of the ambient EDI gun could be focused to about
40 micron with 8 kV acceleration voltage and with the incident beam current of 0.5nA. The design of micro focusing EDI gun will
be presented. Some preliminary data obtained for the biological application will be given.
Reference［ 1 ］K. Hiraoka, D. Asakawa, S. Fujimaki, A. Takamizawa, K. Mori, Eur. Phys. J. D38, 225（2006）［ 2 ］K. Hiraoka,Y. Sakai, Y. Iijima, D. Asakawa and K.Mori , Appl. Surf. Sci. 255, 8947（2009）［ 3 ］Y. Sakai, Y. Iijima, S. Mukou and K. Hiraoka, Surf. Interface Anal. 43, 167（2011）

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 9: Imaging II

PMo-182

13:30 － 14:40

Dyeing Regions of Oxidative Hair Dyes in Human Hair Investigated by
Nanoscale Secondary Ion Mass Spectrometry

Toru Kojima1, Hiromi Yamada1, Toshihiko Yamamoto1, Miyuki Takeuchi2, Yasuyuki Matsushita3,
Kazuhiko Fukushima3
1

Hoyu Co., Ltd., Nagakute, Japan, 2The University of Tokyo, Tokyo, Japan, 3Nagoya University, Nagoya, Japan

Keywords:
NanoSIMS, Hair, dye, distribution, localization,
Novel aspects:
The isotope ion mapping image of colored hair cross-section mesured by NanoSIMS revealed the important dyeing regions, which
are nanometer size, for oxidative hair coloring .
Abstract:
Oxidative or permanent hair colorant, which has many kinds of color tone and brightness, is the most popular hair coloringproduct by people who wish to change their natural hair color and/or cover the gray hair in the world. Colorant, which contains
several types of oxidative hair dyes and alkalizer, is mixed with developer containing oxidizing agent just before use and then the
resulting mixture is applied to hair for dyeing. The general mechanism of oxidative dyeing is simultaneously bleaching of melaninpigment and color development from colorless oxidative dyes in hair fiber.
Human hair is a biological material having a complicated hierarchical structure. Its predominant constituents are proteins whichhave various amino-acid compositions. Also, it contains water, trace elements, small amounts of lipids and pigments which arenanometer sized granule.
Although the penetration and distribution of colored dye generated from oxidative dyes in hair had been investigated by an
optical microscope, it was hard to observe dye localization in fine structure of hair. In order to develop more effective oxidative
hair coloring product and hair care product for colored hair, it is important to understand the dye localization in fine structure of
hair.
Secondary ion mass spectrometry（SIMS）, a mass spectrometric technique, is able to provide information regarding themolecular distribution on surface of the biological material. Nanoscale SIMS（NanoSIMS）is especially able to visualize the
distribution of isotope ion derived from isotopically-labeled molecules with high spatial resolution and sensitivity. In this work, thelocalization of colored dye generated from oxidative dyes in fine structure of human hair was investigated using NanoSIMS.
Human hairs which had never been chemically treated were used. Colorant aqueous solution containing 1,4-phenylenediamine2,3,5,6-d4（pPD-d4）, a stable isotopicallylabeled oxidative hair dye, was prepared. Oxidative dyeing was performed for 60 min at 30°C in mixed aqueous solution of colorant and developer containing hydrogen peroxide. Colored hair were embedded in epoxy
resin and made a smooth surface of hair cross-section with an ultramicrotome. NanoSIMS measurements were performed with a
16 keV Cs+ primary ion beam to detect negative secondary ions.
Deuterium ion mapping was carried out to estimate colored dye distribution and its localization. Deuterium ions were detectedfrom whole hair cross-section and were strongly detected from particulate regions having nanometer size in the colored hair.
These regions correspond to melanin granules and it is confirmed that there are more colored dye generated from oxidative dyes
in melanin granules compared with other hair regions. The NanoSIMS mapping image of deuterium ion on hair cross-section
revealed that melanin granules of human hair were important dyeing regions for oxidative hair coloring.
Therefore, it is considered that NanoSIMS is the effective tool for understanding the interaction of cosmetic ingredients with thefine structure of human hair.

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 9: Imaging II

PMo-183

11:10 － 12:20

Bio-imaging with Swift Heavy Ion Beams

Toshio Seki1,2, Sho Shitomoto1, Shunichiro Nakagawa1, Takaaki Aoki2,3, Jiro Matsuo1,2
1

Kyoto University, Uji, Japan, 2CREST,Tokyo, Japan, 3Kyoto University, Kyoto, Japan

Keywords:
SIMS, Bio-imaging, swift heavy ion
Novel aspects:
The high secondary ion sensitivity of molecules for bio-imaging was realized with swift heavy ions.
Abstract:
The importance of imaging mass spectrometry（MS）for visualizing the spatial distribution of molecular species in biological tissues
and cells is growing. SIMS imaging has been used to visualize elemental distribution at the cellular level because of its low
molecular ion yield. In conventional SIMS with keV-energy ion beams, elastic collisions occur between projectiles and atoms inconstituent molecules. The collisions break the molecules and produce fragments, which makes acquisition of molecular
information difficult. In contrast, MeV-energy ion beams excite electrons and enhance the ionization of high-mass molecules, and a
SIMS spectrum of ionized molecules can be obtained. In a previous study, we have developed a new system for imaging mass
spectrometry using MeV-energy heavy ion beams, termed MeV-secondary ion mass spectrometry（MeV-SIMS）, and demonstrated
more than 1000-fold increase in molecular ion yield from a peptide sample（1154Da）, compared to keV ion irradiation. In addition,
we successfully obtained mass spectrometric imaging of the deprotonated peptides（m/z 1153）without any matrix enhancement［ 1 ］.
To compare MeV-SIMS with conventional SIMS, we obtained molecular imaging of rat cerebellum with MeV-SIMS and conventional
SIMS with Bi3 ions that is know as the best ions to emit secondary molecular ions in convensional probe ions. Both MeV-SIMS andconventional SIMS images of m/z 184 were clearly observed and represented well the structure of the sliced cerebellum in the
optical microscope image. A fragment ion and headgroup of phosphatidylcholine were observed at m/z 184. On the other hand, it
is very difficult to see the distribution of the molecule of m/z 772 in case of Bi3 ion. This is mostly due to the low secondary ion
yields and we could not get much signals with this beam. However, the molecular distribution was clearly observed with swift
heavy ion beam. The high secondary ion sensitivity with swift heavy ions is very useful for biological material analysis.
［ 1 ］Y. Nakata, et al. J. Mass Spectrom.（2009）44, 128-136

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 9: Imaging II

PMo-184

13:30 － 14:40

Secondary Ion Emission with Methanol Gas Cluster Ion Beam Irradiation

Shunichiro Nakagawa1, Tosio Seki1,4, Takaaki Aoki3,4, Jiro Matsuo2,41
Nuclear Engineering, Kyoto University, Uji, Japan, 2Quantum Science and Engineering Center, Kyoto University, Uji,Japan, 3Department of Electronic Science and Engineering, Kyoto University, Kyoto, Japan, 4CREST, Japan Science andTechnology Agency (JST), Tokyo, Japan

Keywords:
Cluster ion, Etching
Novel aspects:
We used a methanol cluster ion to the primary beam of the SIMS.
Abstract:
When etching a substrate such as Si, the chemical etching technique is used to enhance etching. In particular, the effect ofchemical reactivity under ion beam irradiation has been demonstrated when the irradiation energy was below 100 eV. With gas
cluster ions bombardment, the energy is a few eV per atom, and the surface damage is much smaller than with conventional ion
bombardment. Enhancement of sputtering and secondary ion emission, has been reported for organic and inorganic materials
under cluster bombardment, and the effect was called“nonlinear effect“. In this study, we measured the etching depth of Si andinvestigated secondary ion from its surface with GCIB irradiation.
The cluster ion beam SIMS apparatus consisted of a bubbling chamber, a source chamber, an ionization chamber and ananalytical chamber. Neutral gas clusters are formed by adiabatic expansion of high pressure gas through a nozzle in the sourcechamber. The source gas of the ion beam was Ar and a mixture of noble gas（Ar）with methanol was the reactive gas. The sourcegas was generated with the bubbling technique. We expected ethanol to function as a reactive gas during the irradiation. The
cluster was ionized and accelerated to about 10 to 15 eV per cluster in the ionization chamber and the subsequent ionized cluster
was irradiated toward the target.
As a result, the sputter depth was two to three times larger with methanol cluster irradiation than with Ar. This difference
suggests that there may be a chemical etchant effect with irradiation with methanol cluster. In the SIMS spectrum with methanol
cluster ion beam secondary ion emission of the molecule（SiCH3）2 was observed, suggesting that the compound was formed inreaction between the reactive gas and the substrate surface atoms under the irradiation of cluster ion beam. Methanol cluster ion
beam can realize to etch to that reactively and enhance etching speed. This effect of reactive cluster irradiation is useful for highsensitive SIMS imaging.

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 9: Imaging II

PMo-185

11:10 － 12:20

Development of Electrostatic Quadrupole Lens for MeV-SIMS Imaging

Sho Shitomoto1, Toshio Seki1,2, Takaaki Aoki1,2, Jiro Matsuo1,2
1

Kyoto University, Kyoto, Japan, 2CREST, Tokyo, Japan

Keywords:
MeV-SIMS, Electrostatic Quadrupole Lens, Molecular Image
Novel aspects:
We succeeded in developing the focus lens for MeV-SIMS imaging and reducing measurement time.
Abstract:
Imaging mass spectrometry is a highly sensitive, chemically specific, and label-free analytical tool that provides information on thespatial distribution of molecules in biological tissues and cells. This technique is expected to be useful for pharmacokinetic
visualization in vivo, various applications in drug development, and other uses.
Matrix-assisted laser desorption ionization（MALDI）and secondary ion mass spectrometry（SIMS）are soft ionization techniques.
MALDI uses a matrix to induce soft ionization efficiency. This probe is ultraviolet laser radiation. It has been successfully applied in
imaging of large molecules of more than 10 kDa like biomolecules. However, the spatial resolution of MALDI imaging is more thanseveral tens micrometer. Biological molecular imaging in cell needs for spatial resolution of a micrometer. Therefore, MALDI
imaging is mainly used for mapping the molecules in tissue, not cell sections. SIMS analysis uses keV energy ion beams as probe
and not to need matrix. SIMS provides good spatial resolution below several dozen nanometers. In general, SIMS analysis is used
for imaging of elements or molecules below 500 Da, because of the low yields of high mass ions. Thus, we need a soft ionization
technique for biological molecular imaging about more than 500 Da. Moreover, we require spatial resolution below a micrometer.
The technique of SIMS with MeV-energy ion beam has a great potential for providing high resolution molecular images of cells. In
conventional SIMS with keV-energy ion beam, elastic collisions occur between projectiles and constituent atoms in molecules,
and this phenomenon breaks the molecules and produces fragments. In contrast, an MeV-energy ion beam can excite electron
and enhance the ionization of high-mass molecules, and a SIMS spectrum of ionized molecules can be obtained. However, atpresent, obtaining molecular imaging data takes a long time because the current density of the primary beam is quite low. To
focus the swift heavy ion beam and reduce measurement time, we have developed the electrostatic quadrupole lens（Q lens）.
The Q lens is doublet.
To characterize the properties of this lens, we measured the current density of the swift heavy ion passing through the lens. The
primary ion beam was 6 MeV Cu4+ heavy ion beam. The current density increased by a factor of 33. Therefore, the measurement
time can be reduced with the lens by a factor of about 30.
We could get successful secondary ion imaging of cholesterol from the cerebellum of a rat brain. The MeV-SIMS image has 4 mm
´ 4 mm field of view with a pixel size of 40 mm. A clear image was obtained and measurement time was reduced with the lens.
The total measurement time was about 5 min with the lens and about 167 min without this lens. We succeeded in developing the
focus lens for MeV-SIMS imaging and reducing measurement time.

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 9: Imaging II

PMo-186

13:30 － 14:40

Evaluation of the Damage on a Polymer Sample Caused by Shave-off
Section Processing

Teruhiko Tobe, Yoshihiro Morita, Atsuko Yamazaki, Makiko Fujii, Masanori Owari
The University of Tokyo, Tokyo, Japan

Keywords:
ToF-SIMS, FIB, cross section, damage evaluation, organic materials
Novel aspects:
We proposed the novel method for low damage section processing, shave-off scanning, and evaluated the surface roughness andthe amount of ion mixing.
Abstract:
The time-of-flight secondary ion mass spectrometry（ToF-SIMS）is a powerful tool for elemental and/or molecular imaging,
because it has high spatial resolution and high detection sensitivity. Furthermore, it is possible with SIMS to put together a 3 D
image by stacking 2 D images of each cross section fabricated with ion beams. 3 D imaging of organic materials or bio-materials
with ToF-SIMS has been extensively studied in recent years. However, ToF-SIMS 3 D imaging has not been applied to organic
materials or bio-materials practically. One of the significant problems of practical 3 D imaging of these materials with SIMS is thedamage caused by ion beams during etching process. In the case of sample etching with ion beams, the irradiation of beams
causes knock-on-mixing effects（implantation of ions and damage to the sample structure）and the surface roughness. Especiallyin etching of bio-materials or organic materials, the chemical states of samples after etching differ significantly from those of
unirradiated sample. To date, a great deal of efforts has been made to reduce these damage, and some effective methods havebeen developing. For example, the etching with cluster ion sources（SF6 +, C60 +, Ar cluster, etc.）causes less damage than that withatomic ion sources. However, they can not remove the damage and induce surface roughness completely.
We have been developing a novel etching method with Ga+ focused ion beam（Ga+ FIB）, which is called shave-off scanning, inorder to reduce the beam-induced damage. In the conventional method（the raster scanning）, knock-on-mixing effects occur anddamage the sample surfaces and structure seriously because of vertical or oblique beam irradiation to the sample surfaces. In the
shave-off scanning, it is thought that knock-on-mixing effects are suppressed because of horizontal ion beam irradiation to thesample surface. In addition to the suppressing of knock-on-mixing effect, the shave-off scanning is superior to raster scanning insome points； absolute depth scale, and application to rough surface and/or hetero interface. However, due to unique position
between FIB and samples, the beam-induced damage on organic materials with shave-off scanning has been evaluatedinsufficiently.
In our previous study, molecular dynamics simulations were performed to investigate the damage to aluminum model samples
with shave-off scanning. The simulations showed that shave-off scanning had higher sputtering yield and caused lower damage
compared to raster scanning. On the other hand, they indicated that knock-on-mixing effects occurred to some extent even in
shave-off scanning mode. Moreover, these results suggested that shave-off scanning damages organic material surface and thechemical state of its surface are not preserved after shave-off section processing.
In this study, we evaluated the damage on organic samples（ 6 -nylon）after shave-off sectioning with Ga + FIB. The chemical statesof 6 -nylon samples sectioned with Ga+ FIB shave-off scanning were analyzed with XPS, and the roughness of cross sections wasevaluated with AFM. We investigated the effects of some parameters of sectioning（incident energy, scan speed in depth
direction, etc.）on sample damage. In addition, these cross sections were analyzed by ToF-SIMS and measured the secondary ion
yields of molecular ions. From these results, we discussed the optimized Ga + FIB shave-off processing conditions of organic
materials.

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 9: Imaging II

PMo-187

11:10 － 12:20

Study on Depth Resolution Factor of Shave-off Depth Profiling

Makiko FUJII, Yuya HANAOKA, Tatsuya TAKETSUGU, Masanori OWARIThe University of Tokyo, Tokyo, Japan

Keywords:
SIMS, depth profiling, depth resolution, 3 DAP, sputtering
Novel aspects:
We newly investigated the various factors that determine depth resolution of shave-off depth profiling by means of 3 DAP andsome other analytical methods.
Abstract:
In recent years, the establishment of local part analysis methods in a limited area has been aspired for a semiconductor and anelectronic device industry. We have been developing shave-off depth profiling with nano-beam secondary ion mass spectrometer（nano-beam SIMS）, one of the powerful methods that can be applied to the analysis of such materials and devices. Shave-off
depth profiling achieves the highly precise depth profiling with nanometer-scaled depth resolution by utilizing a Focused Ion Beam
（FIB）micro-machining process. We can acquire a depth profile by shave-off scan mode（A fast horizontal sweep of FIB iscombined with the very slow vertical sweep）. Shave-off depth profiling has its own features： absolute depth scale, pin point
depth profiling and application to rough surface and/or hetero interface. In our previous studies, we obtained highly precise depth
profiles of real-world samples. In addition, shave-off scan mode has been applied for low damage section processing to realize thehighly accurate three-dimensional microanalysis.
The various factors that determine the depth resolution of conventional SIMS depth profiling have been investigated in detail formany years（i.e. ripple formation, mixing effect, etc.）. On the other hand, the discussion of the depth resolution factor of shave-off
depth profiling is still insufficient because shave-off scan mode has distinctive position of the primary ion beam against the sample.
In our previous study, the amount of the mixing effects under shave-off scan mode was evaluated using Molecular Dynamics（MD）
simulations. From these results, it was proved that shave-off scan mode had the high sputtering yields and the low mixing effects
compared to conventional raster scan mode. In addition, the impact energy dependence on the sputtering yields and the mixing
effects under shave-off condition was demonstrated. As mentioned above, there are many kinds of depth resolution factors forconventional SIMS depth profiling and the similar factors can be thought to exist for shave-off depth profiling. In this study, we
experimentally investigated the depth resolution factor of shave-off depth profiling by means of various analytical tools.
Three-Dimensional Atom Probe（ 3 DAP）has been developed for highly precise three dimensional reconstruction imaging withnearly atomic level spatial resolution. This method is effective in investigating the damage depth and the mixing effect of thesample after ion bombardment under shave-off scan mode because both 3 DAP and shave-off depth profiling adopt the same
sample preparation method utilizing FIB lift-out method. We prepared the sample of box shape by means of FIB micro-machining
and shaved it off partly under shave-off condition. Then, the sample was processed again to needle shape and 3 DAP
measurement was performed to investigate the damage depth and the mixing effect caused by shave-off process. Some otheranalytical methods were adopted in order to evaluate the sputtered surface. The results obtained from the sample sputtered byshave-off scan, conventional raster scan, and without sputtering were compared and the amount of mixing effects was evaluated.

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 9: Imaging II

PMo-188

13:30 － 14:40

Tracing photosynthetic product accumulation in xylem cell wall using
carbon isotope labeling

Miyuki Takeuchi, Akira Isogai

The university of Tokyo, Tokyo, Japan

Keywords:
cell wall, carbon allocation, NanoSIMS, isotope labeling
Novel aspects:
Carbon flow in a tree was traced using carbon isotope pulse labeling combined with NanoSIMS analysis. Incorporation ofphotosynthetic products into xylem cell wall of each cell was revealed.
Abstract:
Trees fix CO2 in the atmosphere by photosynthesis. A part of the photosynthetic products are accumulated in tree for a long timeand carbon accumulation in the tree stem is an important factor on allocation of assimilated carbon and then, ecosystem functionof the tree. The main storage of carbon in trees is thick cell wall of xylem cells in stem and arrays of the cells form wood with treerings. Environmental conditions are recorded in the tree rings during their growth 1). For example, carbon isotopic ratio reflects
environmental conditions at the time of their photosynthesis. Thus, the information in the tree rings is used for investigation ofplant response to environmental factors, and it can be used as a tool in reconstructing past changes in climate. On these studies,variation of carbon isotope composition in tree rings is focused and seasonal and monthly variation pattern has been shown.
Carbon transport and allocation in trees has been also studied using 14C or 13C labeling, though it was difficult to know time lags
between photosynthesis and carbon accumulation in the stem and effects of photosynthesis on cell wall formation in a single
xylem cell.
In this study, we aimed to trace transfer photosynthate that is produced in a short period, and its incorporation to xylem cells
wall. Stable isotope labeling was combined with a high resolution secondary ion mass spectrometry（NanoSIMS）. 13C labeled
CO2 was applied to a 4 -year-old Populus euramericana for 2 hours and the sample was harvested after 5 days. The small piecesof the xylem was fixed with 3 % glutaraldehyde and embedded in epoxy resin. 12C and 13C analysis on the semi-thin section was
performed using NanoSIMS 50L.
High resolution carbon mapping with NanoSIMS revealed 13C tracer distribution within cell wall and a 13C rich layer was observedin the cell wall of young xylem cells. This 13C rich layer indicates that the layer was accumulated during the 5 days between 13CO2
application and sampling. The position of the layer in the cell wall varied from innermost layer to compound middle lamella, and it
reflects the developmental stage of the cells when they received 13C labeled materials. These results suggest that it is possible toestimate cell wall formation process in a day or a few hours scale and it will help us to understand correlation between cell wall
formation and environmental condition such as diurnal differences.
1 ）D. McCarroll and N. J. Loader（2004）Quaternary Science Reviews. 23, 771-801

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 9: Imaging II

PMo-189

11:10 － 12:20

Acceleration of gluconeogenesis of the host liver bearing human coloncancer metastases revealed by microscopic imaging mass spectrometry

Akiko Kubo1, Mitsuyo Ohmura1, Tsuyoshi Nakanishi3, Shigeki Kajihara3, Kiyoshi Ogawa3,
Makoto Suematsu1,21Keio University, Tokyo, Japan, 2JST, ERATO Suematsu Gas Biology Project, Tokyo, Japan, 3Shimadzu Corporation,
Kyoto, Japan

Keywords:
imaging mass spectrometry, metabolome, CE-MS, gluconeogenesis, tumor metastasis
Novel aspects:
Our results collected by a novel semi-quantitative IMS suggest that tumor bearing-liver utilize gluconeogenic substrates not only
producing glucose to maintain its plasma level, but also stimulated glycogen storage.
Abstract:
Imaging mass spectrometry（IMS）allowed us to collect spatial information of many different metabolites in a single frozen section,and combination with capillary electrophoresis（CE）-MS data collected from the corresponding serial section provided
semiquantitative information of the individual MS signals(1,2). This study aimed to examine alterations in carbohydrate metabolismof the host liver of superimmunodeficient NOG mice that is invaded by metastases of human colon cancer xenografts in vivo；
during the development of metastases, the host mice suffer from systemic hypoglycemia.
NOG mice were intraperitoneally injected with excess amounts of 13C3- L-alanine to determine its conversion to the mass-labeled
gluconeogenic metabolites under overnight-fasted conditions. Livers under fasted conditions were collected at 0, 5 and 15 minutes
and 60 minutes after the L-alanine injection. Liver tissues were removed and snap-frozen in liquid nitrogen, and 5 -micrometercryosections were mounted on ITO-slides followed by being sprayed with 9 -aminoacridine to conduct microscopic MALDI-IMS in
negative ion mode. The adjacent thick section of a frozen tissue was used for metabolome analyses by CE-MS. For semiquantitative analysis, MALDI IMS peaks were automatically extracted from each regions of interest（ROIs）by using IMS
convolution software(3). Extracted intensity values are normalized with quantitative results by CE-MS, and used to assign regionspecific values of individual metabolites (4) . The 13C-labeled gluconeogenic metabolites were produced in both control and tumorbearing livers. Greater than 2 folds of labeled UDP-glucose were detected in parenchyma of the tumor-bearing liver versus thecontrol. MS 2 analyses under loading 13 C 3 -L-alanine provided evidence for the mass-labeled UDP-glucose with 13 C-containinghexose structure in the parenchyma of tumor-bearing liver, suggesting biotransformation of L-alanine is accelerated for glycogensynthesis rather than for supporting glucose delivery to circulation. Our results collected by a novel semi-quantitative IMS suggestthat tumor bearing-liver utilize gluconeogenic substrates not only producing glucose to maintain its plasma level, but also
stimulated glycogen storage, leading to hypoglycemic stress under the tumor-bearing conditions. Overall, IMS combined with CEMS analyses serves as a potentially useful strategem to carry out semi-quantitative analyses of tumor bearing liver in vivo.
References
1. Hattori K, et al.（2010）Antioxid Redox Signal 13, 1157-1167
2. Morikawa T, et al.（2012）Proc Natl Acad Sci USA 109, 1293-1298
3. Hayasaka et al.（2011）Anal Bioanal Chem 401, 183-193
4. Kubo A, et al.（2011）Anal Bioanal Chem 400, 1895-1904
Acknowledgements
This work was supported by JST, ERATO, Suematsu Gas Biology Project as well as by Research and Development of the NextGeneration Integrated Simulation of Living Matter, a part of the Development and Use of the Next-Generation Supercomputer
Project of MEXT（to M.S.）. Imaging mass spectrometry was supported by Grant-in-Aid for SENTAN from JST（A.K.）.

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 9: Imaging II

PMo-190

13:30 － 14:40

Early Detection of Gastric Cancer using ToF-SIMS Metabolomic Imaging

Yong-Chien Ling1, Shiou-Ling Lei1, Chun-Chao Chang2, Chia-Lang Fang3, Fu-Der Mai4

1
Department of Chemistry, National Tsing Hua University, Hsicnhu, Taiwan, 2Department of Internal Medicine, Taipei
Medical University, Taipei, Taiwan, 3Department of Pathology, Taipei Medical University, Taipei, Taiwan, 4Biomedical
Mass Imaging research Center, Taipei Medical University, Taiwan

Keywords:
Metabolomic imaging, ToF-SIMS, Gastric cancer； Early detection, Chemometric processing
Novel aspects:
The first attempt for early detection of gastric cancer using ToF-SIMS metabolomic imaging by ToF-SIMS spatially-resolved
detecting small organic/inorganic species to yield highly specific profile followed by chemometirc grouping.
Abstract:
Metabolomics involves comprehensive, simultaneous, and systematic characterization of small endogenous metabolites in abiological system. ToF-SIMS imaging is capable of detecting organic and inorganic species distribution in gastric tissues, possessing
the potential for early detection of gastric cancer which greatly increases the chances for successful treatment. Herein, we havecollected four gastric tissue samples from patients pathologically classified as at stage from early to late IA（patient 1 ）, II（patient2 ）, and IIIA（patient 1 and 3 ）. A ToF-SIMS IV using a primary ion gun of 25 keV Ga+ at typical 1 pA pulse current was used toacquired ion images from region of interest in both normal mucosa and tumor cell. Statistically low level of Fe, PC, PE and uricacid and high levels of propanoic acid, malic acid, citric acid and hexadecanoic acid were observed in positive ion ToF-SIMS
results； whereas statistically low level of S and CN were observed in negative ion ToF-SIMS results. Similar metabolic profiles
were prevalent among all samples. Principal component analysis（PCA）of metabolic profiles（patients 2,3,4 and 1,2,3,4, based onfrom PI and NI ）reveals distinctive grouping between tumor and normal tissue. Additional PCA of cancer-related metabolic profile
reveals distinct grouping between tumors at different stage, suggesting the potential of metabolomic imaging by ToF-SIMS as early
detection tool for gastric cancer.

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 10: Ion Mobility Spectroscopy Based on Instrument & Theoretical Development

PMo-191

11:10 － 12:20

Separation and identification of Structural isomers of Methylpyrazole byDifferential Mobility Spectrometry

Kazuhide Inoue1, Yasutohi Kawase1, Masazumi Yasumoto1, Seiji Horie1, Shigeru Yamada2,
Ryoma Yamamoto2
1

TAKARA BIO INC. Shiga, Japan, 2K.K. AB SCIEX. Tokyo, Japan

Keywords:
Differential mobility separation, Methylpyrazole
Novel aspects:
Differential mobility separation can separate Structural isomers.
Abstract:［INTRODUCTION］
A lot of compounds are very similar in structure and may have identical chemical composition, making it difficult to separate these
species even by accurate-mass measurements. The analysis of Methylpyrazoles is just such an application, too.
Differential mobility separation（DMS）can separate species based on the difference mobility between their high field and low
field. At a specific separation voltage（SV）, it is possible to ensure transmission of particular species through the DMS device todetector（MS/MS detection in this work）when a compensation voltage（CoV）is applied, while other species will simply be lostto the electrode walls. AB SCIEX SelexION™ device is technology that is using such a differential mobility separation.
In this work, an orthogonal separation technique has been utilized to differentiate between isobaric Methylpyrazoles（ 3 -Methylpyrazole and 4 -Methylpyrazole）, using differential Mobility Spectrometry prior to analysis by MS/MS.
［MATERIALS and METHODS］
HPLC Conditions：
For the analysis was used at a Shimadzu XR LC system. The method was developed using a conventional ODS column with an
isocratic solution of acetonitrile water containing 0.1% formic acid.
MS/MS Conditions：
An AB Sciex QTRAP（R）5500 system with Turbo V（R）source and the electrospray ionization（ESI）probe was used. The source
was operated at 550 degrees with Gas 1 and Gas 2 at 60 and 80 psi, respectively. The ion source region of the mass spectrometer
was modified for incorporation of the AB SCIEX SelexION™ ion mobility separation device.
［RESULTS］
3 -Methylpyrazole and 4 -Methylpyrazole share the same precursor ion mass, and the same MRM transitions, the separation
cannot be performed by tandem mass spectrometry alone. Traditionally the separation is accomplished using longchromatographic gradients, which is effective but which results in a reduction in sample throughput. Using the completely
orthogonal separation provided by AB SCIEX SelexION™ technology, the 3 -Methylpyrazole and 4 -Methylpyrazole have been
completely separated prior to analysis by MS/MS.
In addition it has been shown that the AB SCIEX SelexION™ technology can significantly reduce LC-MS/MS run-times, sincecomplete chromatographic separation of potentially interfering species is no longer required.

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 10: Ion Mobility Spectroscopy Based on Instrument & Theoretical Development

PMo-192

13:30 － 14:40

Investigation of an IMS-TOF MS with a laser based ionisation underatmospheric conditions

Marvin Ihlenborg, Tassilo Muskat, Juergen Grotemeyer
CAU Kiel, Kiel, Germany

Keywords:
IMS, MPI
Novel aspects:
Comparison of the ion yields obtained by a MPI and ionisation by an tritium source.
Abstract:
Introduction
In the past an Ion Mobility Spectrometer（IMS）was purchased from Draegerwerk AG by our group. Under atmospheric pressure
conditions this type of IMS uses a 3H-source to produce ions. To extant the range of analytes, which are not easy to protonate,
the 3 H-source should be replaced by a laser based ionisation method. Additionally other types of ions are formed（e.g. radical
cations instead of protonated species）and due to the wavelength of the laser the ionisation is selective. Furthermore the existing
apparatus（cf. method）should mainly remain the same.
MethodThe mainly used apparatus is described earlier［ 1 ］only a short overview will be given here. A home build and a purchased IMS（Draegerwerk AG）are coupled to a home build time of flight mass spectrometer（TOF-MS）. To transfer the ions from theatmospheric pressure IMS to the high vacuum system of the TOF-MS several different vacuum stages have been incorporated.
In addition a collecting lens is build into the existing setup. Due to this changes the potentials of the different transfer units arealigned to maintain the ion transfer the same.
Preliminary Data
In earlier experiments［ 1 ］it can be seen that during the transfer of the ions from the high pressure system to the high vacuum
stage of the TOF-MS a high loss of ions occurs. To compare the different ionisation methods first measurements were made withthe 3H-source and acetone respectively toluene as analytes. The transferred ions through the IMS were observed with a Faraday
Cup connected to an analogue amplifier. Afterwards the 3H-source was displaced by a laser beam focused directly in front of theentrance of the IMS. The laser beam was obtained by the 4 th harmonic of a Nd：YAG laser. Furthermore a repeller plate wasincorporated in front of the IMS. The measurements with toluene were repeated due to the good ionisation capability by 266 nm
by a multi photon ionisation process（MPI）.
Several effects can be observed by changing the ionisation technique. The obtained signal rises in intensity by the tenfold in
addition to a broadening with a distinctive tailing. Furthermore the signal maximum is shifted by approximately 2,5 ms to later drift
times.

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 10: Ion Mobility Spectroscopy Based on Instrument & Theoretical Development

PMo-193

11:10 － 12:20

Separation of Isobaric Phosphopeptides using Differential Ion Mobility
Spectrometry with Tandem Mass Spectrometry.

Chie Inagaki, Masato Aoshima, Takeshi Shibata, Shigeru Yamada, Sumie AndoKK ABSCIEX, Tokyo , Japan

Keywords:
Ion Mobility, Phosphopeptide, MRM
Novel aspects:
DMS is able to separate and quantitate identical phoshopeptides which have a different phosphorylation site. It helps tounderstand cell signaling pathway.
Abstract:
Introduction
Protein phosphorylation is a key regulator of cellular signaling pathways. This highly controlled biochemical process is involved inmost cellular functions and defects in protein phosphorylation has been implicated with many diseases including cancer,
highlighting the importance of understanding phosphorylation-mediated signaling networks.
Much work has been done over the last few years to develop Multiple Reaction Monitoring（MRM）based strategies to enable
and accelerate the transition from discovery to targeted quantitative MS verification assays because they are not necessarily
dependent on the use of antibodies and can therefore be rapidly and cost-efficiently developed in comparison to traditional
enzyme linked immune sorbent assays（ELISA）. However, it is difficult to identify and resolve phosphopeptides which haveidentical amino acid sequences and differ only in the site of phosphorylation even if using ELISA and MRM.
We report here to use a QTRAP 5500 System equipped with a differential mobility separation device for identification andseparation of identical phoshopeptides which have a different phosphorylation site.
Material and Methods
In this work, we used MS PhosphoMix standards（SIGMA-ALDRICH Corporation ） containing phosphopeptides,
ADEPpSSEESDLEIDK（M 1 ）and ADEPSSEEpSDLEIDK（M 2 ）derived from HeLa cells. A 5500 QTRAP system with Turbo V source
and the electrospray ionization（ESI）probe was used. MRM transitions for M 1 and M 2 of phosphopeptides were developed andused to measure the calibration curves. For the differential mobility separation experiments, a QTRAP 5500 LC/MS/MS systemequipped with SelexION Technology（a differential mobility separation device）was used. Isopropanol was used as a chemical
modifier and was introduced into the curtain gas using an integrated pump.
Results
The SelexION Technology is a planar differential mobility separation（DMS）device that attaches between the curtain plate
andorifice plate of the 5500 QTRAP system. DMS can separate ions in the gas phase based on their different migration rates in highand low electric fields and is orthogonal to both MS and liquid chromatography.

Even if we could detect both M 1 and M 2 phosphopeptides from MS PhosphoMix Standards using MRM mode without DMS, they
were not resolved chromatographically. When using isopropanol as a chemical modifier in the DMS cell, M 1 and M 2 exhibit
different mobility coefficients and were completely separated by using different compensation voltages（CoV）with an optimized
separation voltage（SV）. DMS provided the capability to greatly separate the phosphopeptides where phosphorylation was
different location.
Although both M 1 and M 2 phosphopeptides were not resolved chromatographically in this case, their MRM transitions wereidentical and their separation using DMS was essential to their quantitation. Furthermore, we performed the calibration curve andstatistics in triplicate for M 1 and M 2 in MRM mode with DMS. It was good linearity that correlation coefficient（r）>=0.99 inconcentration range 10-1000 fmol/μL on column and accuracy was between 90% and 110%. The calibration curve was performed
without any internal standard and %CV was within 5 %.
Conclusion

We suggest that SelexION differential ion mobility technology is dramatically able to separate and quantitate localization variantsof phosphopeptides to understand the dynamic changes of the proteome, the crosstalk and selectivity of different
phosphorylation, and their impacts on signaling cascades.

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 10: Ion Mobility Spectroscopy Based on Instrument & Theoretical Development

PMo-194

13:30 － 14:40

Integrative structure determination of protein assemblies using mass
spectrometry, ion mobility and cross-linking.

Argyris Politis1, Zoe Hall1, Florian Stengel2, Ruedi Abersold2, Carol V Robinson1
1

University of Oxford, 2ETH Zurich

Keywords:
IM-MS, CXMS, Integrative structural biology
Novel aspects:
Integrating, for first time, ion mobility-mass spectrometry and cross linking technology for structural determination ofmacromolecular assemblies can make a significant contribution to hybrid structural biology.
Abstract:
Recent advances in the field of mass spectrometry（MS）and proteomics have added a whole new dimension to the study ofprotein-protein interactions and the architecture of protein complexes. The additional coupling of ion mobility（IM）spectrometry
with MS has enabled the two-dimensional separation of gas-phase ions according to ’size’ and mass-to-charge ratio. Here, we
describe an integrative approach to structure characterisation of macromolecular protein assemblies. To this end, we combine
native MS of intact complexes and subcomplexes, IM and cross-linking technology（CXMS）. Subcomplexes are obtained fromsolution-phase disruption experiments in which chaotropic agents or manipulation of ionic strength is used to perturb protein
interfaces. Stable subcomplexes, subunit packing, and pair-wise interactions can be used to reveal the building blocks of protein
complexes. Specifically, MS of the intact complex, with its subcomplexes, can provide information on the stoichiometries andconnectivities of subunits within heterogeneous assemblies. Both CXMS and IM provide information on topology. Whilst CXMS
reveals data on the inter- and intra-protein proximity, information on the overall topology of a protein complex can be attained by
the collision cross section（CCS）from IM. These experiments can be used as spatial restraints in modelling the structures ofprotein assemblies which are difficult to study by conventional structural biology approaches.
Here, we explore in detail the strengths and limitations of using IM-MS for structure determination by disrupting six heteromericcomplexes in solution. Experimental CCS of protein complexes and their subcomplexes agreed well with those calculated fromtheir crystal structures. This indicates minimum perturbation of tertiary structure following the disruption of subunit-subunit
interactions in solution. Next, we benchmarked our modeling approach on several known protein complexes, with different
topologies and complexities, using experimental data from IM-MS. Candidate models for heteromeric complexes tryptophan
synthase（143 kDa, 4 -mer）and nitrobenzene dioxygenase（218 kDa, 6 -mer）were evaluated by incorporating a scoring function
which measures their closeness-of-fit to the experimental data. The best-scored model for these complexes showed goodstructural similarity to the native structure, with centroid RMSD < 9 Angstroms. Finally, we applied our integrative approach on aprotein assembly of high biological interest, the proteasome lid from Saccharomyces cerevisiae, for which no high resolution
structure exists. This was achieved by combining information on connectivity, subcomplex topology and protein-protein
interactions from CXMS and IM-MS. The best-scored model structure was found to be in excellent agreement with recently
published electron microscopy data of the lid（Lander et al., Nature, 2012）, clearly revealing the synergistic effect of combining
multiple techniques.
Overall we show that IM-MS can play a useful role in structural characterisation of assemblies, particularly when integrated withinformation from other sources, such as CXMS. Our MS-based approach can therefore make an important contribution to hybrid
structural biology and integrative modelling approaches, providing insights into both the structure and function of macromolecularmachines.

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 10: Ion Mobility Spectroscopy Based on Instrument & Theoretical Development

PMo-195

11:10 － 12:20

Advances in Chip-based FAIMS/Triple Quadrupole MS: Instrumental
Studies and Analytical Capabilities

Richard A Yost1, Christopher R Beekman1, Chia-Wei Tsai1, Michael Ugarov2, George Stafford2
1

UNIVERSITY OF FLORIDA, Gainesville, Florida USA, 2Agilent Technologies, Santa Clara, California USA

Keywords:
FAIMS, DMS, ion mobility separation, tandem mass spectrometry, triple quadrupole
Novel aspects:
The first demonstration of interfacing a micro-FAIMS cell to a triple quadrupole mass spectrometer for fast-scanning FAIMS.
Abstract:
High-field asymmetric waveform ion mobility spectrometry（FAIMS）separates ions based on differences in ion mobility at high andlow fields, and offers a novel approach to component separation in mass spectrometry. A variety of FAIMS electrode geometries
are available, including planar and curved geometries. A micromachined FAIMS chip, an order of magnitude smaller thanconventional FAIMS electrodes, offers a number of advantages for FAIMS/MS. The chip is composed of multiple micro-channels
with a gap of <100µm； therefore, the field strength required for separation is achieved with lower voltages compared toconventional FAIMS devices. The small size of the FAIMS chip and low operational voltages enable more rapid scanning ofdispersion fields（DF）and compensation fields（CF）than with conventional FAIMS cells.
Data were collected with an Agilent 6460 triple quadrupole mass spectrometer（TQMS）. A custom Owlstone FAIMS chip（microFAIMS）with a gap width of 100µm and thickness of 700µm was mounted in front of the sampling capillary. Electrospray ionization（ESI）and atmospheric pressure chemical ionization（APCI）were employed as ionization sources. The FAIMS waveform was
generated with a custom Owlstone waveform generator providing a DF range of 0 to 350 Townsends（Td）and a CF range of -30
to 30Td（equivalent to a DV range of 0 to ~600V and a CV range of ~-50 to +50V）.
The fast scan ability of the microscale FAIMS device offers unique capabilities. For instance, a complete CF/DF scan can be
performed in a matter of seconds, permitting optimization of DF and CF for individual mixture components. Complete
3 -dimensional CF/DF plots for selected ions or selected MS/MS reactions can identify and differentiate co-eluting isobaric andisomeric compounds. Fast CF/DF scanning permits dynamic programming of FAIMS and MS/MS settings, including rapidly jumping
between selected points in CF/DF space to separate individual mixture components.
For example, the three isomeric phthalate ions（ortho-, meta-, and para-）were analyzed by ESI/FAIMS/MS in full-scan MS mode
and in selected ion monitoring（SIM）mode to characterize scan speed, FAIMS resolution, and transmission. Tandem mass
spectrometry experiments（ESI/FAIMS/MS/MS）were also performed in selected reaction monitoring（SRM）mode. The DF wasscanned from 50 to 350Td（DV of ~85 to 600V）with up to 66 steps of ~4.5Td each, and the CF was scanned with up to 150 steps
from - 3 to + 7 Td（CV range of about 17V）to generate 3 -dimensional CF/DF/intensity plots）. The use of SIM or SRM mode anda smaller number of CV and DV steps dramatically shortens the scan time, providing complete CF/DF scans 10-100x more rapidly
than with a conventional FAIMS cell.
In other experiments, the effect of the addition of carrier-gas modifiers on FAIMS separation has been explored. Adding carbon
dioxide, for instance, dramatically shifts CF values, significantly improving the separation of the isomeric phthalate ions, as well as
separation of dimeric and trimeric ions.
This presentation will report the first experiments interfacing a micro-FAIMS cell to a triple quadrupole mass spectrometer for fastscanning FAIMS.

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 10: Ion Mobility Spectroscopy Based on Instrument & Theoretical Development

PMo-196

13:30 － 14:40

Selected Mobility Accumulation (SMA) in a Trapped Ion Mobility Mass
Spectrometer (TIMS-MS)

Ulrich Giessmann, Melvin A Park, Desmond Kaplan, Mark Ridgeway
Bruker Daltonics, Billerica, MA, USA

Keywords:
Trapped Ion Mobility Spectrometry, Selected Mobility Accumulation, Comparison with conventional IMS, IMS for TOF analyzers,
IMS for FTMS analyzers
Novel aspects:
A new method of selected mobility accumulation（SMA）of ions in a trapped ion mobility spectrometry（TIMS）analyzer.
Abstract:
Introduction:
Recently, a new type of ion mobility analyzer the“Trapped Ion Mobility Spectrometry“（TIMS）analyzer was introduced, which
produces results similar to conventional drift cell analyzers in a physically smaller design（~10X）and at much lower operating
potentials［1, 2 ］. However, an analysis via TIMS as described thus far proceeds too rapidly（~ 2 ms/peak）for any mass analyzer
except time-of-flight（TOF）to follow. In the present a new technique - selected mobility accumulation（SMA）is introduced, whichmatches the timescale of the mobility analysis with that of“low duty cycle“ mass analyzers（e.g. FTMS）and techniques（e.g.
ETD）. SMA is achieved by efficiently accumulating ions of the desired mobility for long（300 ms）periods of time.
Results:
In the present work, a TIMS analyzer is incorporated as a section of an ion funnel in the ion source of a mass spectrometer. As
described previously［1,2］, in“broadband“ mode, ions are initially trapped in the TIMS analyzer wherein gas flow pushing ionsdownstream is counterbalanced by an electric field holding the ions back. After trapping, the electric field is slowly decreased sothat ions are eluted according to mobility. Eluting ions are mass analyzed in the orthogonal time-of-flight analyzer to produce a
TIMS MS spectrum. To perform selected mobility accumulation（SMA）experiments, the electric field is modified to form a“plateau“ within the TIMS trap on which ions of a selected mobility or mobility range can be accumulated.
The mobility analysis in a TIMS device may be viewed as roughly the same as that in a conventional IMS drift cell except that, inthe conventional cell an electric field pushes ions through a stationary gas whereas in the TIMS analyzer a retarding electric fieldholds ion in place against a moving gas. Ions, initially trapped in the TIMS analyzer, elute according to mobility lowest mobility firstas the strength of the retarding electric field is reduced. The“shape“ of the retarding electric field, potential vs. axial position, hasa profound effect on the number and types of ions that are initially trapped and the timing of their elution.
The effects of various retarding field gradient profiles were explored, and a plateau in the gradient profile at about 4 % below thehighest retarding gradient resulted in the optimal SMA results, i.e. mobility selection resolution of greater than 50 and duty cycle >50%. In one series of tests m/z 1222 ions from Agilent tunemix（PN G1969-85000）, which have a a Ko of about 0.72, were able to be
selectively accumulated for 300 ms without signs of saturation. Ejection of these ions required less than 100 ms, thus thefundamental limit on the duty cycle is much better than 50%. In further tests, higher amplitude RF confining fields resulted in longer
accumulation times before the onset of saturation. Also, higher pressures at the entrance to the TIMS analyzer leads to higher
resolution SMA but lower accumulated ion abundances.
［ 1 ］F. Fernandez-Lima, D.A. Kaplan, J. Suetering, and M.A. Park, Int. J. Ion Mobil. Spec. 14, 93（2011）.
［ 2 ］D.A. Kaplan, F. Fernandez-Lima, and M.A. Park, poster MP03 055, Proceedings of the 59th ASMS Conference, June 2011.

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 10: Ion Mobility Spectroscopy Based on Instrument & Theoretical Development

PMo-197

11:10 － 12:20

Evaluation of the performance of microscale FAIMS for enhancement of
quantitative analysis of metabolites and peptides

Robert W Smith2, Lauren Brown1, Danielle E Toutoungi1, James Reynolds2, Ashley Sage3,
Anthony W Bristow4, Andrew Ray4, Daniel Weston5, Ian Wilson5, Billy Boyle1, Colin Creaser2

1
Owlstone Ltd, 2Loughborough University, Loughborough, United Kingdom, 3Agilent Technologies, Stockport, United
Kingdom, 4AstraZeneca, Pharmaceutical Development, Macclesfield, United Kingdom, 5AstraZeneca, DMPK Innovative
Medicines, Alderley Park, United Kingdom

Keywords:
FAIMS, mobility, quantitatation, metabolites, peptides
Novel aspects:
Use of FAIMS pre-selection with LC-MS plus in-source CID for rapid quantitative analysis of metabolites and peptides.
Abstract:
High-field asymmetric waveform ion mobility spectrometry（FAIMS）is an atmospheric pressure technique that separates ions by
their differential mobility under high and low electric fields. In microscale FAIMS, the ions travel through channels with submillimetre dimensions, which enables substantially higher electric fields and faster separation than macroscale differential mobility
devices. Separation on sub-second timescales becomes possible, allowing the device to be used during real-time LC-MS analysis.
The extra separation dimension can be used to improve signal-to-noise in real-time quantitative analysis. This study evaluated theperformance of a microscale FAIMS system in various analyses including the determination of urinary drug metabolites and theidentification and quantification of peptides in plasma.
Microscale FAIMS devices（Owlstone Ltd, Cambridge, UK）were interfaced to an Agilent 6230 series TOF MS with a Jet Stream ESI
source and an Agilent 1200 series LC. The ion channels in these devices consist of an array of parallel electrodes with either 35μm
or 100μm gaps, to which an asymmetric dispersion field（DF）is applied. Ions with a selected differential ion mobility aretransmitted through the device by application of a suitable compensation field（CF）while other ions are eliminated in the filter. At
this scale, DFs of up to 300Td can be applied, and with a typical TOF MS capillary inlet gas flow rate of 1.7L/min, ion residence
times are reduced to 50-250μs. In some cases, in-source collision-induced dissociation of the FAIMS pre-selected ions（LC-FISCIDMS）was also carried out prior to mass analysis.
For the LC-FAIMS-MS analysis of metabolites in urine,（R/S）-ibuprofen 1 -b-O acyl glucoronide（IAG）metabolite was spiked into
filtered, diluted（x 2 ）urine. LC separation of the IAG spiked-urine was carried out on C18 column（Agilent Zorbax Rapid ResolutionHT, 2.1 x 50mm, 1.8μm）using an isocratic 0.2ml/min flow to achieve rapid elution of the metabolite（< 2 mins）.The FAIMS device
was set at a fixed DF and CF（CF = 1.9Td, DF = 260Td）that had been previously determined from a direct infusion of a mixture of
ibuprofen and IAG to give good transmission of the metabolite but not the parent drug. With FAIMS pre-selection, the spiked
standards gave a linear response over two orders of magnitude with an R2 value of > 0.99, showing an improvement in LOQ of afactor of 2 over the same experiment without FAIMS pre-selection. This was due to a doubling of the signal-to-noise ratio for the
metabolite peak with FAIMS pre-selection.
The potential of a quantitative LC-FISCID-MS method was also evaluated. In this technique, the FAIMS device is used to pre-selectcertain peptides, which are then fragmented by setting the TOF fragmentor voltage to produce in-source CID. Aliquots of humanplasma tryptic digest were spiked with the exogenous peptide gramicidin S. A DF of 275Td and CF of 3.0Td were applied to preselect the gramicidin precursor ion. Calibration graphs for the LC-FAIMS-MS analysis of the precursor ion（m/z 571）and the LCFISCID-MS analysis of the four FAIMS-selected in-source CID product ions（m/z 311,424, 685 and 798）show a linear response（R 2>
0.99）in the range 0.45-9.0μg/mL. The %RSD for the peak area of the FAIMS-selected precursor ion（m/z 571）without in-source
fragmentation was 5.1%（n= 6 ； 0.45μg/mL）. LC-FISCID-MS precision for the fragment ions at m/z 311, 424, 685 and 798, was 13.3%,
14.6%, 13.1% and 8.2% respectively, demonstrating good reproducibility for quantitative measurement at this concentration.
The results demonstrate that microscale FAIMS is a quantitative technique applicable to both small and large molecules that can
improve level of quantitation, with good precision and linearity, and that the LC-FISCID-MS method could be an alternative to LCMS/MS for quantitative analysis.

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 10: Ion Mobility Spectroscopy Based on Instrument & Theoretical Development

PMo-198

13:30 － 14:40

Develpment of Ion Trap Mobility Measurement System

Toshiki Sugai, Yoshihiko Sawanishi, Masashi Shinozaki, Sho Osaka
Toho University, Funabashi, Japan

Keywords:
Ion Mobility, Ion Trap, Long term measurement
Novel aspects:
Long term structural measrements have been performed by the newly developed ion trap mobility measurement system.
Abstract:
Ion mobility/mass measurements have been utilized for various bio molecules and nano materials［1,2］. The high sensitivity andhigh throughputs for mixed and unstable materials have advantages to clarify the new structures and the structural changes.
However, the measurement requires high pressure buffer gas which leads to ion dilution and other experimental difficulties. Oneof the approaches to overcome this problem is TWIMS system developed by Waters Co. Ltd.［ 3 ］It utilizes ion traps to reducethe diffusion and to enhance detection sensitivity. However there is potential demand for higher resolution and longer-term trap.
To improve these properties we have developed an ion trap mobility measurement system for charged particles in the first stage.
The system consists of stacked ring electrodes and an RF and LF power supplies. The RF and LF power supplies produced theradio frequency field to trap the charged particles and the low frequency field to move the particles in the trap for the mobility
measurements, respectively. The charged particles of salt water solution and polystyrene were introduced in the trap and weredetected and monitored through their movement by a microscope with a laser irradiation.
The particles were trapped for more than 2 hours where their mobility of the particles was measured by the amplitude of themodulation. The amplitude of the salt solution particles decreased gradually showing the particles growth through water vapor
absorption from air. On the other hand, the amplitude of the polystyrene particles was constant showing steady structures. Those
results show the potential high structural resolution and high traceability of the system.
We are now developing a multi-stage ion trap mobility system with an interface for a mass spectrometer in the second stage
which enables us to realize much higher resolution and application to nano materials.
［ 1 ］T. Sugai et al., J. Am. Chem. Soc. 123, 6427（2001）.
［ 2 ］T. Sugai, J. Mass Spetrsom. Soc. Jpn.58, 47（2010）.
［ 3 ］K. Giles et al., Rapid Commun. Mass Spectrom.18, 2401（2004）.

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 10: Ion Mobility Spectroscopy Based on Instrument & Theoretical Development

PMo-199

11:10 － 12:20

Achieving High Resolution in a Benchtop Resistive Glass Drift Tube Ion
Mobility Spectrometer

WILLIAM C NETOLICKY1, PAULA HOLMES1, FACUNDO FERNANDEZ2, JOE TICE3, BRIAN MUSSELMAN3
1

Photonis USA, Sturbridge, USA, 2Georgia Institute of Technology, Atlanta GA, USA, 3IonSense Inc., Saugus MA, USA

Keywords:
ION MOBILITY SPECTROMETRY, RESISTIVE GLASS, DARTNovel aspects:
An ion mobility spectrometer with very high resolving power was designed and constructed using resistive glass tubes, a photoetched Bradbury-Nielsen gate and a thermally assisted DART ionization source.
Abstract:
Ion mobility spectrometry has become widely accepted for the detection of chemical warfare agents, explosives and narcotics aswell as for pharmaceutical quality control and pesticide screening of food. However, to minimize the frequency of false positiveand false negative results, it is necessary to design and construct an ion mobility spectrometer with high separation power withoutcompromising instrument simplicity, serviceability, and cost. Resistive glass drift tubes provide key benefits such as uniform electricfields with minimal radial inhomogeneities and ease of fabrication. In addition, the single-piece construction allows even counter
flow of drift gas without the need for additional containment. Reaction and drift regions of the ion mobility spectrometer werebuilt using resistive glass technology. The Bradbury-Nielsen-type ion gate was produced using photo-etched grids and wasevaluated for its performance at varying operational voltages and frequencies. Ionization sources included a corona discharge aswell as a novel thermally assisted Direct Analysis in Real Time（TA-DART）source. The DART system provides a non-radioactivemeans for rapid desorption and ionization of both solid and liquid analytes with minimal sample preparation. Identification ofvolatile and solid samples was achieved with great simplicity and excellent sensitivity. Analytical performance was assessed using
several different calibrants and pharmaceutical samples. Resolving power of 70-120 was measured from the mobility spectra
demonstrating the ultra high resolution of the resistive glass drift tube IMS. Reduced mobility values were calculated in order toconfirm the identification of the observed signals with excellent agreement to literature values.

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

OthersPMo-200

13:30 － 14:40

Direct detection of S-nitrosylated peptides with UV-MALDI MS using
porphyrin and retinoic acid as a matrix additive

Makoto Watanabe1, Rie Yamamoto1, Shinichi Iwamoto1, Yuko Fukuyama1, Ritsuko Tanimura1,
Shin-Ichirou Kawabata1, Taka-Aki Sato1, Shunsuke Izumi2, Koichi Tanaka1
1

Shimadzu Corporation, Kyoto, Japan, 2Hiroshima University, Higashi-Hiroshima, Japan

Keywords:
S-nitrosylation, post-translational modification, UV-MALDI, tetraphenylporphyrin, retinoic acid
Novel aspects:
The use of tetraphenylporphyrin（TPP）or retinoic acid（RA）as a matrix additive allows the direct detection of S-nitrosylation by
UV-MALDI MS.
Abstract:
Introduction:
Nitric oxide（NO）exerts a ubiquitous influence on cellular signaling, largely through S-nitrosylation/denitrosylation of cysteines inproteins. Therefore, detailed analysis of protein S-nitrosylation is important for a better understanding the action of NO in various
biological processes. However, S-nitrosylation has never been detected by UV-MALDI MS because of S-NO bond instability.
Methods:
In this study, tetraphenylporphyrin（TPP）and retinoic acid（RA）were used as matrix additives with 2, 5 -dihydroxybenzoic acid
（DHB）to evaluate their suppressive effect on NO group dissociation of S-nitrosylated peptides. The two S-nitrosylated peptides（VFDARDC（NO）RSAQ and EMFTYIC（NO）NHIK）were subjected to MS and MS/MS analysis by MALDI-TOF MS and MALDIQIT-TOF MS, respectively.
Results:
In MS analysis, the predicted intact molecular ions［M+H］+ were observed using TPP or RA as a matrix additive although thecorresponding NO-dissociated ions［M-NO+H］+ were mainly observed. However, these ions［M+H］+ were not observed in theabsence of TPP or RA. Furthermore, MS/MS analysis of these ions indicated that the peptides contained S-nitrosylated cysteines.
Conclusion:
We demonstrated for the first time the possibility that addition of tetraphenylporphyrin（TPP）or retinoic acid（RA）to MALDI
matrix solutions allows the direct detection of S-nitrosylated peptides by UV-MALDI MS. In addition, MS/MS analysis of
S-nitrosylated peptides enables the modification sites to be determined.

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

OthersPMo-201

11:10 － 12:20

Bactericidal effects of atmospheric ions formed in ambient corona
discharges

Rena Gonda, Yuya Yamakawa, Kanako Sekimoto, Mitsuo Takayama
Yokohama City University, Yokohama, Japan

Keywords:
corona discharge, negative ions
Novel aspects:
An ambient corona discharge system which makes it possible to separately form various ionic and neutral species wasestablished, and ionic and neutral species having bactericidal effects were specified.
Abstract:
Atmospheric ions formed in ambient electrical discharges have been applied to various research fields, e.g., the application of
their sterilization effects in plasma medical sciences, as well as the utilization as reagent ions in APCI-like ambient ionization inmodern mass spectrometry. However, it is difficult to regularly produce atmospheric ions and by-product neutral species indischarge area, and therefore, the effects of ionic and/or neutral species on the sterilization have not been specified by now.
Recently we have identified the atmospheric ions and neutral species formed in ambient positive and negative corona discharges,
and found the factors governing the formation of those chemical species in discharge area. Here we attempted to establish an
ambient corona discharge system which makes it possible to separately irradiate various ionic and neutral species to Esherichia
coli（E. coli）and investigate bactericidal effects of individual chemical species.
Discharge experiments were performed in the laboratory ambient air under atmospheric pressure at 25 °C. A highly-curved
electrode used was a needle with 1 µm in the tip curvature radius. An orifice plate and brass plate were utilized as opposite
electrodes to detect ionic and neutral species formed in discharge area using a JMS-LCmate double-focusing MS（JEOL, Tokyo,
Japan）and AXIMA-CFR time-of-flight MS（Shimadzu, Kyoto, Japan）, while in the case of the investigation of bactericidal effects,
agar mediums spread E. coli DH 5 α suspensions of 200 µL was employed. The discharge conditions used were of 3 mm in pointto-plane electrode gap, +4.5 and -3.5 kV in DC needle voltage and 0 to 45°in needle angle with respect to the orifice axis.
The formation of atmospheric negative ions and neutral species in negative corona discharges was attributed to the electric fieldstrength on the needle tip surface, which is determined by the electrode gap, needle voltage and angle. The ionic and neutral
species formed at the tip peripheral region with the low field strength below 107 Vm-1 was hydroxide ion HO- and carbon clusters
Cn. The high field strength above 108 Vm-1 at the tip apex region led to the formation of abundant nitrogen oxide ions NOx -（x =2,3）and neutral species NOx. In the case of positive corona, oxonium ion H3O+ was generated under any electric field strengths,
while neutral species NOx occurred at the tip apex region with high field strength. The relationship between the field strength andthe resulting ionic and neutral species can be interpreted from the standpoint of the field-emitted electron kinetic energy which isthe principal factor governing the sequential progress of successive ion-molecule reactions in the formation of individual chemical
species. The trajectories of the resulting chemical species in discharge area were regulated by the point-to-plane electrodeconfiguration used and electric field distribution established. That is, the ions HO-, NOx -, and H3O+ can move along field lines thatarise from the needle tip surface where individual ions are formed, whereas the motion of the neutral species NOx directs toward
the needle tip axis due to elastic collisions with ions.
By using the discharge system described above, individual ionic and neutral species NOx -, HO-, H3O+, Cn and NOx were separately
irradiated to sample agar mediums with E. coli. When the discharged agar mediums were incubated at 37°C over night, growth of
E. coli was observed at the agar medium area where H3O+ and Cn were irradiated, while E. coli irradiated NOx - and HO- ions andneutral NO x did not grow. These results suggest that the specific chemical species, i.e., NO x - , HO - and NO x have bactericidal
effects.

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

OthersPMo-202

13:30 － 14:40

Thermal Activation of Methane: Why [Ti,O,H]+ and [V,O,H]+ are MoreThan the Sum of Their Atoms.

Robert Kretschmer, Maria Schlangen, Helmut Schwarz
Technical University Berlin

Keywords:
Ligand effects, methane activation, Ion-molecule reactions, vanadium, titanium
Novel aspects:
Discovery of intrinsic properties of transition-metal complexes and ligand effects with respect to methane activaton in the gas
phase
Abstract:
Transition metal catalyzed C-H bond-activation processes constitute an active area of research which can increase the power ofcatalytic organometallic chemistry. Especially the activation and functionalization of methane has been identified as one of the key
challenges to addressing the global energy problem.  While industrial process under environmentally benign and economicallyfeasible conditions have to be developed, the particular role of the electronic configuration of transition-metal complexes in theelementary steps of such processes have been studied in quite some length. Gas-phase studies provide an ideal arena to uncovermechanistic aspects in an unperturbed environment at a strictly molecular level. While“bare“ ground-state transition metals of
the first row do not bring about C-H bond activation of CH4,[1] ligated nickel II species［NiD］+,[2]［NiH］+,[3] and［NiF］+[4] form thecorresponding［Ni（CH 3 ）］+ complexes. In addition, bis-ligated cations are also capable to activate methane under thermal
conditions, i.e.［CrO2］+,[5]［FeO（OH）］+,[6] and［NiH（OH）］+.[7]Here we present the reactions of［M（OH）］+ and［HMO］+（M = Ti, V）with methane under thermal conditions. While［Ti（OH）］+
does not bring about C-H bond activation, the［HTiO］+ /CH 4 couple forms［TiO（CH 3 ）］+ and H 2； the same holds true for theisomers［V（OH）］+ and［HVO］+. These results serve as examples of both the prominent role of the oxidation state and the ligand
effects.

［ 1 ］a）P. B. Armentrout, J. L. Beauchamp, Acc. Chem. Res. 1989, 22, 315； b）J. C. Weisshaar, Acc. Chem. Res. 1993, 26, 213； c）H.
Schwarz, D. Schröder, Pure Appl. Chem. 2000, 72, 2319； d）P. B. Armentrout, Acc. Chem. Res. 1995, 28, 430.
［ 2 ］T. J. Carlin, L. Sallans, C. J. Cassady, D. B. Jacobson, B. S. Freiser, J. Am. Chem. Soc. 1983, 105, 6320.
［ 3 ］a）Q. Zhang, M. T. Bowers, J. Phys. Chem. A 2004, 108, 9755； b）M. Schlangen, H. Schwarz, Angew. Chem., Int. Ed. 2007, 46,5614.
［ 4 ］M. Schlangen, H. Schwarz, Helv. Chim. Acta 2008, 91, 2203.
［ 5 ］A. Fiedler, I. Kretzschmar, D. Schroder, H. Schwarz, J. Am. Chem. Soc. 1996, 118, 9941.
［ 6 ］D. Schröder, H. Schwarz, Angew. Chem. Int. Edit. Engl. 1991, 30, 991.
［ 7 ］M. Schlangen, D. Schröder, H. Schwarz, Angew. Chem. Int. Ed. 2007, 46, 1641.

Poster Session
Monday, 17th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

OthersPMo-203

11:10 － 12:20

Measurements of Proton and Alkali Ion Affinities using CID Spectra ofHigh Performance Tandem Mass Spectrometer

KIMIO ISA1, SHIGEKI MATSUMURA2, RYUJI NAKADA3

Jin-Ai University, Echizen City, Japan, 2Headquarters of Fukui Prefecuture Police, Fukui City, Japan, 3Fukui University,
Fukui City, Japan

1

Keywords:
CID, Proton affinity, Alkali Ion affinity, kinetic method, sector tandem
Novel aspects:
700T BEBE tandem mass spectrometer was high performance, especially ion intensity relatively strong and mass resolution was
also good and affinity determination was easily done.
Abstract:1990 years day, the measurements of CID spectra of a protonated dimer ion, (M1+H+M2)+ were determined the proton affinity
values or proton affinity (PA) order of amino acids M1 and M2 1) (20 amino acids) using kinetic method. JEOL 300 doublefocusing mass spectrometer (EBE type with cylindrical electrostatic analyzer: one sector) was used. The following conditions
were adapted: mass resolution 1000; FAB ion source, FAB gun was argon, and accelerating voltage of fast atom beam 3 kV; andCID gas argon at a pressure at 2*10-6   Torr at the Penning gauge (to attenuate the intensity of the gas pressure did not have asignificant effect on the CID spectra.) Hetero dimer ions was accerelated at 3 kV.
1996 years day, the proton affinity order of H2SO4 and 14 solvent molecules (acetone, methyl alcohol, ethyl alcohol, 1-propyl
alcohol, 2-propyl alcohol, 1-butyl alcohol, 2-butyl alcohol, t-butyl alcohol, 1-pentyl alcohol, 1-hexyl alcohol, ethylene glycol,
acetic acid, dimethyl sulfoxide, and water) were measured and CID spectra of the hetero dimer cluster ions were also measured.
The order of proton of H2SO4 was also confirmed to be PA(H2SO4)=707kJ/mol2) .
2003 years day, measurements of proton and alkali ion affinities using CID spectra of high performance tandem mass
spectrometer JEOL 700T (BEBE geometry: two sectors tandem) operating under FAB ionization conditions (Xe beam of 6 keVtranslational energy, gun current 3 mA). Ion accelerating voltage was 10 kV. The amine cluster ion including HCl, was measured
and the proton affinity of methamphetamine was determined to be 965 kJ/mol using the hetero dimer ions (A 1+HCl+H+A2)+
type ions including HCl, r value is 0.96.3)
2009 years day, dicision of proton affinity of amino acids and dipeptide including glycine using kinetic method4) by FABMS andFABMS/MS. And the behavior of two types samples are compared. The r values are almost 0.99. The measured samples are 9
amino acids (Cys, Ser, Asp, Val, Thr, Leu, Ile, Phe, and Met) and 12 dipeptides (Gly-Ser, Gly-Val, Gly-Leu, Gly-Asp, Gly-Thr, GlyIle, Gly-Phe, Gly-Met, Ser-Gly, Thr-Gly, Phe-Gly, and Ala-Gly). The comparison and the examination of these PAs are done.
2010 years day, alkali metal ion affinity of glycerol was determined by means of MS/MS. The experimental results wereanalyzed by kinetic method (Cooks et al. proposed 1978). Both Na + and Li + ion affinitys of glycerol were determined to be
181.8(kJ/mol) and 261.7(kJ/mol), respectively.
700T BEBE tandem mass spectrometer was high performance, especially ion intensity relatively strong and mass resolution was
also good and FAB ionization was long life time available ion continuing to decide PA. But reference samples was not easy to
get reference samples in case of alkali metal ions. Furthermore, the calculated affinity valued (Molecular orbital theory) aresometimes used to decide experimental affinity.
References
1) K.Isa et al., Org. Mass Spectrom. 25,620-628 (1990).
2) K.Isa et al., J. Mass Spectrom. Soc. Jpn. 44, 197-209(1996).
3) S.Matsumura et al., J. Mass Spectrom. Soc.Jpn 51, 196-200 (2003).

Plenary Lecture
Tuesday, 18th September

Main Hall

08:00 － 08:45

Plenary Lecture 3: Albert J R Heck
Chair:

Alma L Burlingame（UCSF, USA）

PL3-0800
08:00 － 08:45

A personal perspective on the role of mass spectrometry for protein
analysis in proteomics and structural biology

Albert J R Heck1,2
1

Utrecht University, Utrecht, The Netherlands, 2Netherlands Proteomics Centre

Keywords:
mass spectrometry, stem cells, viruses
Novel aspects:
novel mass analyzers, novel quantification methods
Abstract:
Mass spectrometry is nowadays everywhere! In the life sciences mass spectrometry is heavily used especially to analyze proteins,with the aim to identify them, count their numbers present in a given living system and to characterize them in detail, which
includes the analysis of protein modifications and interaction partners.
In our laboratory we focus on developing novel approaches to further analyze and characterize proteins using mass spectrometry
as the primary tool. Through recent examples I will highlight how such new tools assist in the better understanding of living
biological systems.
In more detail, I will describe the use of different quantification methods in MS-based proteomics to profile in-depth difference inprotein expression in between embryonic and induced pluripotent stem cells and in following cellular reprogramming offibroblasts into induced pluripotent stem cells, and how we can also use such sensitive methods to define the stemness of frommurine intestine isolated low amounts of adult stem cells.
Switching gears, I will also present new data on using native mass spectrometry to structurally characterize protein assemblies,
including viruses and bacterial clock systems.

Oral Session
Tuesday, 18th September

Main Hall

09:00 － 11:00

Session 11: Glycomics: From Disease Markers to Therapeutic Antibody Products
Chair:

Hyun joo An（ChungNam National University, Korea）

S11-0900
09:00 － 09:40[Keynote Lecture] Is high throughput glycomics possible?

Carlito B Lebrilla

University of California, Davis

Keywords:
Glycans, glycomics, oligosaccahrides, nanoLC
Novel aspects:
High throughput glycan analysis
Abstract:
The role of glycosylation and their biological functions whether as free oligosaccharides or glycoconjugates has been madepossible by the recent advancements in the analyses of these compounds. The heterogeneity and the large structural diversity
have made oligosaccharide analysis significantly more difficult than other biopolymers. The next stage of development is toachieve high throughput analysis. However, the structural elucidation of oligosaccharides remains an extremely difficult task.
Recent reports reveal that the diversity of structures in a given biological system is finite and may not be large. It may be possible
to create a database of structures that can be used to determine the identity of known compounds. This capability would
therefore make high throughput glycomics possible. Achieving this task depends on the proper selection of chemical
characteristics to identify the compound. In this presentation, nanoflow liquid chromatography retention times, accurate mass,and tandem MS is used to determine structure with a high degree of certainty. The method is used to determine the biological
function of milk oligosaccharides as well as to discover glycan-based biomarkers for diseases.

Oral Session
Tuesday, 18th September

Main Hall

09:00 － 11:00

Session 11: Glycomics: From Disease Markers to Therapeutic Antibody Products
Chair:

Hyun joo An（ChungNam National University, Korea）

S11-0940
09:40 － 10:00

Site specific characterization of O-GlcNAcylation on cytosolic and nuclear
proteins using electron transfer dissociation mass spectrometry.

Alma L Burlingame1, Samuel Myers1, Jonathan C Trinidad1, Giselle Knudsen1, Barbara Panning1,
Agnes Thalhammer2, Ralf Schoepfer2
1

UCSF, 2University College, London

Keywords:
O-GlcNAcylation, electron transfer dissociation
Novel aspects:
Developed a platform for separation of both O-GlcNAc and phosphopeptides from the SAME samples. Have investigatedregulation of pluripotency in stem cells and signal transduction system at synapse by O-GlcNAcylation.
Abstract:
O-GlcNAcylation is the reversible, posttranslational modification of serine and threonine residues on cytosolic and nuclear
proteins by β-N-acetylglucosamine. O-GlcNAc transferase and OGlcNAc-ase are the enzymes responsible for this carrying out thisprocess, respectively. Alterations in the O-GlcNAcylation sites and stoichiometries are modulated by many stimuli, including UDPGlcNAc levels and stress. The transferase is associated with a variety of adapter proteins, chromatin re-modeling complexes andis highly expressed in the brain. O-GlcNAcylation has been implicated in many important physiological processes including thediseases diabetes, Alzheimer and cancer. It may be particularly important in the central nervous system. OGT is present in
dendrites and axon terminals； it is also associated with microtubules. Neuron-specific deletion of OGT results in neonatal lethality
due in part to abnormal neuronal development and motor deficits. Since the transferase targets protein serine and threonine
residues, cross-talk with protein phosphorylation has been postulated.
We have developed a lectin based strategy to enrich native O-GlcNAcylated and multiply modified peptides from proteolytic
digests of mixtures of proteins and have employed electron transfer dissociation（ETD）mass spectrometry to assign sites ofmodification. We have also developed a platform for separation of both O-GlcNAc and phosphopeptides from the SAME
biological samples.
We have employed these methods to investigate regulation of pluripotency in mouse embryonic stem cells and the signaltransduction system at the synapse. This presentation will focus on our most recent results from these investigations.
Financial support was provided by NIH NIGMS Grants P41RR001614/ P41GM103481 and P50 GM081879-02（to ALB）.

Oral Session
Tuesday, 18th September

Main Hall

09:00 － 11:00

Session 11: Glycomics: From Disease Markers to Therapeutic Antibody Products
Chair:

Hyun joo An（ChungNam National University, Korea）

S11-1000
10:00 － 10:20

Carbonyl trapping activity of pyridoxal 5’-phosphate in vitro and in vivo

Roumyana Mironova1, Ivaylo Ivanov2, Valentin Lozanov3, Silvia Russeva3, Svetlana Simova4, Ivan Ivanov1,
Toshimitsu Niwa5
Institute of Molecular Biology Roumen Tsanev, Sofia, Bulgaria, 2University of Sofia St. Kliment Ohridski, Sofia, Bulgaria,
Medical University, Sofia, Bulgaria, 4Institute of Organic Chemistry with Centre of Phytochemistry, Sofia, Bulgaria,
5
Nagoya University Graduate School of Medicine, Nagoya, Japan1
3

Keywords:
glycation, diabetes, carbonyl stress, vitamin-B 6
Novel aspects:
A novel physiological role for vitamin B 6 is postulated： The free, protein unbound form of pyridoxal 5 ’-phosphate traps highly
toxic carbonyl compounds and thus alleviates carbonyl stress.
Abstract:
Glycation is a spontaneous chemical reaction, first discovered a century ago by the French chemist Louis Camille Maillard. After
him the reaction was called the Maillard reaction. In this reaction carbonyl compounds such as reducing sugars react nonenzymatically with primary amines including proteins, DNA and amino lipids to compromise their physiological functions. In thelast decades it has been recognized that the Maillard reaction is implicated in pathological conditions, such as diabetic
complications, renal failure and Alzheimer’s disease. The deleterious consequences of the Maillard reaction have instigated thesearch for compounds with carbonyl trapping activity that can protect tissues from carbonyl stress and glycation damage. In
previous studies we have shown that the physiologically active form of vitamin B6, pyridoxal 5 ’-phosphate（PLP）, prevents the
progression of diabetic nephropathy and dialysate-induced peritoneal damage. We have also demonstrated by gas
chromatography mass spectrometry that PLP scavenges the highly toxic dicarbonyl compound 3 -deoxyglucosone（ 3 DG）underphysiological conditions in vitro. The aim of the present study was to solve the structure of the PLP- 3 DG reaction product and tolook for its potential formation under physiological conditions in vivo.
In order to provide insight into the mechanism of PLP- 3 DG interaction, we have studied the reaction of PLP with three structural
analogues of 3 DG. These were glycerol（propane-1,2,3,-triol（PTO））, methylglyoxal（MGO）and D-glucose. Reactions between
PLP and the model compounds including 3 DG were carried out under physiological conditions in vitro（37 o C, pH 7.4）and the
products were analyzed by high performance liquid chromatography, tandem electrospray ionization mass spectrometry coupled
to liquid chromatography（LC/ESI-MS/MS）and nuclear magnetic resonance（NMR）including HRMS, 1 H-NMR, 13 C-NMR and
2 D-NMR techniques. Under these conditions PLP reacted poorly with PTO and D-glucose. In the case of PTO, glycerol-1,3-acetal（（ 5 -hydroxy- 4 -（ 5 -hydroxy-1,3-dioxan- 2 -yl）- 6 -methylpyridin- 3 -yl）methyl dihydrogen phosphate）and glycerol-1,2-acetals（cisand trans-（ 5 -hydroxy- 4 -（ 4 -（hydroxymethyl）-1,3-dioxolan- 2 -yl）- 6 -methylpyridin- 3 -yl）methyl dihydrogen phosphate）of PLP
were formed. PLP reacted slowly with D-glucose to form many isomers of aldol addition（m/z 428）and aldol condensation（m/z410）products, the reaction involving the enediol form of D-glucose as a nucleophile and the PLP aldehyde group as anelectrophile. The alpha-oxoaldehydes MGO and especially 3 DG demonstrated strong reactivity towards PLP with a yield of nearly
50% in the case of 3 DG. The reaction between PLP and MGO produced the hemiacetal 4 H-［1,3］-dioxino［4,5-c］pyridine- 4 -ol
as well as PLP-MGO aldol addition product（（ 2 -formyl-2,4-dihydroxy- 8 -methyl-3,4-dihydro- 2 H-pyrano［2,3-c］pyridin- 5 -yl）methyl
dihydrogen phosphate）and its hydrate form. In the reaction mixture of PLP with MGO we detected also an hemiacetal formed
between PLP and formaldehyde, the latter resulting most probably from partial decomposition of MGO. The reaction of PLP with
3 DG resembled that of PLP with D-glucose in that both aldol addition （m/z 410）（two isomers of ［（ 3 S,4R）-1,4,5,10a-tetrahydroxy- 3 -（hydroxymethyl）- 9 -methyl-1,3,4,4a,5,10a-hexahydropyrano［ 4 ’,3’：5,6］pyrano［2,3-c］pyridin- 6 -yl］
methyl dihydrogen phosphate - major products and two isomers of {（ 1 S）- 1 -［（ 1 R）-1,2-dihydroxyethyl］-3,3a,9-trihydroxy- 5 methyl-3,3a,9,9a-tetrahydro- 1 H-furo［ 3 ’,4’：5,6］pyrano［2,3-c］pyridin- 8 -yl}methyl dihydrogen phosphate - minor products）andaldol condensation（m/z 392）products were formed.
In order to evaluate the physiological significance of the PLP- 3 DG interaction, we have studied rats with streptozotocin-induced
diabetes mellitus, which were treated with oral administration of PLP（600 mg/kg/day）for 16 weeks. By using LC/ESI-MS/MS wehave detected PLP- 3 DG reaction products in sera and urine from the experimental animals with the aldol condensation product（m/z 392）prevailing over that of the aldol addition reaction（m/z 410）. It is noteworthy that these products were detected not
only in body fluids of the PLP-treated diabetic rats but also in the control group of healthy non-PLP treated animals. Based onthese experimental data we are tempted to postulate a novel physiological function for PLP： Besides its coenzyme activity, thefree, protein unbound form of PLP seems to play in vivo a role in detoxification of the toxic alpha-oxoaldehyde 3 -deoxyglucosone
and perhaps of other carbonyl compounds.

Oral Session
Tuesday, 18th September

Main Hall

09:00 － 11:00

Session 11: Glycomics: From Disease Markers to Therapeutic Antibody Products
Chair:

Hyun joo An（ChungNam National University, Korea）

S11-1020
10:20 － 10:40

Bioinformatics Platform for the Glycan Marker Discovery by Mass
Spectrometry

Jaehan Kim1, Hyunjoo An1, Carlito Lebrilla2
1

Chungnam National University, Daejeon, Korea, 2University of California, Davis, USA

Keywords:
Glycomics, Biomarker, Two-dimensional data analysis, Glycan correlation
Novel aspects:
We sugget the bioinformatics platform for the high throughput analysis of MS dataset for biomarker diacovery. In addiion, theglycan correlation was emplyed to validate the glycan marker
Abstract:
Despite the fast growing interest, mass spectrometry is still not well accepted as a primary discovery tool, particularly inglycobiology. Recent advancement of mass spectrometric instrument enable to provide high accuracy of molecular mass of
glycans, however, the wide range of their stereochemical and structural diversities hampers to conclude the glycan informationthat observed from unknown samples. Despite the rapid advancement, the elucidation of the structural（or compositional）
information of glycans by MS/MS tandem mass still necessitate the labor intense work and exhibit various limitations. Lack ofbioinformatical platform for the analysis of glycan causes the uncertainty in the data interpretation obtained from the mass
spectrometry and makes the conclusive judgment of experimental observation difficult. Particularly, the statistical tools used forproteomics such as PCA, ANOVA, or hierarchical cluster analysis are easily employed without the consideration for the uniquebiological properties of glycans and the statistical characteristics of glycan profiling by MS. The lack of an appropriately
customized bioinformatics platform for the analysis of MS data hampers the progress in glycan marker discovery. To overcome thecurrent limitations, we employed two-dimensional analysis using frequency and intensity domain after a series of data filtration.
Glycan correlation was applied to evaluate the biological relevance as a glycan as well as enabling a comprehensive glycomics
that can examine the glycans and modified glycans. With this approach we have found a set of potential glycan markers with thehigh sensitivity and high specificity in patient sera with ovarian cancer.

Oral Session
Tuesday, 18th September

Main Hall

09:00 － 11:00

Session 11: Glycomics: From Disease Markers to Therapeutic Antibody Products
Chair:

Hyun joo An（ChungNam National University, Korea）

S11-1040
10:40 － 11:00

Characterization of Elastin Degraded Peptides in Chronic Obstructive
Pulmonary Disease (COPD)

Yong Y Lin1, Toyonobu Usuki2

St.Luke-Roosevelt Hospital Center, Columbia University College of Physicians and Surgeons, 2Faculty of Materials andSciences, Sophia University

1

Keywords:
Elastin degraded peptides, COPD, Biomarkers, LC-ESI/MSMS, Peptidpmic
Novel aspects:
Elastn degradation which occurs in prevalent diseases have been studied. LC-MS/MS characterization of elastin degraded peptides
was performed to study disease development and search for potential biomarkers.
Abstract:
Characterization of Elastin Degraded Peptides in Chronic Obstructive Pulmonary Disease (COPD)
Yong Y Lin*
Department of Medicine, St. Luke’s Roosevelt Hospital Centers
Columbia University College of Physicians and Surgeons, New York, NY 10019, USA
Toyonobu Usuki
Department of Materials and Life Sciences, Faculty of Science and Technology
Sophia University, Tokyo 102-8554, Japan
Elastin is a highly crosslinked insoluble protein responsible for elasticity and resilience in various tissues including arterial bloodvessels, lung, and the skin. The degradation of elastin-containing tissues occurs in several widely prevalent diseases, such as
pulmonary emphysema, chronic obstructive pulmonary disease, cystic fibrosis, atherosclerosis, aortic aneurysm, etc. By utilizing
mass spectrometric characterization we have been investigating the degradation of lung elastic tissues involved in chronic
obstructive lung disease（COPD）. COPD is well recognized as one of the major health problem in the world. In the US it has
moved to the third leading cause of death.
An early insight into the mechanisms leading to lung alveolar destruction in patients with COPD is that lung elastin is a target forprotease degradation by cellular elastases. Elastin is a highly crosslinked insoluble protein formed by condensation of lysyl
residues in the soluble precursor, tropoelastin（786 amino acids）, which can be degraded into soluble peptide fragments by theelastolytic enzymes produced by neutrophils and macrophages. Two pyridinium compounds, desmosine（D）and isodesmosine（I）
were known as the major crosslinking molecules of insoluble elastin. We have developed a specific and sensitive LC-MS/MS using
a novel d4-desmosine as the internal standard, which can measure D and I in biofluids and has shown to be an effective biomarker
in COPD.
We have also studied degradations of lung elastin into peptide fragments by the specific proteases neutrophil elastase andmacrophage elastase, two major elastases involved in lung elastin degradation in COPD. Utilizing peptidomic LC-MS/MS we havecharacterized over 50 of elastin degraded peptides（EDPs）, some of them have been identified in plasma of COPD patients.
Inflammation mediated by T cells in the lung has been considered a possible perpetuating disease mechanism in COPD. TheseEDPs generated during lung injuries may modulate inflammation and lead to the progression of COPD. The detection andcharacterization of such peptides will allow us to investigate further enzymatic degradation process involved in pathogenesis in
COPD and search for effective peptide biomarkers and target for therapy.
In addition, we have synthesized and studied LC-MS/MS fragmentography of several desmosine peptides as models for the
crosslink structure of elastin.
1 ）Shuren Ma, Seymour Lieberman, Gerard M Turino, Yong Y. Lin. The detection andquantitation of free desmosine and isodesmosine in human urine and their peptide-boundforms in sputum. PNAS 2003；100：12941-12943.
2 ）Shuren M, Yong Y Lin, Gerard M Turino. Measurements of desmosine and isodesmosine by mass spectrometry in COPD. Chest
2007；131：1363-1371.
3 ）Jiangtao He, Gerard M Turino, Yong Y Lin. Characterization of peptide fragments fromlung elastin degradation in COPD. Exp Lung Res 2010； 36：545-557
4 ）Toyonobu Usuki, Haruka Yamada, Takahiro Hayashi, Hiroto Yamada, Yohei Koseki,
Noriyuki Suzuki, Yoshiro Masuyama, Yong Y Lin. Total synthesis of COPD biomarker

desmosine that crosslinks elastin. Chem Commun 2012； 48： 3233-3235.

Oral Session
Tuesday, 18th SeptemberRoom A

09:00 － 11:00

Session 12: On-site Mass Spectrometry -Miniaturized Instruments and Allied TechnologiesChair:

Shuichi Shimma（National Cancer Center Research Institute, Japan）

S12-0900
09:00 － 09:40[Keynote Lecture] In-vivo, Real-time Identification of Tissues in Human
Surgical Environment by Rapid Evaporative Ionization Mass Spectrometry

Zoltan Takats1, Julia Balog2, James Kinross1, Christian Schafer1, Laura Muirhead1, Ottmar Golf4,
Laszlo Sasi-Szabo3, Balazs Dezso3, Kirill Veselkov1

Imperial College, London, UK, 2Medimass Ltd., Budapest, Hungary, 3University of Debrecen, Debrecen, Hungary, 4Justus
Liebig University, Giessen, Germany

1

Keywords:
ambient ionization, ion transfer, lipidomics
Novel aspects:
Presentation of a mass spectrometric method for the in-vivo identification of tissues during surgical interventions, together with theresults of first clinical study involvig 250 subjects.
Abstract:
Rapid identification of biological tissues is a long-standing problem on various fields of interventional medicine, with special regard
to cancer diagnostics and cancer surgery. While healthy tissue types（including benign tumors）show complete anatomical
separation, malignant proliferations infiltrate surrounding healthy tissue parts, making visual localization extremely hard, or even
impossible. While histological techniques provide the ultimate solution for the cellular-level identification of cancer cells, theapproach is extremely complex and time consuming. Nevertheless, accelerated version of histopathology（so-called ’frozen
section’ method）is widely used for the intraoperative characterization of tissue samples removed from the surgical area. Since
frozen section histology is less reliable than the traditional approaches, and the accelerated procedure still takes approx. 30
minutes for a single sample, there has been ongoing research for the development of more accurate and faster methods.
Molecular spectroscopy techniques including IR, Raman, solid state NMR and mass spectrometry have been used for thecharacterization of intact biological tissues and showed enormous potential for the differentiation of tissues with various
histologies, including multiple different types of cancer. While all of these techniques are able to provide rapid information, theunderlying experimental setups have not allowed the utilization of these techniques in surgical or diagnostic environments（e.g. incase of mass spectrometric imaging of tissue sections）.
Rapid Evaporative Ionization Mass Spectrometry is based on the observation that electrosurgical dissection of vital tissues involves
the ionization of various tissue constituents, with special emphasis on membrane lipids. Electrosurgical methods employ electric
current for the rapid heating and evaporation of tissue material and they are widely used both for dissection and coagulation on
practically all fields of surgery. Hence, the direct combination of electrosurgery with mass spectrometry provides a tissue
identification methodology, where the tissue manipulation part is already widely used by surgeons and fully approved from
regulatory point of view. Electrosurgical handpieces have been coupled to mass spectrometers using flexible polymer tubing and
Venturi air-jet pump. The experimental setup allows the transfer of ionic species from surgical site to mass spectrometer installedin the operating theatre. Mass spectrometers were equipped with special, noise insulated airtight chassis for surgical applications.
REIMS data of human tissues were found to show high histological specificity and excellent patient-to-patient reproducibility.
While the detected complex lipid-type species generally occur in most tissue types, their distribution pattern is unique for the
given histological types. Spectral data was analyzed using multivariate statistical approaches including principal component
analysis and linear discriminant analysis. Based on these approaches, a rapid tissue identification system was implemented, whichis capable of the identification of unknown tissues within 1 sec, during surgical interventions. The technique has been tested incase of gastrointestinal adenocarcinoma and its liver metastases, involving 250patients. Tumor tissue has been identified
successfully in all cases either in-vivo or using fresh ex-vivo tissues. Keeping false negative rates at 0 %, false positive rate < 0.5 %has been achieved.
While the tissue identification process requires large number of histologically assigned authentic spectra, collection of databaseentries in a parallel fashion with histopathological examination is time and sample consuming process. Nevertheless, more than
40,000 individual database entries were collected and stored in the REIMS database. Since imaging mass spectrometric methods
are able to provide histologically resolved data on tissue lipidome, we have developed a spectral pre-processin galgorithm tobring DESI/MALDI imaging and REIMS data to a statistically comparable level. The combination of different mass spectrometricanalysis modalities results in 3 orders of magnitude faster data collection rate and significantly improved histological accuracy.

Oral Session
Tuesday, 18th SeptemberRoom A

09:00 － 11:00

Session 12: On-site Mass Spectrometry -Miniaturized Instruments and Allied TechnologiesChair:

Shuichi Shimma（National Cancer Center Research Institute, Japan）

S12-0940
09:40 － 10:00

Miniaturization of a Toroidal Ion Trap Mass Analyzer Composed of
Cylindrical Electrodes

Daniel E Austin, Nicholas Taylor, Jessica Higgs
Brigham Young University, Provo, Utah, USA

Keywords:
toroidal, ion trap, quadrupole
Novel aspects:
first demonstration of toroidal ion trap using cylindrical electrodes
Abstract:
Simplification of electrode geometry allows ion traps to be miniaturized without tolerance complications associated withmachining hyperbolic surfaces. Cylindrical and rectilinear ion traps exemplify this process： each uses electrodes that are
geometrically simpler than those used in the quadrupole and linear ion traps, respectively. A challenge of accomplishing thisprocess with toroidal ion traps is the asymmetry that is needed to counteract the curvature of the trapping region. We present a
novel toroidal ion trap design employing cylindrical electrodes. Asymmetric overlap of the electrodes compensates for thecurvature of the toroidal trapping region. Mass spectra of several organics demonstrate instrument performance, including mass
resolution better than that reported in other toroidal devices.
A full-size（r0 = 6 mm）prototype device was constructed, consisting of four electrodes： one inner cylinder（ground）, one outercylinder（AC）, and two end-cap plates（RF）. Mass analysis is accomplished using resonance ejection and a ramp of the applied
RF amplitude. Mass-analyzed ions are radially ejected to a single point at the center of the device by an ac signal applied to theouter cylinder electrode. Headspace vapor samples were ionized in situ using an electron gun aimed at a small slit in the outercylinder electrode. Higher-order components of the electric field were optimized using SIMION. Several different values of higherorder field components were studied experimentally by varying the spacing between the two end-cap plates（RF）. Both forward
and reverse scans were explored.
For toluene samples, the 91-92 peaks are baseline resolved, with mass resolution of 0.32. Mass resolution as a function of the RF
end-cap spacing is best when the octopole-component of the trapping field is slightly positive, +0.4 % , and gets progressively
worse above and below this value. Evaluation of both forward（mass selective instability scan）and reverse（resonance ejectionscan）scans give fairly similar resolution for a given octopole value with the optimum being at +0.4 % for the reverse scan withresonance ejection. Simulations indicate that ejection efficiency is close to unity： all ions are ejected radially toward the center,and none are lost in the opposite direction. Tandem mass analysis（MS2）was also demonstrated using this device.
Under current development are two miniaturized versions of this device（r 0 = 1 and 2 mm）, in which the RF and AC electrodes
are made using sheets of stainless steel cut using wire EDM. This approach greatly reduces machining costs, and also allows
uniformity of trapping dimensions along the toroidal trapping region. Because the major toroidal radius is unchanged from the fullsize version, the trapping capacity is not significantly reduced in these miniaturized versions. In addition, an ion guide is integrated
on the trap, forming a“p“ shape, to facilitate introduction of ions into the trap. Simulations show that this eliminates the RF
barrier for ion introduction.

Oral Session
Tuesday, 18th SeptemberRoom A

09:00 － 11:00

Session 12: On-site Mass Spectrometry -Miniaturized Instruments and Allied TechnologiesChair:

Shuichi Shimma（National Cancer Center Research Institute, Japan）

S12-1000
10:00 － 10:20

Auto-sampling Explosives Trace Detection System using Mass
Spectrometry

Yuichiro Hashimoto, Hisashi Nagano, Yasuaki Takada, Yasutaka Suzuki, Hideo Kashima,
Masakazu Sugaya, Yasunori Doi, Koichi Terada, Minoru Sakairi
Hitachi, Ltd. Central Research Laboratory

Keywords:
Explosives Trace Detection, Cyclone-type Concentrator
Novel aspects:
Development of a novel auto-sampling explosives trace detection system using mass spectrometry that achieved high-throughput
detection within a few seconds for various types of explosives.
Abstract:
Introduction
Due to the ease of obtaining procedures for making explosive devices via the Internet, the threat of improvised explosive devices（IEDs）has increased in many countries. Since explosives trace detector（ETD）is a powerful tool to help prevent terror attacks,
many types of ETDs are being developed currently. We developed a high-throughput auto-sampling explosives trace detector（ETD）portal using mass spectrometry［ 1 ］. The ETD portal achieved high-speed detection of volatile explosives within a few
seconds without human hands. We also reported a false-positive rate of less than 0.1 % when the prototype detector was
deployed in at ticket gates at train stations in Japan［ 2 ］. In this paper, I’ll review our recent developments of a novel autosampling ETD system, which achieved less volatile explosives detection using a cyclone particle concentrator.
MethodTo extend the analysis targets to less volatile explosives such as TNT, PETN, and RDX, particle sampling as well as vapor samplingis required. We’ve been developing a novel particle sampler using a cyclone-type particle concentrator that can quickly andeffectively collect and concentrate explosive particles that are attached to objects. This sampler consists of detachment,
evacuation, condensation, and heating components. First, explosive particles on a card are detached by air jet pulses and thencollected by evacuation. Particles are collected by a cyclone particle separator, where only particles are collected near thebottom of the container. The collected particles are heated, and then the explosive vapor is introduced into a mass spectrometer.
Preliminary data
We tested a prototype of the particle sampler using NIST SRM 2905, trace particulate explosive simulants. All the signals from theug-level explosive particles, which included a percent ng level of TNT and RDX, were detected within a few seconds after an air
jet pulse was injected from a spray nozzle onto a plastic card. An appropriate evacuation flow rate is over 50 L/min because
anything lower will not be sufficient to collect the detached particles in the sample collector. However, the typical flow rate into a
mass analyzer is about 1 L/min. Therefore, the cyclone separator, which is positioned between the sample collector and themass spectrometer, is used to condense the explosive vapor from the particles to a volume 50 times smaller compared to thecase without it. The collection efficiency of the cyclone separator is over 50%, which means the sensitivity was enhanced by morethan 25 times using the cyclone separator compared to the case without it. After a TNT signal was detected, the signal level
returned to the background level in less than 20 seconds. The recovery time strongly depends on the heating temperature of theparticle heater.
This work was supported by“R&D Program for Implementation of Anti-Crime and Anti-Terrorism Technologies for a Safe andSecure Society“, Strategic Funds for the Promotion of Science and Technology of the Ministry of Education, Culture, Sports,
Science and Technology, Japan.
References
1 ）Y. Takada et al., Rapid Commun. Mass Spectrom. 2011； 25（17）： 2448.
2 ）Y. Hashimoto et al,“Field test evaluation of a walkthrough portal detector of improvised explosive devices at a train station“,
Trace Explosives Detection Workshop, April, 2011, Portland.

Oral Session
Tuesday, 18th SeptemberRoom A

09:00 － 11:00

Session 12: On-site Mass Spectrometry -Miniaturized Instruments and Allied TechnologiesChair:

Shuichi Shimma（National Cancer Center Research Institute, Japan）

S12-1020
10:20 － 10:40

Rapid Screening Chemicals in the Liquids and Solids via Mobile Ambient
Mass Spectrometry (MAMS)

Min-Zong Huang, Chu-Nian Cheng, Hung Su, Jentaie Shiea
National Sun Yat-Sen University, Kaohsiung, Taiwan

Keywords:
Mobile Ambient Mass Spectrometry；thermal desorption； miniaturized mass spectrometry
Novel aspects:
Mobile ambient mass spectrometry was used to rapidly screen chemicals in the liquids and solids samples.
Abstract:
A mobile ambient mass spectrometry（MAMS）was developed and constructed for rapid characterization of chemicals in theliquids and solids. The MAMS system is comprised of a commercial mass spectrometer equipped with an ambient ionization
source, gas generator, and power supply. The MAMS was set on a moving cart for expanded mobility； shock absorbers wereadded to protect the system from damage when the entire apparatus was on the move. Three steps are usually involved ina
typical MAMS analysis - sampling, thermal desorption, and electrospray ionization. The liquid or solid sample attached on a metalprobe was inserted in a preheated oven. The analytes in the sample were subsequently desorbed and conducted to the tip of acapillary where ions and charged droplets were continuously generated by electrospraying an acidic methanol solution. The
desorbed analytes reacted with the charged methanol species or fused with the charged solvent droplets to produce analyteions. The analytes could be protonated by the ion-molecule reactions between analytes and solvent ions, or fused in the charged
solvent droplets and then electrospray ionized. Since the total analytical time neededis only three seconds per sample, a highthroughput screening strategy can be made by this technique.
The MAMS system is integrated delicately on a golf cart； therefore, its moveability is non-restricted that the analysis can be
performed wherever is needed. In addition, because the conventional mass spectrometer is employed, the MAMS system has
wider applicability and higher specificity than that of the portable MS. This feature has broadened its practical use in on-site
analysis, and made itself as a powerful analytical means in the areas of public safety, environmental protection, and pollution
monitoring.

Oral Session
Tuesday, 18th SeptemberRoom A

09:00 － 11:00

Session 12: On-site Mass Spectrometry -Miniaturized Instruments and Allied TechnologiesChair:

Shuichi Shimma（National Cancer Center Research Institute, Japan）

S12-1040
10:40 － 11:00

Development of portable vacuum ultraviolet single photon ionization
mass spectrometer for trace measurement of volatile organic compounds

Yukio Yamamoto1, Hiroyuki Yamada2, Kenichi Tonokura1
1

The University of Tokyo, Chiba, Japan, 2National Traffic Safety and Environmental Laboratory, Tokyo, Japan

Keywords:
photoionization, time-of-flight mass spectrometer, volatile organic compounds, vehicle emission
Novel aspects:
Portable VUV-SPI-TOFMS was applied to real-time measurement of VOCs in the vehicle exhaust gas.
Abstract:
Volatile organic compounds（VOCs）play an important role in the tropospheric chemistry in terms of production of ozone and theorganic aerosols. It is necessary to understand the composition and concentration of numerous VOCs in the environment since
the mechanisms for ozone and organic aerosols formation are different for every VOC. New techniques that can perform in-situ
and real-time measurement of each VOC are needed for the identification of the sources and the distributions of VOCs. Time-offlight mass spectrometry（TOFMS）is a suitable technique for quantitative and qualitative analysis of trace VOCs in anenvironment. The advantages of MS for air analysis are excellent sensitivity and real-time response. TOFMS has a merit of its
compact size. Single photon ionization（SPI）using vacuum ultraviolet（VUV）light is a threshold ionization method for analyticalmass spectrometry. VUV-SPI is also a soft ionization technique that can be adjusted to be nearly fragmentation free and themolecular ion peaks of most organic molecules can be observed. For real-time measurement of VOCs with high sensitivity, we
have developed VUV-SPI-TOFMS1.
The compact VUV-SPI-TOFMS that we developed is 0.5 m × 0.5 m × 0.5 m in size and about 30 kg in weight, excluding the power
supply for the Nd：YAG laser. Ionization is conducted by photoionization at 10.5 eV（wavelength： 118 nm）VUV light generated by
the 9 th harmonics of a Nd：YAG laser. Accelerated ions are introduced to field-free drift region and are reflected by reflector. The
ion signals are detected by microchannel plate. The TOF mass spectra can be recorded in real time or stored on the hard disk to
be analyzed by home-written LabVIEW software programs. We achieved a limit of detection of 8, 11, and 18 parts per billion by
volume（ppbv）for benzene, toluene, and chlorobenzene, respectively, with an integration time of 5 s（100 measurements
averaging）. A mass resolution of 800 at 112 m/z was obtained in a 0.35 m long reflectron TOFMS instrument1.
The measurements of vehicle exhaust gases were demonstrated2. The vehicle emission is one of the major sources of VOCs in
atmosphere. Because the vehicle exhaust constantly changes in synchronization with engine operation, real-time measurement isrequired to analyze the species in exhaust gases. The exhaust gases were sampled in an exhaust pipe of tested vehicles driven ona chassis dynamometer. The TOF mass spectra of the sampled gas were averaged and recorded every 1 second（20 single-shot
spectra）. The obtained signals were converted into concentration profiles using premixed standard gas. Emission profiles of
VOCs from both gasoline and diesel engine vehicles were measured. Alkylbenzens, alkenes, alkanes, and dienes were measured
in addition to ppmv-level aromatics such as benzene and toluene in the vehicle exhaust gases. The changes in concentrations of
VOCs in the exhaust gases were different between gasoline and diesel engine vehicles. We obtained the mass spectra of theexhaust during each driving time and the temporal profiles of every m/z in one measurement. Furthermore, from themeasurements of hydrocarbons using the VUV-SPI-TOFMS, we obtained time profiles of the emitted weights of VOCs and their
total emissions. We have shown that VUV-SPI-TOFMS is useful for real-time and simultaneous measurements of VOCs in thevehicle exhaust with high sensitivity and real-time response.
References：［ 1 ］K. Tonokura, N. Kanno, Y. Yamamoto, and H. Yamada, Int. J. Mass Spectrom., 290, 9 -13（2010）.
［ 2 ］Y. Yamamoto, Y. Kambe, H. Yamada, and K. Tonokura, Anal. Sci., 28, 385-390（2012）.

Oral Session
Tuesday, 18th SeptemberRoom B-1

09:00 － 11:00

Session 13: Accelerator Mass Spectrometry
Chair:

Hiroyuki Matsuzaki（The University of Tokyo, Japan）

S13-0900
09:00 － 09:40[Keynote Lecture] Accelerator Mass Spectrometry - analysis of the rarest
atom species for earth and environmental science

Peter Steier1

University of Vienna, Faculty of Physics

1

Keywords:
Accelerator mass spectrometry, Anthropogenic nuclides, Beryllium-10, Iodine-129, Uranium-236, Ocean Tracers, Fission products,
Isobar separation
Novel aspects:
Many new AMS facilities emerge for the "traditional" AMS isotopes Be-10, C-14, Al-26, Cl-36, and I-129, while the development
proceeds towards more efficient measurements with always smaller machines.
Additionally, in the next years, improved isobar separation will allow to extend the AMS technique to the middle mass range from60 to 200 amu. Since many long-lived anthropogenic and natural fission products are present in the environment, many new
applications will show up.
Abstract:
Accelerator Mass Spectrometry (AMS) excels over other methods of analytical isotope research by its extraordinary abundance
sensitivity, i.e. the ability to separate out the rarest atom species from abundant stable isotopes. This makes the method theperfect fit for long-lived radionuclides with half-lives between 1 kyr and 100 Myr, of both natural and anthropogenic origin. During
the last 30 years, AMS has made significant contributions to practically all fields of science.
Dating based on natural C-14 for archaeology and palaeontology is still one major application. With increasing precision,
radiocarbon dating starts to compete with traditional historical dating, e.g. connected to the ancient Thera eruption in the Eastern
Mediterranean Sea. Anthropogenic C-14, produced by the atmospheric nuclear weapons tests, allows for so-called bomb-peakdating with a precision of about 1 year. This development has fallen on fertile ground in biomedicine. Dating of human tissue andespecially DNA reveals new insights into turnover rates and regeneration. Pharmaceutical research benefits also from so called"microdosing" which studies the human metabolism with C-14 labeled, but nonhazardous small amounts of new substances. New
dedicated C-14 AMS systems usually have reduced size and complexity, providing competitive performance with a footprint of
only a few square meters.
A complementary trend to larger size accelerators is observed for the upcoming facilities aiming at the other "traditional" AMS
isotopes Be-10, Al-26, Cl-36, and I-129. Their detection in surface rocks or in natural archives like ice cores allows to investigate
the processes shaping our planet, and to assess their time scales, which is the basis for the prediction of the impact ofanthropogenic activities on a global scale. The new facilities are often designated as working horses for earth and environmental
science, and are not necessarily affiliated at physics departments.
Recently, U-236 and the plutonium isotopes from global fallout have demonstrated their potential as environmental tracers. U-236
shows conservative behavior in the ocean. Pu, on the other hand, binds to soil particles, and can thus serves as a tracer for
sediment transport. Both uranium and plutonium can be used to detect releases of nuclear fuel.
The main technical challenge for AMS is imposed by stable isobars from other elements, which are always present at the ppm
level even in purified samples. While the higher energy of AMS allows their suppression via the different energy loss in matter forthe lighter masses, even the largest machines cannot generally separate isobars above Fe-60. Many isotopes in the middle mass
range with suitable half-life are present in the environment as natural and anthropogenic fission products. New methods based on
chemical reaction cells or laser detachment are developed to remove the interfering isobars. Once successful, a whole newspectrum of applications will be opened.

Oral Session
Tuesday, 18th SeptemberRoom B-1

09:00 － 11:00

Session 13: Accelerator Mass Spectrometry
Chair:

Hiroyuki Matsuzaki（The University of Tokyo, Japan）

S13-0940
09:40 － 10:00

Small scale radiocarbon dating and its applications to understandAntarctic ice sheet

Yusuke Yokoyama1, Yosuke Miyairi1, Suga Hisami2, Matsuzaki Hiroyuki3, Ohkouchi Naohiko2
1

University of Tokyo, Chiba, Japan, 2JAMSTEC, Yokosuka, Japan, 3University of Tokyo, Tokyo, Japan

Keywords:
AMS, Radiocarbon, Small Scale, Antarctica
Novel aspects:
Small scale radiocarbon dating on compound specific measuremens conducted by AMS allows us to understand Earth’s surface
dynamics. Antarctic ice sheet melting history is clearly depicted.
Abstract:
Recent advancement of accelerator mass spectrometry（AMS）enables us to conduct small scale radiocarbon dating. This allows
us to measure compound specific measurements for understanding dynamics of Earth surface systems in detail. We havedeveloped AMS measurement techniques for small- scale samples ranging from 0.01 to 0.10 mg C（Yokoyama et al., 2010）with a
new type of MC- SNICS ion source system（Southon and Santos, 2007）. We can generate 4 times higher ion beam current
intensity for ultra-small samples by optimization of graphite position in the target holder with the new ionizer geometry. CO 2 gas
graphitized in the newly developed vacuum line is pressed to a depth of 1.5 mm from the front of the target holder. This is much
deeper than the previous position at 0.35 mm depth. We measured 12C 4 + beam currents generated by small standards and ion
beam currents（15-30 μA）from the targets in optimized position, lasting 20 min for 0.01 mg C and 65 min for 0.10 mg C. We
observed that the measured 14C/12C ratios are unaffected by the difference of ion beam currents ranging from 5 to 30 μA,
enabling measurement of small size samples with high precision. Examination of the background samples revealed 1.1 μg ofmodern and 1 μg of dead carbon contaminations during target graphite preparation. We make corrections for the contaminationfrom both the modern and background components. Reduction of the contamination is necessary for conducting more accurate
measurement.
In this presentation, we will introduce our system at our lab and recent developments regarding further efforts of reducing
background. Then present some geological applications using sediment samples obtained around Antarctica coupling also with
10Be measurements.
References
Southon JR, Santos GM. 2007. Life with MC-SNICS. Part II： further ion source development at the Keck carbon cycle AMS facility.
Nuclear Instruments and Methods in Physics Research B 259（ 1 ）： 8893.
Yokoyama, Y., Koizumi, M., Matsuzaki, H., Miyairi, Y., and Ohkouchi, N., 2010. Developing ultra small-scale radiocarbon sample
measurement at the University of Tokyo. Radiocarbon, 52, 310-318.

Oral Session
Tuesday, 18th SeptemberRoom B-1

09:00 － 11:00

Session 13: Accelerator Mass Spectrometry
Chair:

Hiroyuki Matsuzaki（The University of Tokyo, Japan）

S13-1000
10:00 － 10:20

Beryllium 10 analysis for the Dome Fuji ice cores and cosmic-ray
stratigraphy

Kazuho Horiuchi1, Shota Suguchi1, Kensuke Suda1, Tomoko Uchida2, Takahiro Aze3, Yusuke Yokoyama4,
Yasuyuki Muramatsu5, Hiroyuki Matsuzaki4, Hideaki Motoyama6
Hirosaki University, Hirosaki, Japan, 2IAA, Shirakawa, Japan, 3Tokyo Institute of Technology, Tokyo, Japan, 4The
University of Tokyo, Tokyo, Japan, 5Gakushuin University, Tokyo, Japan, 6National Institute of Polar Research, Tokyo,
Japan
1

Keywords:
Beryllium 10, Cosmic ray, Paleointensity, Stratigraphy
Novel aspects:
A novel long-term record of cosmogenic beryllium 10 from Antarctica that serves as a proxy of the paleo-cosmic-ray flux and as astandard curve for cosmic-ray stratigraphy.
Abstract:Long-term records of cosmogenic radionuclides（such as 10Be, 14C, and 36Cl）provide a clue to understand the history of cosmic
ray variations and climate-driven changes in the fallout/depositional systems in the atmosphere, land and ocean. They can be alsoutilized for radiometric and/or stratigraphic dating back to the past ten million years.
We have been analyzing the cosmogenic radionuclide 10Be in polar ice cores by employing one of the state-of-the-art systems of
Accelerator Mass Spectrometry（AMS）in Japan. As a good example of the results of our project, we show here a 300-kyr recordof 10Be in the Dome Fuji ice cores with a time resolution of 1 kyr. The Dome Fuji ice cores were drilled at the Dome Fuji station（77°19’S, 39°42’E）, inland East Antarctica. The concentration of 10Be in the ice samples was determined by using an AMS system
at The University of Tokyo（MALT-AMS system）. The measured concentration was converted to the flux by multiplying the snow
accumulation rate estimated by Parrenin et al.（2007）. The chronology of the cores is based on a direct and precise orbital tuningwith the variations in the ratios of O2 and N2 in air trapped in the ice cores（Kawamura et al., 2007）.
The 10Be flux over the past 300 kyr recorded in the Dome Fuji ice cores showed distinct increasing peaks at around 41, 114, and 189
ka, which are correlative to the well-known geomagnetic excursions Laschamp, Blake, and Iceland Basin, respectively. Empiricaland semi-quantitative estimation of geomagnetic paleointensity from the 10 Be flux indicates that most of the variations in 10 Be
were attributable to the variations in cosmic-ray intensity modulated by geomagnetic effects for incoming cosmic-ray particles.
This fact strongly suggests that our 10Be record properly serves as a proxy of the paleo-cosmic-ray flux and as a standard curve forthe stratigraphic correlation based on the cosmic-ray variations（i.e. as a standard curve for cosmic-ray stratigraphy）. In thispresentation, we will also explore the possibility of the cosmic-ray stratigraphy using our 300-kyr record of the 10Be flux.
References：
Parrenin, F. et al.（2007） 1 -D-ice flow modelling at EPICA Dome C and Dome Fuji, East Antarctica. Clim. Past , 3, 243-259.
Kawamura, K et al.（2007）Northern Hemisphere forcing of climatic cycles in Antarctica over the past 360,000 years, Nature, 448, 912916.

Oral Session
Tuesday, 18th SeptemberRoom B-1

09:00 － 11:00

Session 13: Accelerator Mass Spectrometry
Chair:

Hiroyuki Matsuzaki（The University of Tokyo, Japan）

S13-1020
10:20 － 10:40

Quantifying soil formation and sediment yield in mountains watersheds
using terrestrial cosmogenic nuclides

Yuki Matsushi1, Hiroyuki Matsuzaki2
1

Kyoto University, Uji, Japan, 2The University of Tokyo, Tokyo, Japan

Keywords:
terrestrial cosmogenic nuclides, sediment yield, soil production, denudation rate, geomorphology
Novel aspects:
Advances in sensitivity and accuracy in measurements, and evolution in methodology in applying isotopes in mountaingeomorphology.
Abstract:
This report overviews principle and potential for use of terrestrial cosmogenic nuclides in mountain geomorphology, especially forquantification of earth surface processes. Technological advance in accelerator mass spectrometry enables us to measurecosmogenic nuclides in rock minerals such as 10 Be and 26 Al in quartz, and 36 Cl in calcite, which provide exposure ages ordenudation rates of land surfaces. In recent decades, the nuclide analysis offers not only the minimum exposure age or maximum
denudation rate of a single rock surface, but spatially-averaged denudation rate of a watershed, soil production rates in hill slopes,
and also information for reconstructing basin development in mountainous regions under humid temperate climates. We present
example applications in Japan, which are determination of sediment yield from watersheds by analyzing fluvial sand, and soil
formation and transport by analyzing saprolite（weathered bedrock beneath soil layer）in mountain catchments underlain by
granitic rocks. These applications offer potential usage of terrestrial cosmogenic nuclides for quantitative assessment of mountainhazards with comprehensive understanding of long-term sediment dynamics in mountainous terrains.

Oral Session
Tuesday, 18th SeptemberRoom B-1

09:00 － 11:00

Session 13: Accelerator Mass Spectrometry
Chair:

Hiroyuki Matsuzaki（The University of Tokyo, Japan）

S13-1040
10:40 － 11:00

Iodine isotope system in environment: Natural system, anthropogenic
system and influence from NPP accident

Hiroyuki Matsuzaki1, Maki Honda2, Yasuto Miyake1, Takeyasu Yamagata2, Hironori Tokuyama1,
Yasuyuki Muramatsu3
1

The University of Tokyo, Tokyo, Japan, 2Nihon University, Tokyo, Japan, 3Gakushuin University,Tokyo, Japan

Keywords:
Iodine-129, Iodine, Accelerator Mass Spectrometry, Cosmogenic radio nuclide, Fukushima Dai-ichi
Novel aspects:
Natural equilibrium iodine isotopic ratio in marine environment was found to be 2 x10-13, which is lower by one order previously
proposed.
Abstract:
Iodine isotope system, concerning with wide ranges of isotopic ratio（10 -14 to 10 -6 as 129 I/ 127 I）has great potential to investigate
various aspects of earth environmental systems. Before 1950’s there had exist only“natural iodine isotope system“ where 129I had
been provided by two sources, cosmogenic and fissiogenic. The half life of the 129I is 1.57 x107 yr which is, though negligibly shortcompared to the earth history, much longer than ordinary material circulation in the earth system such as marine circulation. The
iodine isotopic ratio in a certain compartment is determined by the balance between the turnover time and the production rate.
Generally this balance is different with site to site. But if the circulation is well quick between different sites, the isotopic ratio is
identical everywhere. If this hypothesis is true, or within the region this is true, the dating of the closed system should be possible
from the isotopic ratio. The earth surface environment, especially marine, seems to be the case. The iodine isotopic ratio of 129I/127I= 1.5 x 10-12 had been proposed and used as the initial value for dating（J. Moran, 1998）. It was shown that, using this initial ratio,
the age of the methane hydrate could be determined（U.Fehn, 2000）. Afterwards the ages of many geologic samples especially
iodine containing fluids and sedimentary rocks have been determined. However some samples showed discrepancy between theiodine isotopic age and geological determined age.
To examine whether the natural iodine is fully mixed in the ocean and whether the initial value previously proposed（ 129I/127I = 1.5
x 10-12）is valid enough, we measured depth profiles from surface to bottom of 129I/127Iratio for seawater samples collected fromseveral points in the Indian ocean. The results showed significantly low 129I/127I ratio（around 2 x10-13）in the middle to deep layer.
According to our results, the initial value should be one order lower than that of previously proposed. Serious difficulty, then,arises for the interpretation of samples having isotopic ratio higher than 2 x10-13 but lower than 1.5 x 10-12. It should be interpreted
as the anthropogenic contamination.
The other possibility is that the iodine is not mixed enough between different sites. To examine this issue, the elemental iodine
dynamics（circulation path, rate, turnover time）should be examined for several compartments in the earth surface environment
including atmosphere, soil, fluvial system, underground water, and marine environments. In this context, anthropogenic 129I can beused as the tracer of the iodine dynamics. After human began to utilize the nuclear power, 129 I as a byproduct of the fission
nuclear reaction entered to the earth surface environment overwhelming the naturally produce 129I. As a result, the isotopic ratioof the surface environment increased by several orders. The spatial distribution of 129 I/ 127 I tells us the iodine dynamics in theenvironment. The accident at Fukushima Dai-ichi nuclear power plant also made a spike of 129 I onto the land surface, which
provides an useful tracer for observation of the iodine diffusion process. As one of important sites for iodine reservoirs we havebeen focusing on the soil. The depth profile of 129I/127I ratio in the soil indicates that there are at least two different modes ofmoving way, quick diffusion and slow movement.

Oral Session
Tuesday, 18th SeptemberRoom D

09:00 － 11:00

Session 14: Ion-surface Collisions: Collision-induced Dissociation and Soft Landing
Chair:

Jean H Futrell（Pacific Northwest National Laboratory, USA）

S14-0900
09:00 － 09:40[Keynote Lecture] Ion-Surface Collisions in Mass Spectrometry: Activation,
Dissociation and Soft-Landing

Julia Laskin

Pacific Northwest National Laboratory, Richland, WA, USA

Keywords:
surface-induced dissociation, soft-landing, reactive landing, kinetics, self-assembled monolayer surfaces
Novel aspects:
Overview of the field
Abstract:
Collisions of hyperthermal（ 1 -200 eV）complex ions with surfaces are utilized in mass spectrometry studies for structuralcharacterization of large molecules and for highly selective modification of surfaces. Two major processes are dominant in thisrange of collision energies： reactive and non-reactive scattering of ions and ion loss on the surface as a result of neutralization or
soft-landing of projectile ions. Ion activation by collisions with a surface followed by surface-induced dissociation（SID）ofvibrationally excited ions offers unique advantages both for analytical applications and for fundamental studies of gas-phasefragmentation of complex ions. Measuring the time dependence of surface induced dissociation（SID）of complex ions withspecially prepared surfaces as a function of ion kinetic energy uniquely defines the energetics, dynamics and mechanisms offragmentation of complex molecules in the absence of solvent. This approach provides important information on the strength ofcovalent bonds in complex molecules and has been recently extended to understanding factors that determine dissociation of
non-covalent complexes. Ion capture on surfaces is the principal competing reaction channel to scattering and SID. Depending on
the experimental conditions, ions are captured as intact charged species, exchange charges with the substrate, evaporate or reactwith functional groups on the surface. Many studies demonstrated that preparatory mass spectrometry is a unique tool for highly
selective preparation of novel materials and for obtaining molecular level understanding of the effect of the surface on the
structure, charge state, and conformations of trapped ions. Several examples of controlled deposition of complex ions on selfassembled monolayer（SAM）surfaces demonstrating the utility of preparatory mass spectrometry for highly-selective preparationof biological and catalytic surfaces will be presented and factors affecting the competition between physical and chemical
processes accompanying ion deposition will be discussed.

Oral Session
Tuesday, 18th SeptemberRoom D

09:00 － 11:00

Session 14: Ion-surface Collisions: Collision-induced Dissociation and Soft Landing
Chair:

Jean H Futrell（Pacific Northwest National Laboratory, USA）

S14-0940
09:40 － 10:00

Surface-induced Dissociation of Non-covalent Macromolecular Complexes

Vicki H Wysocki1,2, Mowei Zhou1,2, Anne Blackwell2, Royston Quintyn1,2, Xin Ma1,2, Shai Dagan2
1

Ohio State University, Columbus, OH, USA, 2University of Arizona, Tucson, AZ, USA

Keywords:
surface-induced dissociation； collision-induced dissociation； protein complex； ion mobility
Novel aspects:
Surface-induced dissociation is an effective activation method for large non-covalent macromolecular assemblies, sampling well
the original structure of the complex and causing less unfolding than collision-induced dissociation.
Abstract:
Surface-induced dissociation is an activation method in tandem mass spectrometry that involves collisions of ions with a surface
target to induce their dissociation. This activiation method is especially useful for the dissociation of large macromolecularcomplexes because it provides more extensive fragmentation that collision-induced dissociation（CID）, presumably because of
the large mass of the target relative the size of the complex. Examples will be presented that illustrate the surface-induced
dissociation of refractory systems such as phosphorylase B that do not dissociate well by CID. SID also samples and respresents
the original structure of the complex better than CID, with results reflecting whether a complex has undergone source unfolding
or whether the use of different solution additives have changed the maximum charge state and structure of a complex. In CID,
unfolding is common whether the activation is carried out in source or in a typical collision cell. Results for both homooligomeric（GroEL, phosphorylase B, small heat shock proteins）and heterooligomeric（toycamin nitrile hydratase, BrnT/BrnA toxin-antitoxin,
tryptophan synthase）complexes will be presented. In all cases, surface induced dissociation better reflects the original structureof the complex. Both the precursor ion and the product ions remain more compact after SID than after CID, as measured bycoupling ion mobility with surface-induced dissociation.

Oral Session
Tuesday, 18th SeptemberRoom D

09:00 － 11:00

Session 14: Ion-surface Collisions: Collision-induced Dissociation and Soft Landing
Chair:

Jean H Futrell（Pacific Northwest National Laboratory, USA）

S14-1000
10:00 － 10:20

Mimicking redox protein function by soft landing Co(Salen) ions on selfassembled monolayer surfaces

Wen-Ping Peng1, Grant Johnson2, Julia Laskin2
1

Department of Physics, National Dong Hwa University, Taiwan, 2Pacific Northwest National Laboratory, Richland, WA

Keywords:
Co（Salen）, redox reaction, self assembly monolayer, secondary ion mass spectrometry
Novel aspects:
Charge neutralization andredox properties of Co（salen）complexes are demonstrated on self-assembledmonolayer（SAM）
surfaces prepared by soft landing of mass selectedions
Abstract:
INTRODUCTION
Transition metal ions are the active sites in many naturally occurring oxygen carrier and storage proteins and, therefore, play animportant role in regulating the redox balance and reactive oxygen needed for proper signaling. Co（salen）（salen = N,N’-dis（salicylidene）ethylenediamino ligand）is a common“model system“ used to understand how proteins（e.g. myoglobin）function
in redox signaling. Herein, soft landing of mass-selected ions is employed to achieve immobilization of catalytically active Co（salen）metal complexes on distinct self-assembled monolayer（SAM）surfaces. Using in-situ secondary ion mass spectrometry
we demonstrate that the relative abundance of protonated Co（salen）H+ compared to the native ion Co（salen）+ following softlanding onto surfaces increases in the order FSAM <COOH-SAM< HSAM. In addition, we investigated the redox behavior of softlanded Co（salen）complexes on the different SAM surfaces. The results indicate that oxidation of Co（salen）+ occurs on the
FSAM and COOH-SAM surfaces and but not on the HSAM surface, possibly due to neutralization of Co（salen）+ to Co（salen）.
METHODTo investigate the immobilization and reactivity of these model protein active sites, monomers and multimers of protonatedCobalt Salen［Co（salen）+H］1-3 + were soft landed onto the three distinct self-assembled monolayer（SAM）surfaces consisting offluorinated alkanethiol（FSAM）, alkanethiol（HSAM）and hydrophilic carboxyl-terminated alkanethiol（COOH-SAM）on gold. The
in-situ time-of-flight secondary ion mass spectrometry（TOF-SIMS）technique was employed to examine the surface composition
before and after oxygen uptake by［Co（salen）+H］+ on three SAMs surfaces. The surfaces were exposed to molecular oxygen ina vacuum environment for 5 minutes and subsequently reanalyzed using TOF-SIMS to monitor the redox reactivity of theimmobilized Co（salen）complexes and the influence of the self assembled monolayer on the observed behavior.
PRELIMINARY DATA
Ions were generated by electrospray ionization and guided to a mass filter where species with m/z values corresponding to thedesired size and composition（monomer, dimer, trimer）were selected and gently deposited onto three distinct SAM surfaces of
FSAM, HSAM and COOH-SAM coated on gold. In the first experiment, protonated monomers of Co（salen）were soft landed on
the surfaces. Then the treated surface was examined using in situ SIMS before and after exposure to molecular oxygen. The sameprocedure was employed for the dimer and trimer of［Co（Salen）+H］2-3 + species in a second and third experiment on same typeof surfaces, respectively. SIMS spectra reveal that the relative abundance of protonated Co（salen）H+ compared to Co（salen）+
following soft landing increases in the order FSAM <COOH-SAM< HSAM. Thisv suggest that the positive charge of the Co（salen）+
ion is retained on the FSAM surface. In contrast, on the HSAM surface, the Co（salen）+ is rapidly neutralized following soft landing
and is reionized through protonation during the SIMS analysis. The COOHSAM surface exhibits peaks consistent with both charge
retention and partial protonation of some neutralized ions. As further evidence of this charge neutralization, for Co（salen）,
oxidation is observed following O2 exposure on both the FSAM and COOHSAM surface with the reaction being most pronounced
on the FSAM. In contrast, no oxidation is observed on the HSAM surface. This indicates that neutralization of Co（salen）+ andreduction of the Co oxidation state reduces oxidation reactivity with O2. Comparing the isotope ratios for the dimer of Co（salen）+
on FSAM, COOHSAM, and HSAM shows that on FSAM and COOHSAM the dominant ion is［Co（salen）］2 H + at m/z = 651.
Comparing the isotope ratios for the trimer of Co（salen）+ on FSAM, COOHSAM, and HSAM shows that on FSAM and COOHSAM
the dominant ion is［Co（salen）］3H+ m/z= 976 and［Co（salen）］3H2 + m/z = 977,respectively.

Oral Session
Tuesday, 18th SeptemberRoom D

09:00 － 11:00

Session 14: Ion-surface Collisions: Collision-induced Dissociation and Soft Landing
Chair:

Jean H Futrell（Pacific Northwest National Laboratory, USA）

S14-1020
10:20 － 10:40

Epitaxy and Nanostructure Growth by Electrospray Ion Beam Deposition:
Nonvolatile Molecules, Peptides, Proteins

Stephan Rauschenbach1, Gordon Rinke1, Matthias Pauly1, Ludger Harnau2,3, Klaus Kern1,4

1
Max-Planck-Institute for Solid State Research, Stuttgart, Germany, 2Max-Planck-Institute for Intelligente Systeme,
Stuttgart, Germany, 3Institute for Theoretical und Applied Physics, Univ. Stuttgart, Germany, 4Institut de Physique de laMatiere Condensee, Ecole Polytechnique Federale de Lausanne, Switzerland

Keywords:
soft landing, scanning tunneling microscopy, electrospray ion beam deposition, protein, peptide
Novel aspects:
We show that soft landing/ion beam deposition can indeed be used equivalent to molecular beam epitaxy. It is further shown,that for large molecules the growth kinetics becomes very important.
Abstract:
Ordering phenomena can lead to materials with unique and often very useful properties. For instance, a long-range ordered pieceof silicon is a semiconductor, perfectly suited for electronic devices. Another example, an ordered-which means foldedpolypeptide chain is a functional protein. Clearly, the control of ordering is of pivotal importance in material science.
Traditionally, molecular beam epitaxy（MBE）is the vacuum deposition approach to fabricate highly ordered thin films and selforganized nanostructures. Atoms and molecules are evaporated and the vapor is condensed on a surface in ultrahigh vacuum（UHV, 10-10 mbar）to avoid contamination. Here we show that for nonvolatile substances like large organic molecules, peptides orproteins, electrospray ion beam deposition（ES-IBD）can serve as an equivalent vacuum deposition method to produce highlyordered coatings and nanostructures. In ES-IBD intact molecular gas phase ions, generated by electrospray ionization（ESI）, areconveyed to a surface in UHV, using ion optics within a differentially pumped vacuum system.
Despite the similarities, the differences between MBE and ES-IBDare significant. Specifically in MBE neutral particles are deposited
with thermal energies, in ES-IBD the particles are charged and have hyperthermal energies. The advantage of using charged
particles instead of neutrals is that this offers a maximum level of process control, specifically by mass spectrometry, mass- andcharge state selection, energy selection and quantitative coverage monitoring. The deposition energy of the hyperthermal particles
however, often exceeds the interaction energies that govern the ordering phenomena at the surface.
Using scanning tunneling microscopy in ultrahigh vacuum, we show for several small nonvolatile molecules that ES-IBD can beused to create crystalline coatings at the surface that can be extended into the third dimension.[1]For larger molecules, specifically
peptides, we show ordering of the internal degrees of freedom at the surface after ES-IBD. They are found to self-assemble into
one preferred configuration, while other configurations appear less abundant. Another step up in size from peptides, unfolded
proteins deposited to a surface are not found to reach a preferred, two-dimensional equilibrium structure at the surface.[2]［ 1 ］S. Rauschenbach, G. Rinke, N.Malinowski,T. Weitz, N. Thontasen, Z. Deng, T. Lutz, T. P. M. de Almeida Rollo,G. Costantini, L.
Harnau, K. Kern： Crystalline Inverted Membranes Grown on Surfaces by Electrospray Ion Beam Deposition inVacuum. Adv.Mater.
(2012）［ 2 ］Z. Deng, N. Thontasen, N. Malinowski, G.Rinke, L. Harnau, S. Rauschenbach, K. Kern： A Close Look at Proteins: Submolecular
Resolution of Two- and Three-Dimensionally Folded Cytochrome-c at Surfaces. Nano Lett.（2012）

Oral Session
Tuesday, 18th SeptemberRoom D

09:00 － 11:00

Session 14: Ion-surface Collisions: Collision-induced Dissociation and Soft Landing
Chair:

Jean H Futrell（Pacific Northwest National Laboratory, USA）

S14-1040
10:40 － 11:00

Soft and Reactive Landing of Ions for the Fabrication of Bioactive Metal
Surfaces

Michael Volny, Joelle M Rolfs, Tim Elam, Frantisek Turecek
University of Washington, Seattle, USA

Keywords:
soft landing, reactive landing, sensors, surface modification
Novel aspects:
application of reactive landing for unique surface modification and for development of sensors
Abstract:
Ion soft landing（SL）and reactive landing（RL）are techniques in which hyperthermal polyatomic ions are nondestructively
deposited onto a surface under controlled conditions of kinetic energy, surface composition, roughness, etc. RL refers to nondestructive immobilization of（bio）molecular ions on the surface following hyperthermal impact during SL. Conceived originally asa physical method for deposition of atomic ions and later applied to fundamental studies of gas-phase ions SL studies have been
extended to achieve various goals.
SL and RL were used to deposit proteins, large organic molecules, or to modify self-assembled monolayers. Previous research has
established SL as a method for multiplex separation of biomolecules and demonstrated that biomolecular ions soft-landed on dry
surfaces have been shown to retain their original molecular structure and/or bioactivity. The reactively landed species adhere to
the surface without losing their specific properties（enzymatic activity, molecular recognition, etc.）even after prolonged washingby solvents, buffers, and detergents. RL is a special type of a surface modification by molecular ions that, as experimentallydemonstrated, most likely represents a unique transition between physisorption and chemisorption phenomena on metal/
metaloxide surfaces. Although the amount of material that can be processed by SL and RL is practically limited to high picomoles
or low nanomoles, the continuing miniaturization of scientific instruments and overall downscaling of experiments in molecularsciences make soft landing perfectly compatible with current methods of life-science research.

Several research groups have developed methods to characterize the soft and reactively landed molecules that included specificenzyme and binding assays, fluorescence imaging, secondary ion mass spectrometry, IR spectroscopy and surface enhanced
Raman scattering（SERS）spectroscopy. SERS was shown to allow detection of soft-landed material at sub-attomole levels.
This presentation will be focused on application of RL for unique surface modifications that are difficult or impossible to beachieved by other means. Fabrication process based on reactive landing of ions has been developed to produce surfaces withspecific activity. For instance, hyaluronic acid（MW 420,000）, a polysaccharide that prevents protein adhesion to surfaces andactivation of blood platelets by foreign bodies, was reactively landed on plasma-treated 316 stainless steel and shown to prevent
blood platelet activation when exposed to whole blood during platelet adhesion studies. Stainless steel and other conductive
surfaces were also modified by reactive landing of titanium and zirconium propoxide for selective enrichment of phosphopeptides
from complex peptide mixtures. The latest application of RL is for surface coating of metal and semiconductor materials tofabricate biosensors based on conductivity measurements.

Oral Session
Tuesday, 18th SeptemberRoom E

09:00 － 11:00

Session 15: Mass Spectrometry for Nuclear Applications and Safety
Chair:

Nobuo Takaoka（Kyushu Univertsity, Japan）

S15-0900
09:00 － 09:40[Keynote Lecture] Extractive Electrospray Ionization Mass Spectrometryfor Uranium Chemistry Studies

Huanwen Chen, Mingbiao Luo, Saijin Xiao, Yongzhong Ouyang, Yafei Zhou, Xinglei Zhang
East China Institute of Technology, Nanchang, P.R. China

Keywords:
EESI, uranium, chemistry, rapid detection
Novel aspects:
Progress of EESI-MS on uranium chemistry including detection, synthesis and characterization will be presented.
Abstract:
Uranium chemistry is of sustainable interest. Breakthroughs in uranium studies make serious impacts in many fields including
chemistry, physics, energy and biology, because uranium plays fundamentally important roles in these fields. Substantial progressin uranium studies normally requires development of novel analytical tools.
Extractive electrospray ionization mass spectrometry（EESI-MS）is a sensitive technique for trace detection of various analytes incomplex matrices without sample pretreatment1-5. EESI-MS shows excellent performance for monitoring uranium species in various
samples at trace levels since it tolerates extremely complex matrices. Therefore, EESI-MS is an alternative choice for studying
uranium chemistry, especially when it combines ion trap mass spectrometry.
In this presentation, examples of EESI-MS for uranium chemistry studies will be given, illustrating the potential applications of EESIMS in synthesis chemistry, physical chemistry, and analytical chemistry of uranium. More specifically, case studies on EESI-MS forsynthesis and characterization of novel uranium species, and for rapid detection of uranium4 and its isotope ratios5 in various
samples will be presented. Novel methods based on EESI-MS for screening uranium ores and radioactive iodine-129 will bepresented.
Acknowledgement(s)This work is jointly supported by NNSFC（No.21175019）and MOST（No.2011QY14015008）of China.
References（ 1 ）Xue Li, Bin Hu, Jianhua Ding, HuanwenChen, Rapid characterization of complex viscous samples at molecular levels by
neutral desorption extractive electrospray ionization mass spectrometry, Nature Protocols,2011, 6, 10101025.
（ 2 ）HuanwenChen, Shuiping Yang, Ming Li, Bin Hu, Jiang Wang, Sensitive detection of nativeproteins using extractive electrosprayionization mass spectrometry, Angew.Chem. Int. Ed., 2010, 49, 3053-3056.
（ 3 ）Huanwen Chen, Shuiping Yang,Arno Wortmann, Renato Zenobi, Neutral desorption sampling of living objects for rapid
analysis by extractive electrospray ionization mass spectrometry, Angew. Chem. Int. Ed., 2007, 46, 7591-7594.
（ 4 ）Mingbiao Luo, Bin Hu, Xie Zhang, Daofeng Peng,Huanwen Chen, LIli Zhang and Yanfu Huan. Extractive electrospray ionization
mass spectrometry for sensitive detection of Uranyl Species in Natural water samples. Anal.Chem., 2010, 82,282-289（ 5 ）Chunxiao Liu, Bin Hu, Jianbo Shi, jianqiangLi, Xinglei Zhang and Huanwen Chen. Determination of uranium isotopic ratio（235U/238U）using extractive electrospray ionization tandem mass spectrometry. J. Anal. At. Spectrom., 2011, 26,2045-2051

Oral Session
Tuesday, 18th SeptemberRoom E

09:00 － 11:00

Session 15: Mass Spectrometry for Nuclear Applications and Safety
Chair:

Nobuo Takaoka（Kyushu Univertsity, Japan）

S15-0940
09:40 － 10:00

Importance of process blank in isotope ratio determination of uranium
and plutonium in ultra trace levels

Sunyoung Lee, Young Geun Ha, Jong-Ho Park, Kyuseok Song
Korea atomic energy research institute, Daejeon, Korea

Keywords:
MC-ICP-MS, Uranium, Plutonium, Nuclear safeguard, Isotopic analysis
Novel aspects:
We demonstrated that U and Pu can be separated from the isotopes blend and quantitative（few ng of U and pg of Pu）andisotopic analyses were performed by MC-ICP-MS.
Abstract:
Highly accurate and precise analysis for determining isotope ratios of uranium（U）and plutonium（Pu）plays a key role inmonitoring undeclared nuclear activities for safeguards and nuclear forensics. Multi-collector inductively coupled plasma mass
spectrometery（MC-ICP-MS）which allows simultaneous measurements for the isotopes of one or more elements has emerged asa powerful analytical tool to determine U and Pu isotopes in ultra-trace levels. Prior to the mass spectrometric analysis, anadditional chemical separation is required to separate individual elements. However, we observed variation of process blankderived from the chemical separation. Several factors such as lab environment, separation methods, and chemical equipment may
influence on the process blank that becomes a critical issue for the accurate isotopic ratio determination in ultra trace levels. The
separation process followed by an acid treatment provides pico-gram level of process background of U. Finally, we demonstrated
that U and Pu can be separated from the isotopes blend and quantitative（few ng of U and pg of Pu）and isotopic analyses weresuccessfully performed by MC-ICP-MS.

Oral Session
Tuesday, 18th SeptemberRoom E

09:00 － 11:00

Session 15: Mass Spectrometry for Nuclear Applications and Safety
Chair:

Nobuo Takaoka（Kyushu Univertsity, Japan）

S15-1000
10:00 － 10:20

Fissiogenic noble gases anomaly associated with Fukushima-daiichi
nuclear power plant disaster after the 3.11 Northeast Japan Earthquake

Keiko Sato1, Hidenori Kumagai1, Naoyoshi Iwata2, Hironobu Hyodo3, Katsuhiko Suzuki1

IFREE, JAMSTEC, Yokosuka, Japan, 2Faculty of Science,Yamagata University, Yamagata, Japan, 3RINS, Okayama
University of Science, Okayama, JAPAN1

Keywords:
noble gas, fissiogenic, 3.11 Northeast Japan Earthquake, nuclear power plant disaster
Novel aspects:
In our series of sampled atmosphere covering over Japanese islands, relative elemental abundances and isotope ratio in heavier
noble gases were changed； Xenon and Krypton were enriched to pre-disaster atmosphere.
Abstract:
Noble gases have unique characteristics that they are rarely combined with other chemicals as their very stable nature. Because
its main reservoir is atmosphere, their isotopic composition is well defined and believed to be uniform all over the world
insensitive to disturbance from anthropogenic and/or natural emission of geologically trapped noble gases in the earth interior.
Based on our preliminary friction experiment, however, detectable amount of noble gases seem to be emitted accompanied witha fault motion（Sato et al., 2009）. After the extreme Northeast Japan Earthquake occurred on March 11, 2011, extraordinary
increase of seismic activity as numerous aftershocks e.g. over 4000 felt earthquakes in four months, which may be a source of nonatmospheric component preserved in the earth interior.
In terms of anthropogenic component, NPP（Nuclear Power Plant）is a potential source, which is frequently monitored byradioactive species, e.g. Xe-133, Xe-135. A serious NPP accident has occurred due to 3.11 Northeast Japan Earthquake andaccompanying extreme tsunami.
We widely collected atmosphere samples all over Japan： two from Hokkaido Island（Sapporo and Akan）, eight from Tohoku
and Kanto District（Sendai, Yamagata, Koriyama, Miharu, Tsukuba, Akiruno, Chiba and Yokosuka）, three from Southeast Japan（Osaka, Okayama and Nangoku）, two from Kyushu Island（Beppu and Aso）. The atmospheres were sampled into vacuumed
containers, Isotube@, at each sampling site. Further sampling to evaluate time-series change still continues.
The elemental and isotopic compositions of the samples were analyzed mainly by quadrupole residual gas analyzers（RGA-200,
SRS Co.）and partly confirmed by sector-type mass spectrometers（GVI-5400, GV instruments）. In the duplicated analyses of theselected a few samples, the measured elemental and isotopic compositions were consistent within analytical uncertainties.
In our series of sampled atmosphere covering over Japanese islands, from Kyushu to Hokkaido, relative elemental abundances inheavier noble gases were changed； Xenon and Krypton were enriched to pre-disaster atmosphere. Further, radioactive andfissiogenic Xenon and Krypton isotopes are significantly higher than those in“pre“ 3.11 Earthquake atmospheres. It includes
unstable isotopes with short half-lives（e.g. Kr-85, Xe-133, Xe-135）and stable（daughter）isotopes（e.g. Kr-86, Xe-134, Xe-136）, whichsuggests by the nuclear plant disaster as their source. Thus, the atmospheric xenon isotopic composition in Eastern Japan area
until last typhoon season is similar to those reported as in the geological samples taken from the vicinity of Natural Nuclear
Reactor in the Oklo（e.g. Kuroda, 1992； Meshik et al., 2004）and Okelobonde（e.g. Meshik et al., 2000； Meshik et al., 2004）.
Additionally, Ar-41, Ar-42, Ar-39 are also detected in the sampled atmosphere as observed in neutron irradiated geological samples
in Ar-Ar dating.

Oral Session
Tuesday, 18th SeptemberRoom E

09:00 － 11:00

Session 15: Mass Spectrometry for Nuclear Applications and Safety
Chair:

Nobuo Takaoka（Kyushu Univertsity, Japan）

S15-1020
10:20 － 10:40

Investigations of Different Sample Matrices in Uranium Abundance
Analysis Using the MTE Methodology

Claudie K Black, Evelyn Zuleger, Joan Horta-Domenech, Martin Vargas Zuniga

Institute for Transuranium Elements, Joint Research Centre, European Commission, Karlsruhe, Germany

Keywords:
TIMS, Nuclear, Safeguards, Uranium, Plutonium
Novel aspects:
Insight into differing run parameters and preparations for different sample matrices.
Abstract:
The ITU in Karlsruhe routinely analyses a multitude of samples from a wide range of internal and external customers. High throughput analysis techniques are employed using meticulous care to ensure accurate, precise and timely results are provided. Whilst
the samples are generally the same there is a diversity of sample matrices, each of which requires individual chemical processing
for analysis. This can give rise to differences in analytical run performance. TIMS is used for isotopic analysis of Uranium andPlutonium to determine abundance and concentration information. More recently there has been an increase in requests for
Uranium minor isotope analysis for the U234 and U236 isotopes. At ITU we employ the MTE methodology［ 1 ］for minor isotope
analysis and have observed varying run quality between samples of differing matrix. This presentation will provide some insight
into the investigations carried out into the differences observed and how they can be improved for consistent quality routine
analysis.［ 1 ］S. Richter et al, J. Anal. At. Spectrom., 2011, 26, 550564

Oral Session
Tuesday, 18th SeptemberRoom E

09:00 － 11:00

Session 15: Mass Spectrometry for Nuclear Applications and Safety
Chair:

Nobuo Takaoka（Kyushu Univertsity, Japan）

S15-1040
10:40 － 11:00

Selective analysis of individual uranium particles with higher U-235
abundance by secondary ion mass spectrometry

Fumitaka Esaka, Chi-Gyu Lee, Masaaki Magara, Takaumi Kimura
Japan Atomic Energy Agency, Ibaraki, Japan

Keywords:
SIMS； Uranium； Particles； Fission-track
Novel aspects:
Analytical techniques to select and measure individual uranium particles with higher U-235 abundance in environmental samples
have been developed.
Abstract:
Uranium isotopic signature in dust samples taken at nuclear facilities gives important information on nuclear activities. The
presence of undeclared nuclear activities related to the production of nuclear weapons can be deduced by analyzing isotope
ratios of individual uranium particles. In particular, the detection of uranium particles with higher 235U abundance has a crucial roleto unveil undeclared nuclear activities. Secondary ion mass spectrometry（SIMS）is commonly used for measuring uranium isotope
ratios in individual particles［1,2］. Since uranium particles in the sample are randomly measured with SIMS, it is difficult to detecta small number of uranium particles with higher 235U abundance in a large number of natural and depleted uranium particles. In
this work, we have studied on the SIMS technique combined with pre-screening of uranium particles by a fission track（FT）
technique［ 3 ］or an automated particle measurement（APM）to analyze uranium particles with higher 235U abundance selectively.
Uranium standard materials（NBL CRM U950a and U100）and inspection swipe samples taken at nuclear facilities were used in thiswork. Particles in the sample were recovered onto a glassy-carbon planchet for APM-SIMS analysis, which allows to perform prescreening of isotopic composition for individual particles［ 4 ］. For the FT technique, particles in the sample were recovered onto
polycarbonate membrane filters. The filters were dissolved to make polycarbonate films containing particles. The films were thenirradiated with thermal neutrons in a nuclear research reactor. As the result, fission tracks were created and observed with an
optical microscope. The uranium particles having a large number of fission tracks were selected for subsequent analysis, because
the number of fission tracks depends on the amount of 235U in the particle. Finally, the uranium isotope ratios of the selectedparticles were measured with SIMS.
In the analysis of an inspection sample with APM-SIMS, we could detect uranium particles by obtaining 235U and 238U ion images.
The uranium particles with higher 235U abundance ratio were then selected and the isotope ratios were measured by SIMS withmicro-beam to obtain precise isotope ratios. In the FT-SIMS analysis for an inspection sample, we measured 20 particles having
the 235U abundance ratio between 2.8 and 4.6. The range was higher than that measured by SIMS without using the FT technique.
In conclusion, both methods examined in this work are effective to identify and analyze uranium particles with higher 235 U
abundance.
This work was supported by the Ministry of Education, Culture, Sports, Science and Technology of Japan.
References［ 1 ］G. Tamborini et al., Spectrochim. Acta B 53, 1289（1998）.
［ 2 ］F. Esaka et al., Talanta 71, 1011（2007）.
［ 3 ］F. Esaka et al., Anal. Chim. Acta 721, 122（2012）.
［ 4 ］M. Hedberg et al., J. Anal. At. Spectrom. 26, 406（2011）.

Oral Session
Tuesday, 18th September

Main Hall

15:00 － 17:00

Session 16: Glycoanalytical Technology for Systems Glycobiology and Functional
Glycomics
Chair:

Jane Thomas-Oates（University of York, UK）

S16-1500
15:00 － 15:40[Keynote Lecture] Glycoanalytical technologies for systems glycobiology,
biomakrer discovery & therapies

Radka Fahey, Eugune Dempsey, Karna Marino, Amanda McCann, Pauline M Rudd
NIBRT - Ireland

Keywords:
Systems Glycobiology, Glycan analyics, Biomarkers, Genomics/Glycomics
Novel aspects:
First demenstration of carmelations between gemone and glycome GWAS study of more than 75,000 people using Hight Theraputic
glycoanalysis
Abstract:
Glycoanalytical technologies for systems glycobiology, biomarker discovery and therapies
Pauline M Rudd. Glycobiology Group, National Institute for Bioprocessing Research and Training, Fosters Avenue, Mount Merrion,
Blackrock, Co.Dublin, IrelandOver half of all proteins are glycosylated, and alterations in glycosylation are common in physiological and pathological processes.
Glycan structures are, in the first instance, controlled by genes, however the complex pathways and epigenetic factors that
regulate their processing provide a further mechanism for fine tuning and diversifying the glycans and the functions of the proteinsto which they are attached. Thus in the determining changes of glycosylation in disease it is imperative to understand the systems
which are perturbed to generate the altered glycan processing. To understand the relationship between the glycome and otheromics data as well as to identify and validate potential glycosylated biomarkers a robotic platform capable of releasing andlabelling glycoproteins in a 96 well plate format has been developed as a front end to various glycan separations technologies
including HILC and RP HPLC, MS and capillary electrophoresis, chip technologies and on-line combinations of these techniques.
Data bases for all of these technologies are currently under construction（http：//glycobase.nibrt.ie/tools.html）. This platform
allows the glycan structures to be profiled, characterised or analysed in detail. By combining genome-wide association studies
and high-throughput glycomics analysis of 3000 individuals we identified SNPs in genes encoding Hepatocyte Nuclear Factor 1 α（HNF 1 α ）and fucosyltransferases FUT 6 and FUT 8 that modulate the fucosylation of human plasma glycoproteins, including
IgG. HNF 1 α and its downstream target HNF 4 α are both necessary and sufficient to regulate the expression of key
fucosyltransferase and fucose biosynthesis genes in hepatic cells. Interestingly, levels of outer arm fucosylated glycans proved to
be an effective marker for Mid-Onset Diabetes of the Young（MODY）. Now that it is possible to link different -omics data
together in a systems approach to Glycobiology, new insights are emerging that suggest new control points for glycosylation,
relevant to clinical marker discovery.
5 -AZA- 2 ’-deoxycytidine induced demethylation influences N-glycosylation of secreted glycoproteins in ovarian cancer. Saldova R,
Dempsey E, Pérez-Garay M, Mariño K, Watson JA, Blanco-Fernández A, Struwe WB, Harvey DJ, Madden SF, Peracaula R, McCann
A, Rudd PM. Epigenetics. 2011 Nov 1 ； 6 （11）.
Genomics meets glycomics-the first GWAS study of human N-Glycome identifies HNF 1 α as a master regulator of plasma protein
fucosylation. Lauc G, Essafi A, Huffman JE, Hayward Rudd PM, Rudan I. et al., PLoS Genet. 2010； 6 （12）Glycomic and glycoproteomic analysis of serum from patients with stomach cancer reveals potential markers arising from hostdefense response mechanisms. Bones J, Byrne JC, O’Donoghue N, McManus C, Scaife C, Boissin H, Nastase A, Rudd PM. J
Proteome Res. 2011 Mar 4 ；10（ 3 ）：1246-65.
Ultra performance liquid chromatographic profiling of serum N-glycans for fast and efficient identification of cancer associatedalterations in glycosylation. Bones J, Mittermayr S, O’Donoghue N, Guttman A, Rudd PM. Anal Chem. 2010 Dec 15；82（24）：1020815.
Function and glycosylation of plant-derived antiviral monoclonal antibody. Ko K, Tekoah Y, Rudd PM, Harvey DJ, Dwek RA, Spitsin
S, Hanlon CA, Rupprecht C, Dietzschold B, Golovkin M, Koprowski H. Proc Natl Acad Sci U S A. 2003 100（13）：8013-8.
Identification of N-glycosylation changes in the CSF and serum in patients with schizophrenia. Stanta JL, Saldova R, Struwe WB,
Byrne JC, Leweke FM, Rothermund M, Rahmoune H, Levin Y, Guest PC, Bahn S, Rudd PM. J Proteome Res. 2010 Sep 3 ； 9 （ 9 ）：4476-89. Erratum in： J Proteome Res. 2010 Oct 1 ； 9 （10）：5510.
A systematic approach to protein glycosylation analysis： a path through the maze. Mariño K, Bones J, Kattla JJ, Rudd PM. Nat
Chem Biol. 2010 Oct； 6 （10）：713-23.

Oral Session
Tuesday, 18th September

Main Hall

15:00 － 17:00

Session 16: Glycoanalytical Technology for Systems Glycobiology and Functional
Glycomics
Chair:

Jane Thomas-Oates（University of York, UK）

S16-1540
15:40 － 16:00

A Rapid-Throughput Platform for Quantitation of Site-Specific
Glycosylation with Differentiation of Structural Isomers

Serenus Hua1, Chloe Hu2, Charles C Nwosu2, John S Strum2, Carlito B Lebrilla2, Hyun Joo An1
1

Chungnam National University, Daejeon, South Korea, 2University of California, Davis, USA

Keywords:site-specific, glycoproteomic, quantitation, biomarkers, biotherapeutics
Novel aspects:
Automated workflow for rapid yet highly reproducible quantitation of site-specific glycosylation
Abstract:
The type, extent, and location of glycosyl modifications on protein therapeutics mediates their in vivo activity, their rate ofclearance from the body, and ultimately, their effectiveness. However, few methods are available to simultaneously monitor all ofthese characteristics. We have developed a highly reproducible method of rapidly characterizing and quantifying both N- and Olinked glycosylation within the context of their location within a protein or protein mixture. Our method provides comprehensive
analysis of glycan structure, glycosylation site, and relative quantities of all significant glycoforms in a system.
Glycoproteins are rapidly digested in a microwave reactor using controlled broad-specificity proteolysis in order to produce
informative glycopeptides. Due to the effects of microwave energy, our method is very quick, with digestion times measured inmere minutes rather than the hours or days required for conventional protease digests.
Following enrichment by graphitized carbon or hydrophilic interaction（HILIC）solid-phase extraction, glycopeptides are separated
and analyzed by chip-based porous graphitized carbon（PGC）nano-LC/MS and nano-LC/MS/MS. Automated data extraction,
processing, and interpretation facilitates site-specific characterization and quantitation of the glycoprotein and associatedglycoforms. The rapid-throughput workflow takes, on average, about 40 minutes per sample（36 samples per day）including
sample digestion, clean-up, and nano-LC separation.
In general, chip-based PGC nano-LC/MS identified hundreds of chromatographic peaks per glycopeptide injection. Of these, over
100 glycopeptide peaks per injection could be confirmed and identified by MS/MS（limited mainly by the per-spectrum acquisition
time of the mass spectrometer）. Combined with the structure-sensitive properties of PGC, chip-based nano-LC enabled baseline
separation of pairs, triplets, and quadruplets of glycopeptide structural isomers. Structural differences between isomers werecharacterized by accurate-mass MS/MS.
The method was found to be extremely sensitive. Glycopeptides were detected and successfully profiled from as little as 10
nanograms（or approximately 600 femtomoles）of starting glycoprotein. Dynamic range spanned five orders of magnitude, so
glycopeptides from low-occupancy glycosylation sites could be detected alongside those from higher-occupancy sites. Using thismethod, up to 12 glycosylation sites have been simultaneously monitored for glycosylation thus far.
The abundance and species of glycopeptides generated by this method were found to be highly reproducible, given the samedigestion conditions. Calibration curves were successfully fit to digestions of various concentrations, enabling accurate quantitative
analysis of site-specific glycosylation.
Several N- and O- glycosylated proteins have been digested and analyzed using this method, including prostate-specific antigen（acancer biomarker）, recombinant erythropoietin（EPO）, and several therapeutic monoclonal antibodies（mAbs）. Protein
mixtures have also been analyzed successfully. Combined with the automated data extraction and spectral interpretation software
package recently developed by our lab, this method can be used to rapidly and quantitatively profile large glycosylated sample
sets, with clear applications to quality control of therapeutic glycoproteins； drug testing and screening； glycoproteomic
biomarker discovery； and a host of other functions.

Oral Session
Tuesday, 18th September

Main Hall

15:00 － 17:00

Session 16: Glycoanalytical Technology for Systems Glycobiology and Functional
Glycomics
Chair:

Jane Thomas-Oates（University of York, UK）

S16-1600
16:00 － 16:20

Glycome Signature for Cancer Cell Identification

Hyun joo An1, Lauren Dimapasoc2, Mary Saunders2, Seunghyup Jeong1, Jaehan Kim1, Kit Lam2,
Carlito B Lebrilla2
1

ChungNam National University, 2University of California, Davis

Keywords:
cell surface, glycosylation, mass spectometry, glycan mapping
Novel aspects:
Comprehensive characterization of cancer cell membrane glycomes performed for the first time by mass spectrometry
Abstract:
Every cell in an organism synthesizes a heterogeneous array of glycans in the form of various structures. Cell surface glycosylation
may play an important role in development and may provide important new sources of markers for differentiation. However,studies regarding the glycosylation of cell surfaces are limited due to the lack of sensitive analytical methods. We have recently
developed a method to measure cell surface glycosylation and provide extensive characterization of the heterogeneity of thehuman embryonic stem cells（hESC）membrane proteins. Enrichment of membrane proteins followed by high performance mass
spectrometry and chromatography makes this approach both highly specific and sensitive. The resulting approach provides
comprehensive and highly quantitative structural information including isomer separation.
Using this new method, we have profiled surface glycans from more than 40 cancer cell lines. Each cancer cell displayed not only
common glycans of cancer cells but also unique glycans that have the characteristic features of specific cell types. Interestingly we
discovered that most of the cancer cells such as breast cancer（MCF 7 -her2, MCF 7 -C6, MCF 7 -WT, ZR751）, ovarian cancer（SKOV- 3 and ES- 2 ）, intestine cancer（HT-29 and Caco- 2 ）, leukemia（Jurkat, TK6, molt 4 ）, prostate cancer（PC 3 ）, andcervical cancer（CCL 2 ）have high levels of high mannose glycans on the cell surface. These findings suggest that high-mannose
glycans are the major component of cancer cell surface glycosylation. The amount of high mannose provide indications thatmisglycosylation in cancer occurs mainly in the endoplasmic reticulum. High mannose glycans are not commonly presented on thesurfaces of normal mammalian cells or in human serum, yet they may play important roles in cancer cell biology. Hierarchicalcluster and PCA analysis of human cancer cell lines were also performed to examine the correlation of cancer cells based on
glycan profile.
The results represent that glycomic signatures on cell membrane may be used for the identification of specific cancer cells. The
results also mean that distinguishing cancer cells from other mammalian cells may be facilitated by the major difference in theglycosylation of the cell membrane.

Oral Session
Tuesday, 18th September

Main Hall

15:00 － 17:00

Session 16: Glycoanalytical Technology for Systems Glycobiology and Functional
Glycomics
Chair:

Jane Thomas-Oates（University of York, UK）

S16-1620
16:20 － 16:40

Enhancement of carbohydrate signal by diamond nanoparticles in MALDIMS

Chieh-Lin Wu1, Chia-Chen Wang2, Yin-Hung Lai1, Yuan Tseh Lee1, Yi-Sheng Wang1,2
1

Academia Sinica, 2National Yang-Ming University and Academia Sinica

Keywords:
nanodiamond, carbohydrate, MALDI-MS, triple-layer
Novel aspects:
Incorporation of diamond nanoparticles in MALDI sample enhances the ion yield of carbohydrates. The method allows thedetection of carbohydrates when they are mixed with comparable amount of proteins.
Abstract:
Enhancements of carbohydrate signal by incorporating nanodiamond（ND）into the samples of matrix-assisted laser desorption/
ionization mass spectrometry（MALDI-MS）are demonstrated. In this work, we used ND as an additive of 2,5-dihydroxybenzoicacid（2,5-DHB）and developed a new sample preparation method. Our new approaches revealed that ND improves carbohydratesignal and enhanced signal-to-noise ratio（S/N）by decreasing noise.
The ND was selected as the additive in this study because it is chemically inert and it possesses many advantages over othernanoparticles. First, ND is transparent in the UV-A region, which is the most employed laser wavelength range in MALDI-MS.
Therefore, the presence of ND in matrix crystals does not attenuate the laser energy for matrix ionization. Furthermore, because
ND is an electrically insulator and is chemically inert, it does not react with matrices or analytes under the high temperature
condition of MALDI. Other metallic nanoparticles may induce side reactions under a harsh ionization environment. The superior
thermal conductivity of diamond may also contribute to the enhancement of ion yield of thermal labile molecules. A detail
discussion of the method will be presented.
The best S/N was obtained using a triple-layer preparation method, in which the sample was prepared and vacuum-driedsuccessively with the matrix in the bottom, ND in the middle and analyte in the upper layer. The S/N of carbohydrates wasincreased with increasing the particle size of ND. In comparison with non-ND samples, the use of ND increased the S/N of dextran（1.5 KDa）and β-cyclodextrin by an order of magnitude and more than fifty times, respectively. For a large polysaccharide,
pullulan（9.6 KDa）, higher signal intensity than non-ND samples was also observed. The method also facilitated the detection of
dextran that was mixed with equal amount of insulin B chain. Systematic comparisons between the data obtained with various
sample conditions are made to study the reaction mechanism, including the removal of ND, the size, amount and distribution of
ND. The morphology of the triple-layer sample was studied using fluorescence lifetime imaging microscopy（FLIM）. In conclusion,
the new sample preparation method improves the detection efficiency of carbohydrates and it can be further applied to theglycomic studies.

Oral Session
Tuesday, 18th September

Main Hall

15:00 － 17:00

Session 16: Glycoanalytical Technology for Systems Glycobiology and Functional
Glycomics
Chair:

Jane Thomas-Oates（University of York, UK）

S16-1640
16:40 － 17:00

Electron-based fragmentation approaches for glycans andglycoconjugates

Catherine E Costello, Joseph Zaia, Cheng Lin
Boston University School of Medicine

Keywords:
glycans, tandem mass spectrometry, cross-ring fragments, ExD, glycoconjugates
Novel aspects:
Increased fragmentation for glycans enables rapid, efficient and highly sensitive structural assignments
Abstract:
For the full structural determination of glycan structures, glycosidic fragmentation that defines sequence and cross-ringfragmentation that specifies linkage positions are both required. Under low-energy collision and IRMPD conditions, glycosidic
fragments dominate the spectra. We are exploring various electron-based fragmentation approaches（ExD）and have establishedconditions that meet both criteria, usually requiring only MS2 dissociations, with data acquisition rates that are compatible with
LCMS, even for high-performance FTMS experiments. These spectra are information-rich and can be utilized for automatedinterpretation. Development of the techniques and application to structural problems will be discussed.
This research is supported by NIH Grants P41 RR10888/GM104603, S10 RR020946 and S10 RR025082 and NIH-NHLBI contract N01
HV00239.

Oral Session
Tuesday, 18th SeptemberRoom A

15:00 － 17:00

Session 17: Non-Covalent Ion-Molecule Interactions
Chair:

Seung-Koo Shin（Pohang University of Schence and Technology, Korea）

S17-1500
15:00 － 15:40[Keynote Lecture] The Thermochemistry of Non-Covalent Ion-MoleculePeter B Armentrout

University of Utah, Salt Lake City, USA

Keywords:
Molecular recognition, Collision-induced dissociation, Bond-energies
Novel aspects:
Thermodynamic information about a wide variety of non-covalent complexes is obtained and reveals the details controlling thestrength of these interactions.
Abstract:
The use of mass spectrometry to study non-covalent interactions has a long history and pertains to phenomena ranging from ion
solvation to host-guest complexes to protein folding. In all of these cases, electrostatic interactions often dominate the formationof the complexes of interest but more subtle interactions are also at play, changing the probabilities that Coulomb fission
processes can occur, that host-guest complexes have selective interactions useful in environmental remediation, and that protein
folding is directed and not stochastic.
Energy-resolved collision-induced dissociation in a guided ion beam tandem mass spectrometer is a method developed over the
past years that permits the measurement of the energetics for a variety of non-covalent interactions. Experiments involvemeasuring the probability for dissociation of an appropriate complex induced by collisions with an inert gas as a function of the
kinetic energy of the ion, which can be varied over a wide range. Analysis of such absolute cross section data, taking into accountmultiple collisions, the lifetimes for dissociation, and the energy distributions of the reagents, allows the extraction of the intrinsic
affinity between the ion and various ligands, both simple and complex. Such absolute data can often be compared with quantum
chemical reactions, thereby clearly identifying the structures of the reactant complexes and the mechanisms for their association/
dissociation.
In this presentation, examples of such measurements as applied to metal dication hydration and metal cation complexes withamino acids and crown ethers will be examined. The trends in such data reveal some of the complexities that control theenergetics of these interactions.

Oral Session
Tuesday, 18th SeptemberRoom A

15:00 － 17:00

Session 17: Non-Covalent Ion-Molecule Interactions
Chair:

Seung-Koo Shin（Pohang University of Schence and Technology, Korea）

S17-1540
15:40 － 16:00

Atomic metal anions: one-step dehydrogenation and defluoronation of
linear and cyclic hydrocarbons, alcohols and their fluoronated analogues

Paul M Mayer1, Alex Mungham1, Jeffery Butson1, Jaleh Halvachizadeh1, Dhiya Hassan1, Sharon Curtis1
1

University of Ottawa, Ottawa, Canada

Keywords:
metal, anions, dehydrogenation, defluorination, ligand
Novel aspects:
This work is the first extensive examination of the reactions of atomic metal anions, and demonstrates new reactions withalcohols, alkanes and fluorinated systems.
Abstract:
The generation and reactivity of gas-phase metal cations has been a focus of mass spectrometry related studies for quite sometime. This has mostly been related to adduct formation and their involvement as reagents in chemical ionization processes. Our
research group has recently discovered a way to generate a wide variety of metal anions (Na−, K−, Rb−, Cs−, Fe−, Ni−, Co−, Cu−,Ag−) using a simple commercial electrospray mass spectrometer. The metal anions were all generated by the decomposition of
metal cation carboxylate complexes. Owing to the previous difficulties associated with generating these unique species, the ideaof reactive gas phase atomic metal anions has been relatively untouched. We will first present the results of theoretical
calculations on the precursor metal-oxalate anions with a view to understanding the formation of the metal anion upon CID of thecomplex. The performance of a wide selection of methods was assessed and a robust computational approach to these systems
was established. Clear from the calculations is that the situation for the transition metal anions is complicated by several low-lying
electronic states, making the computational estimate of their properties a multi-determinant problem. None-the-less, evidence isseen for the insertion of metal atoms into the C-C bond in the oxalate dianion, a reaction that leaves the metal already primarily
negatively charged. In some cases, the calculated structure is essentially a MCO2 anion interacting with a neutral CO2 molecule atsome distance. Experimentally, this study investigates the previously unexplored reactivity of atomic metal anions produced in thesource of a triple quadrupole mass spectrometer with several volatile organic molecules. One class consisted of the reaction of
Fe- with the primary alcohols methanol through heptanol, secondary alcohols 2-propanol and 2butanol and the tertiary alcoholt-butanol. Dehydrogenation of the alcohols (except for methanol) was demonstrated through the observation of FeH2− in the
resulting mass spectrum. Labelling studies showed that this dehydrogenation takes place in the reaction of Fe − with a single
alcohol molecule, and not in the two-step process previously observed for metal carbonyl anions such as Fe(CO)2−. In the case of
the smaller alcohols methanol, ethanol and propanol, FeOR − and FeOH− anions are observed. Fe − was also observed todehydrogenate alkanes in a similar manner. Theory has been applied to chart the mechanism of the dehydrogenation reaction
and the results will be presented in conjunction with rate constant measurements on an ion trap mass spectrometer. Metal anions
have also been allowed to react with fluorinated ring-systems such as per-fluorophenol and hexafluorobenzene. Here there isevidence for competitive deprotonation of the phenol OH group and F+ abstraction from the ring.

Oral Session
Tuesday, 18th SeptemberRoom A

15:00 － 17:00

Session 17: Non-Covalent Ion-Molecule Interactions
Chair:

Seung-Koo Shin（Pohang University of Schence and Technology, Korea）

S17-1600
16:00 － 16:20

Structural and Energetic Effects in the Molecular Recognition of Peptides
and Proteins by 18-Crown-6: Threshold Collision-Induced Dissociation andTheoretical Studies

Mary T Rodgers, Ranran Wu
Wayne State University

Keywords:
amino acids； binding affinities； collision-induced dissociation； crown ethers； molecular recognition
Novel aspects:
The first determination of the absolute 18-crown- 6 binding affinities of a variety of protonated peptidomimetic bases, amino acids,
N-acetylated amino acids, and side-chain acetylated amino acids.
Abstract:
The SNAPP（selective noncovalent adduct protein probing）technique relies on selective binding of 18-crown- 6 （18C 6 ）or othercrown ethers to peptides and proteins to facilitate identification and characterization of protein sequence, structure, andconformational changes. Accurate structural and thermochemical information regarding the binding between 18C 6 and related
model systems may provide insight into the factors that lead to selectivity in the complexation process. Unfortunately, very limited
structural and thermochemical data has been reported in the literature. Therefore, we use energy-resolved collision-induced
dissociation（CID）measurements coupled with threshold analyses of the measured CID cross sections and density functional
theory calculations to characterize the structures anad 18C 6 affinities of a variety of models systems including protonatedpeptidomimetic bases and natural and acetylated amino acids to elucidate structural and energetic effects that lead to selectivity
in the molecular recognition of peptides and proteins by 18C6. Nine peptidomimetic bases were investigated that include a seriesof primary amines as models for the n-terminal amino group and the side chain of lysine（Lys）residues including methylamine,
ethylamine, n-propylamine, i-propylamine, n-butylamine and 1,5-diaminopentane； imidazole and 4 -methylimidazole as models forthe side chain of histidine（His）； and 1 -methylguanidine（MGD）as a model for the side chain of arginine（Arg）. In addition, fiveamino acids including glycine（Gly）, alanine（Ala）, Lys, His, and Arg as well as the backbone acetylated forms of Lys, His andArg, and the side-chain acetylated form of Lys, were also investigated. Collision-induced dissociation of these complexes results inloss of intact 18C 6 in all systems allowing the absolute 18C 6 affinities of these species to be determined. In addition, loss of theintact base or amino acid is observed in competition with loss of 18C 6 for many systems, allowing the absolute base or aminoacid affinities of protonated 18C 6 to be determined. Competitive analyses of these latter systems also allow the relative proton
affinities（PAs）of 18C 6 and the associated base or amino acid to be extracted. This data is also employed to re-evaluate the PAof 18C 6 as the value reported in the NIST webbook is inconsistent with our experimental observations and theoretical
calculations, and clearly needs to be adjusted. Trends in the computed structures and measured and calculated 18C 6 binding
affinities are examined to provide useful insight into the processes that occur in the molecular recognition of amino acids by 18C 6
and the implications for binding to peptides and proteins.

Oral Session
Tuesday, 18th SeptemberRoom A

15:00 － 17:00

Session 17: Non-Covalent Ion-Molecule Interactions
Chair:

Seung-Koo Shin（Pohang University of Schence and Technology, Korea）

S17-1620
16:20 － 16:40

Cation and solvent effects on G-quadruplex nucleic acid structure: fromthe solution to the gas phase

Valerie Gabelica1, Adrien Marchand1, Rubén Ferreira2, Frédéric Rosu1, Hisae Tateishi-Karimata3,
Daisuke Miyoshi3,4, Naoki Sugimoto3,4, Edwin De Pauw1

University of Liege, Liège, Belgium, 2Institute for Research in Biomedicine (IRB), Barcelona, Spain, 3Frontier Institute for
Biomolecular Engineering Research (FIBER), Konan University, Japan, 4Faculty of Frontiers of Innovative Research in
Science and Technology (FIRST), Konan University, Japan
1

Keywords:
ion mobility spectrometry, supramolecular complexes, nucleic acids, folding, dissociation
Novel aspects:
Special DNA structures like G-quadruplexes are actually stabilized by a decrease in water activity. However, cation coordination isessential to preserve the structure from the solution to the gas phase.
Abstract:
Introduction:“Native“ mass spectrometry requires preparing the sample in solvents and buffers that preserve the desired fold, and tuning themass spectrometer so as to just desolvate the complexes and preserve them until they reach the mass analyzer. G-quadruplex
nucleic acid structures are peculiar because they require cation coordination between tetrads of guanines in order to be stable insolution. A current limitation of ESI-MS for nucleic acid studies is its incompatibility with native solution conditions, i.e.
physiologically relevant salt concentrations（> 100 mM KCl, 5 mM Mg2+）. Ammonium ions can properly stabilize G-quadruplexesin solution, but the resulting structure may differ from the potassium-induced structure. In an effort to render the G-quadruplex
structures amenable to investigation by ESI-MS more native-like, we explored G-quadruplex structural changes in ammonium
solutions when electrospray-compatible co-solvents are added, thereby decreasing the water activity. This will lead us todiscussing the effects of solvent（or absence thereof, i.e. gas phase conditions）and cations on G-quadruplex structure andstability.
Methods：
Oligodeoxynucleotides d（TTAGGG）2, d（TAGGGT）2, d（AGGGTT）2 and d（GGGTTA）2 were purchased from Eurogentec（Belgium）.
G-quadruplex dimers were formed by preparing the oligonucleotides in aqueous ammonium acetate, supplemented with variousorganic co-solvents（methanol,ethanol, isopropanol or acetonitrile）. The water activity in these solvent mixtures was determinedby the osmotic stressing method via freezing point depression osmometry using aTyp Dig. L osmometer（KNAUER, Berlin,
Germany）. The amount of G-quadruplex dimer formed was quantified using ESI-MS, taking into account the relative response
factors［Anal. Chem., 2009, 81, 6708］. The structure of the dimer was determined in solution using circular dichroism spectroscopy
on a Jobin Yvon CD 6 dichrograph, and in the gas phase using ion mobility spectrometry on both SynaptG 1 and Synapt G 2
HDMS（Waters, Manchester, UK）, which were mobility-calibrated using other known G-quadruplexes. By varying the injectionenergy, we also probed collision-induced loss of ammonia and concomitant structural changes.
Results：
The addition of organic co-solvents to aqueous ammonium acetate promotes and accelerates the formation of dimericG-quadruplexes from 12-mer telomeric sequences. For example, the telomeric sequence d（TAGGGT）2 does not form significant
amounts of dimer when annealed at 5 µM strand concentration in purely aqueous 100 mM NH4 OAc, but dimer formation isobserved when common electrospray co-solvents like methanol（MeOH）, ethanol（EtOH）, isopropanol（iPrOH）or acetonitrile（ACN）are added to this solution. Both the total amount of dimer and the rate of dimer formation increase with the percentage
of co-solvent. Interestingly, not only the fraction of co-solvent, but also the nature of the co-solvent drives the folding： conversion
into parallel folds is much less favored in methanol than in the other co-solvents. We also performed a detailed thermodynamic
and kinetic study of dimeric G-quadruplex formation as a function of the water activity in the different co-solvents. Fasterassociation rates are observed in low water activity conditions, and we propose that the rate limiting factor for G-quadruplex
formation is cation desolvation upon coordination to the G-quadruplex cavity. In solution as well as in the gas-phase, thedissociation rates depend on the structure in a similar way： parallel G-quadruplexes dissociate more slowly than antiparallel
G-quadruplexes. We also demonstrate that ammonium ion coordination is indispensable to maintain the native structure in thegas phase.

Oral Session
Tuesday, 18th SeptemberRoom A

15:00 － 17:00

Session 17: Non-Covalent Ion-Molecule Interactions
Chair:

Seung-Koo Shin（Pohang University of Schence and Technology, Korea）

S17-1640
16:40 － 17:00

Noncovalent Binding of Tetracationic Porphyrin to Thrombin-Binding
Aptamer DNA Destabilizes the Guanine-Quadruplex Structure

Jongcheol Seo, Eun Sun Hong, Hye-Joo Yoon, Seung Koo Shin
Pohang University of Science and Technology (POSTECH)

Keywords:
thrombin-binding aptamer, guanine-quadruplex, hydrogen/deuterium exchange
Novel aspects:
Hydrogen/deuterium exchange experiment and infrared multiphoton dissociation spectra show that tetracationic porphyrin binds
to the TGT loop of thrombin-binding aptamer DNA and destabilizes the guanine-quadruplex structure
Abstract:
A thrombin-binding aptamer（TBA）DNA d（GGTTGGTGTGGTTGG）forms the antiparallel chair-type guanine-quadruplex（G 4 ）structure in the presence of potassium ion, which involves two stacked G-tetrads with an intercalated potassium ion. This G 4
structure is an active form of the aptamer, and noncovalent interactions between negatively charged TBA and organic cation can
induce a significant structural change in the G 4 structure. Here we studied the accessibility of solvent to the bases of TBA in thepresence or absence of tetracationic 5,10,15,20-tetrakis（N-methyl- 4 -pyridyl）porphyrin and potassium ions by hydrogen/deuterium exchange（HDX）experiment. The extent of HDX was measured by electrospray ionization（ESI）mass spectrometry（MS）. The mass spectra of TBA and 1 ： 1 TBA-porphyrin complex were obtained at various H/D ratios of ESI solvent（ 3 ： 1
water/isopropanol, vol/vol）in the presence or absence of potassium ion. HDX results showed that porphyrin-bound TBA was
more flexible than potassium-bound TBA or free TBA, indicating that porphyrin destabilized the G 4 structure to allow easieraccess of solvent to the bases of TBA. To support this finding, we measured the binding constant of potassium ion to TBA and 1：
1 TBA-porphyrin complex by titration method. We also examined the circular dichroism（CD）spectra of TBA. Porphyrin binding
on TBA slightly reduced the potassium-binding constant from 8600 to 7900 M-  1 and attenuated the characteristic G 4 peaks in the
CD spectra. These results are in line with the HDX data. Furthermore, we determined the binding site of porphyrin on TBA byinfrared multiphoton dissociation（IRMPD）of the 1 ： 1 TBA-porphyrin complex using Fourier-transform ion cyclotron resonance（FT-ICR）MS. The product ion distribution showed that the porphyrin is on the central TGT region of TBA. Thus, the planar
porphyrin cation binds to the TGT loop region of the negatively charged TBA through electrostatic interactions, thereby weakening
noncovalent interactions between the G-tetrads and the potassium ion and destabilizing the G 4 structure of TBA.

Oral Session
Tuesday, 18th SeptemberRoom B-1

15:00 － 17:00

Session 18: Advances in Resolution and Accuracy of Isotope Ratio Analyses
Chair:

Takafumi Hirata（Kyoto University, Japan）

S18-1500
15:00 － 15:40[Keynote Lecture] Advances in Isotope Ratio Mass Spectrometry andRequired Isotope Reference Materials

Jochen Vogl

BAM Federal Institute for Materials Research and Testing, Berlin, Germany

Keywords:
reference material, traceability, measurement uncertainty
Novel aspects:
The approach of using two delta-reference materials, one at delta= 0 and one at delta=x, enables the validation of the analyticalprocedure and the estimation of a measurement uncertainty.
Abstract:
Outside the isotope community the isotopic composition of an element is often considered as invariant in nature, because thevariations usually are too small to detect with conventional techniques. With precise mass spectrometric techniques thesevariations can be detected and the determined isotopic composition can be used to obtain specific information of a sample likethe formation conditions, age or provenance.
In the past isotope ratio determinations to a large extent have been the domain of gas mass spectrometers and thermal ionization
mass spectrometers. The invention of an inductively coupled plasma mass spectrometer（ICPMS）in the mid 1980s and its
commercial introduction a few years later triggered the idea that the determination of isotope ratios will change in the future.
However, it took more than a decade until competitive ICPMS instruments have been available. Especially the introduction ofmulti-collector（MC）ICPMS instruments and their broader availability enabled a large number of new isotope systems to beinvestigated.
Like any other mass spectrometric technique ICPMS is affected by mass fractionation or mass discrimination effects, which lead toa bias in the isotope ratio determination. This bias depends strongly on the type of instrument, the optimization of it and theapplied measurement procedure. Therefore mass fractionation or discrimination effects need to be corrected for by applying
suitable reference materials（RM）. This correction together with a proper uncertainty statement enables isotope data being
traceable to the international system of units（SI）or to a commonly accepted standard. Traceability in turn enables comparabilityof the isotope data.
Usually this correction is carried out by applying RMs being certified for the isotopic composition of the element underinvestigation. Such isotope reference materials（IRMs）featuring“absolute“ or“true“ isotope data can only be certified via
synthetic isotope mixtures, which enable the calibration of the applied mass spectrometer. This certification approach, however,is costly and labor-intensive and the achievable uncertainties are mainly limited by the purity of the enriched isotopes used.
Nowadays relative standard uncertainties between 0.005 % and 0.01 % can be achieved for the isotope amount ratio of an IRM by
using synthetic isotope mixtures. The reproducibility of isotope ratios determined with a MC-ICPMS of the present generation,
however, can reach values of 0.001 %. It is obvious that IRMs being certified via synthetic isotope mixtures cannot fully meet theneeds of present and future isotope research and therefore an alternative approach is needed.
In many fields where the interpretation of isotopic variations is of interest, isotope ratios are not required on a“true“ basis, but
the difference of the isotope ratio between two or more samples is of interest, which can be expressed as a
δ-value.
Consequently, δ-reference materials are required, preferably one material defining δ=0, the so-called δ-zero material, and one
material with an offset, δ=x. It is desirable that the δ-zero material in parallel is certified for its“true“ isotopic composition. The
offset material is required to validate the whole analytical procedure, especially to prove the absence of isotope fractionation
during the matrix separation step. As an example the situation of boron isotope measurements and available reference materials
will be described. Combining both approaches, the δ-reference materials and IRM certified via synthetic isotope mixtures, theneeds in isotope analysis can be met to a large extent.

Oral Session
Tuesday, 18th SeptemberRoom B-1

15:00 － 17:00

Session 18: Advances in Resolution and Accuracy of Isotope Ratio Analyses
Chair:

Takafumi Hirata（Kyoto University, Japan）

S18-1540
15:40 － 16:00

An experimental study on stable isotopic fractionation of rare earth
elements during the adsorption on iron and manganeseoxides

Ryoichi Nakada1, Masaharu Tanimizu1,2, Yoshio Takahashi1,21
Hiroshima University, Higashi-Hiroshima, Japan, 2Japan Agency for Marine-Earth Science and Technology (JAMSTEC),
Nankoku, Koch

Keywords:
isotopic fractionation, MC-ICP-MS, XAFSNovel aspects:
The slight change in coordination environment affects the direction and degrees of mass-dependent isotope fractionations among
REE.
Abstract:
Recent development of analytical instruments, especially multiple collector（MC）ICP-MS, has enabled us to discuss the massdependent isotopic fractionation of heavy elements. Although several rules controlling the isotopic fractionation has have beensuggested, it is still insufficient to discuss which chemical properties are the most important factor to evaluate bond stiffness inthe equilibrium isotopic fractionation. This study, therefore, exhibits the results of stable isotopic fractionation of rare earth
element（REE）during the adsorption experiment to discuss the cause of the isotopic fractionation among REE.
Lanthanum, Ce, Nd, and Sm chloride solutions with various concentrations were respectively added to both synthesized
ferrihydrite and d-MnO 2 suspensions. In all the systems, pH was adjusted to 5.00（+0.05）and shaken for 6 hours before thefiltration. Stable isotope ratios in both liquid and solid phases were determined using MC-ICP-MS. REE-Cl 3 solutions used in theadsorption experiment were employed as standard solutions and the isotope ratio of each element was expressed in epsilon
notation relative to the average standards, which is shown in the equation as follows： e =（Rsample /R STD - 1 ）*10 4 , where R was
defined as 139La/138La, 142Ce/140Ce, 145Nd/143Nd, or 149Sm/147Sm, respectively. For the solid phase, K-edge EXAFS of filtered samples
was measured at BL01B 1 in SPring- 8 to obtain the information of the coordination environment.
Though accurate determination of La isotope ratio was difficult due to the large difference in the isotopic abundance
between 138La and 139La, a broad trend obtained in this study suggested that the lighter La isotope was selectively partitioned into
the solid phase. In Ce system, it is clearly shown that the lighter isotope was partitioned into solid phase, whereas in Nd and Sm
systems, lighter isotopes remained in the liquid phase, suggesting that physicochemical factors have been changed between Ce
and Nd. According to the EXAFS analysis, split first shell（La-O bond）was observed for La-adsorption system, suggesting that thefirst coordination sphere is distorted in the system. Such distortion was also expected for Ce-adsorption system from their EXAFS
results. On the other hand, split first shell was not observed for Nd and Sm systems. Thus, it is expected that the slight change incoordination environment, which can also cause the difference in their hydration numbers, affects the direction and degrees ofmass-dependent isotope fractionations among REE.

Oral Session
Tuesday, 18th SeptemberRoom B-1

15:00 － 17:00

Session 18: Advances in Resolution and Accuracy of Isotope Ratio Analyses
Chair:

Takafumi Hirata（Kyoto University, Japan）

S18-1600
16:00 － 16:20

The influence of the extreme climate conditions on Romanian fruit juices
isotopic composition

Dana Alina I Magdas1, Romulus Puscas1, Gabriela Cristea1, Nicoleta Vedeanu212National Institute for Research and Development of Isotopic and Molecular Technologies, Cluj-Napoca, ROMANIA,
Iuliu Hatiegan“University of Medicine and Pharmacy“, Biophysics Department, Cluj-Napoca, Romania

Keywords:
IRMS, food quality, fruit juices
Novel aspects:
The characterization of 70 Romanian fruit juices IRMS are discussed. We measured isotopic ratios to see how meteorologicalpeculiarities of year 2011 influenced the isotopic composition of investigated fruit juices.
Abstract:
Isotope ratio mass spectrometry（IRMS）is a very useful tool for origin assignation of food, thus 2 H/ 1 H, 18 O/ 16 O and 13 C/ 12 C；
measurements are intensively used in forensic study to prove product authenticity. This application has been particularly useful infood quality control, because it allows the detection of added sugar and water in fruit juices and in tracing the geographical originof food. One of the greatest limitations of this technique applicability in origin assignation is the lack of large databases of isotopic
abundance in food items. The interpretation of such analysis requires a sufficient number of data for authentic juices of the same
seasonal and regional origin, especially since the isotopic parameters of fruit juices show remarkable variability depending on
climatologically factors.
The characterization of 70 Romanian single strength fruit juices（apples, pears, plums and grapes）collected from different
Transylvanian areas, Romania, by mean of stable isotope approach are presented and discussed in this work. We
measured 2H/1H, 18O/16O ratios from water juice and 13C/12C from pulp and we compared the isotopic compositions of fruit juices
from 2010 harvest with those from 2011 one, in order to see how meteorological peculiarities of year 2011 influenced the isotopic
composition of the investigated fruit juices. We correlated the 2H/1H, 18O/16O isotopic ratios from water juice with those fromprecipitations in Transylvania aria to understand better the naturally isotopic differences that appear between these fruits
produced in the mentioned two years. These differences arise from the specific meteorological conditions of year 2010, which wascharacterized by higher humidity in comparison with 2011. The average annual rainfall of 2010 was 57.6 l/m 2 while thosecorresponding to 2011 were about 37.6 l/m2.
The obtained results have shown that beside the meteorological and climate conditions, the isotopic enrichment degree depends
on the fruit species, even for the species originated from the same geographical area. The most notable differences in isotopic
content appear in the case of grapes.
Our data presents significant differences in isotopic composition between fruits produced in the same areas but in two
consecutive years, which are characterized by very different meteorological conditions. Thus, these results may serve in thedetection of added water or C4 type sugars（cane or corn derived sugar syrups）to commercial fruit juices.

References
1. N. Ogrinc, K. Bat, I.J. Kosir, T. Golob, and R. Kokkinofta, Characterization of Commercial Slovenian and Cypriot Fruit Juices Using
Stable Isotopes, J. Agric. Food Chem. 57, 6764（2009）.
2. F. Camin, M. Perini, L. Bontempo, S. Fabroni, W. Faedi, S. Magnani, G. Baruzzi, M. Bonoli, M.R. Tabilio, S. Musmeci, A. Rossmann,
S.D. Kelly, P. Rapisarda, Potential Isotopic and Chemical Markers for Characterizing Organic Fruits, Food Chem. 125, 1072（2011）.
3. D. A. Magdas, R. Puscas, Stable isotopes determination in some Romanian fruit juices, Isotopes in Environmental and HealthStudy, 47, 372378（2011）.

Oral Session
Tuesday, 18th SeptemberRoom B-1

15:00 － 17:00

Session 18: Advances in Resolution and Accuracy of Isotope Ratio Analyses
Chair:

Takafumi Hirata（Kyoto University, Japan）

S18-1620
16:20 － 16:40

The Fe isotopic analysis using LAL-MC-ICPMS technique

Satoki Okabayashi1, Tetsuya Yokoyama2, Takafumi Hirata1
1

Kyoto University, Kyoto, Japan, 2Tokyo Institute of Technology

Keywords:
MC-ICPMS, laser ablation in liquid, LAL, Fe isotope
Novel aspects:
We have developed a new sampling technique, laser ablation in liquid（LAL）. This technique achieve the reliable isotopic analysis
using MC-ICPMS.
Abstract:
After the development of the multi-collector ICP-MS（MC-ICPMS）, the accuracy and the precision of the isotopic analysis havebeen dramatically improved. Especially the sample-standard bracketing technique makes it much easier to produce isotopic data.
To obtain the isotopic data from the solid sample, especially from the specific region or phase in the solid sample such as amineral in a geological rock, in most cases the solid sample is crashed in the agate mortar and hand-picked or micro-mill
technique is applied to dissolve the sample into the solution. It should be noted that these techniques have the risk of thecontamination from the equipments or apparatus used for the sample handling. The laser ablation（LA）is one of the mostsensitive sampling techniques for in-situ elemental and isotopic analysis of solid samples. The LA technique has been widely
applied for the sampling technique for the ICP-MS technique to determine the isotopic compositions. The LA-ICPMS techniqueachieves rapid and precise isotopic analyses under the spatial resolution of better than 30 μm. Nevertheless, it should be notedthat the resultant precision in the isotopic ratio measurements is significantly poorer than those achieved by the conventional
solution introduction technique. Moreover, preparation of matrix matched standard is highly desired to achieve the accurate
isotopic ratio measurements for complex minerals or phases. This indicates that the isotopic data obtained by the LA-ICPMS could
be erroneous when no proper isotopic standards were available.
To overcome these problems, our group developed a new sampling technique, laser ablation in liquid（LAL）（Okabayashi et al.,2011； Douglas et al., 2011）. This sampling technique is very simple and effective for the isotopic analysis using MC-ICPMS because（ 1 ）it has row risk of the contamination since any equipment does not touch the sample surface（ 2 ）the positional information
can be kept during the LAL sampling procedure（ 3 ）the solution analysis is applied for the LAL-sampled material.
In this study, we have evaluated the Fe isotopic fractionation during the spot sampling. The pure Fe metal（IRMM-014）wasablated under the ~ 3 mm thickness deionized water. A femtosecond laser（780 nm wave length）was used for the LAL sampling.
During the LAL procedure, the fluence was set to 20 Jcm-2 and the various repetition rates（10-250 Hz）were applied. As the result,
we could not found the Fe isotopic fractionation during the spot sampling of LAL procedure. Our recent study（Okabayashi et al.,2011）also revealed that Fe isotopic fractionation did not occur during the raster sampling of LAL procedure. These results
demonstrate clearly that LAL sampling technique can be applied for the isotopic analysis using MC-ICPMS.
In addition, Fe-Ni metal and FeS phase in the ordinary chondrite were sampled using present LAL technique to measure the Fe
isotopic ratios of them. In the case of the LA-ICPMS technique, the reliable Fe isotopic measurement of FeS phase was difficult
because of the non-spectroscopic interference from S to Fe isotopes. In this study, after the LAL sampling of the FeS phase, thesuspension of the sample was decomposed by conc. HCl to volatilize S as H2S, so the reliable Fe isotopic measurement could beachieved. The details of the Fe isotopic ratios obtained by LAL-MC-ICPMS technique will be discussed in this presentation.

Oral Session
Tuesday, 18th SeptemberRoom B-1

15:00 － 17:00

Session 18: Advances in Resolution and Accuracy of Isotope Ratio Analyses
Chair:

Takafumi Hirata（Kyoto University, Japan）

S18-1640
16:40 － 17:00

A novel technique for correction of instrumental mass bias in isotope ratio
measurements

Gina Chew, Thomas Walczyk

NUS Graduate School for Integrative Sciences and Engineering, National University of Singapore, Singapore

Keywords:
instrument mass bias correction
Novel aspects:
This work will open up new doors to measure fractionation effects at high accuracy that occur in nature for elements with threeof more isotopes.
Abstract:
Isotope ratio measurements by inorganic mass spectrometric techniques are always subject to mass bias effects which needs tobe corrected for in order to determine the true isotope ratios of the sample under study. This is necessary in particular for themeasurement of small natural isotope abundance variations of metals induced by mass dependent processes in nature as an
emerging tool in the Earth, Environmental and Life Sciences.
The double spike technique is the reference technique for the correction of such instrument mass bias effects in Thermal
Ionization Mass Spectrometry（TIMS）. It is applicable for elements with four or more isotopes. This is because there are three
unknowns, namely the sample fractionation factor α, instrument fractionation factor β and the proportion of the double spike tothe sample p in the blend. To solve the system, there have to be at least three isotope ratios that have the same reference
isotope in the denominator. To date, there is no method available to correct for instrument mass bias effects by double spiking forelements such as magnesium which has only three stable isotopes（24Mg, 25Mg,26Mg）.
Our novel technique is based on mixing the spike with the sample in different weight ratios. The mole ratio of spike to sample will
be proportional to their weight ratio and a linear plot can be established. The mole ratio for each blend is calculated following
principles of isotope dilution mass spectrometry whereas the weight ratio is measured using a high-precision analytical balance.
Since two isotope ratios can be measured for Mg（26Mg/24Mg and 25Mg/24Mg）, the mole ratio calculated using each isotope ratio
will be slightly different as the measured ratios are subject to instrument fractionation effects. The unknown variables are theinstrument fractionation factor of the individual blends and the sample fractionation factor. The system is solved by finding
solutions to the variables that will give the same mole ratio of spike to sample calculated from both isotope ratios for the different
mixes. Optimization of the spike composition and the weight ratios for linear regression analysis was done by Monte Carlo
Simulation, a numerical technique used to simulate measurement results on the basis of probability density functions（PDFs）of
the input quantities. This work will open up new doors to measure fractionation effects at high accuracy that occur in nature forelements with three of more isotopes.

Oral Session
Tuesday, 18th SeptemberRoom D

15:00 － 17:00

Session 19: Mass Spectrometric Diagnosis
Chair:

Toyofumi Nakanishi（Osaka Medical College, Japan）

S19-1500
15:00 － 15:40[Keynote Lecture] Ambient Mass Spectrometry for Detecting Biomarkers
in Breath

Renato Zenobi
ETH Zurich

Keywords:
Ambient MS； SESI； EESI； Breath
Novel aspects:
MS-based Diagnosis by Sensitive Detection of Biomarkers in Exhaled Breath
Abstract:
Ambient mass spectrometry stands for a series of recently introduced methods for analyzing complex samples with very little orno sample preparation. Ambient MS includes methods such as Desorption Electrospray Ionization（DESI）and Direct Analysis in
Real Time（DART）. We have been particularly active in the use of Secondary Electrospray Ionization（SESI）and Extractive
Electrospray Ionization（EESI）for the analysis of complex liquids, surfaces（following a neutral desorption step）as well as gases
and aerosols.
We have recently concentrated on breath analysis by EESI and SESI, and found the detection of exhaled drugs and metabolites
with molecular weights >200 g/mol to be challenging, due to the low breath concentration. Using an ion funnel operating at
ambient pressure that contains a SESI / EESI source, we are able to boost the sensitivity by a factor or 100 ... 1000, which should,
for example, allow the detection of opioids in exhaled breath of anesthesized patients. Very recently, we have also employed SESI
and EESI for monitoring drug dosage（in the case of valproic acid, VPA, an antiepileptic drug）and for medical diagnosis（fordistinguishing patients with chronic obstructive pulmonary disease, COPD, from smokers and from healthy controls）. The latest
results from these studies will be discussed.

Oral Session
Tuesday, 18th SeptemberRoom D

15:00 － 17:00

Session 19: Mass Spectrometric Diagnosis
Chair:

Toyofumi Nakanishi（Osaka Medical College, Japan）

S19-1540
15:40 － 16:00

A Validation Assay for Cardiac Ischemia Biomarkers

Robert J Cotter1, Christine Jelinek1, Jennifer Van Eyk1, Kevin W Meyer2, David Taggart3

Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA, 2Perfinity Biosciences, West Lafayette, IN,
USA, 3John Radcliffe Hospital, Oxford, England
1

Keywords:
selected reaction monitoring, cardiac ischemia, biomarker, post-translational modification
Novel aspects:
Use of the post-translational modifications of albumin as biomarkers, thereby developing a test from blood that does not require
albumin removal.
Abstract:
Using a triple quadrapole mass spectrometer combined with an automated sample preparation system, we have developed aselected reaction monitoring（SRM）assay to quantify Albumin PTMs（phosphorylations and cysteinylations）implicated in cardiac
ischemia. The multiplex SRM assay was designed for twenty-eight HSA peptides using Skyline. Four of the most abundanttransitions for each HSA peptide were selected, including one specific to the observed PTM（or lack thereof）. Relative
quantification was facilitated by an internal HSA peptide and an external E.coli β-galactosidase peptide. Analyses were initially
carried out on control sera to determine reproducibility and gender differences. It was then expanded to include a plasma based
longitudinal mild-ischemic patient cohort. A Perfinity workstation was used to automate all sample preparation for the assay.
Tryptic digestion of 1 ：50 diluted sera or plasma occured in six minutes, desalt is performed on-column, followed by a 20 minute
LC-SRM analysis.
Initial results using manual sample preparation suggested that the MRM-MS approach was sufficiently sensitive to determine if anyof the peptides could signal a disease pathology, though there were discrete differences between samples as a result of
inconsistencies within the sample preparation pipeline. To minimize run-to-run variability, all bench-top preparation steps were
eliminated and replaced with column-mediated tryptic digestion, desalting, and reverse phase HPLC separation performed in-line
on a Perfinity Workstation. Incorporating the Perfinity Workstation into the assay pipeline dramatically decreased the %CVs（tounder 10% for all peptides screened）. The total throughput of the assay was also increased. Sample digestion on a bench-top
heat block had required 18 hours of incubation with the enzyme reaction mixture. Using an immobilized enzyme reaction chamber,complete digestion of each plasma sample now occurs in six minutes and the entire SRM-assay could be completed in less than
35 minutes, compatible with the time frame required for diagnosis. If an MI induced by cardiac ischemia remains untreated for fourhours, the patient’s risk of mortality increases to 90%. To validate the 28 selected HSA peptides for use as clinical cardiac ischemia
markers, a fifteen patient cohort was screened. The sample cohort consists of six peripheral plasma samples per patient taken
during and after aortic bypass surgery. Initial results suggest several HSA PTMs are sensitive to cardiac ischemia and may haveclinical utility as ischemic biomarkers.

Oral Session
Tuesday, 18th SeptemberRoom D

15:00 － 17:00

Session 19: Mass Spectrometric Diagnosis
Chair:

Toyofumi Nakanishi（Osaka Medical College, Japan）

S19-1600
16:00 － 16:20

Diagnosis, analysis of pathogenesis and therapies for transthyretin form ofamyloidosis

Yukio Ando

Department of Diagnostic Medicine, Graduate School of Medical Sciences, Kumamoto, Japan

Keywords:
amyloidosis, TTR, FAP, mass, hereditary
Novel aspects:
We have established screening system for transthyretin related amyloidosis.
Abstract:
Progress in molecular genetics and biochemical methodologies has led to the identification of various types of amyloidosis andtheir amyloidogenic precursor proteins. So far, 27 different amyloidogenic proteins including lactoferrin which we first reported
have been identified, although the mechanism of amyloid formation in amyloidosis remains to be elucidated except for a few
types of amyloidosis. One of the most important steps in the diagnosis of the specific type of amyloidosis is detection of amyloid
deposits in tissues. It is not easy to predict the presence of these amyloid deposits on the basis of patients’ clinical manifestations,that is, without histopathological materials, so amyloidosis is sometimes diagnosed after the death of the patients. A real-timemonitoring of amyloid fibril deposition in the amyloid targeted tissues is very important.
In Japan, we have big foci of familial amyloidotic polyneuropathy（FAP）patients. As of today, reports of 123 different points ofmutations or a deletion in the TTR gene have been identified. Because transthyretin（TTR）, amyloid precursor protein for FAP, issynthesized by the liver, liver transplantation has been performed for the patients to save their lives. In addition, senile systemic
amyloidosis induced by wild type TTR has been focused in the recent attention. Early diagnosis of FAP is absolutely imperative forthe treatments, because duration of the disease is one of the most important prognostic factors for patients’ survival after
treatments including liver transplantation. To make a diagnosis of FAP sooner, we established a novel rapid diagnostic system. It
includes histopathological, genetic and proteomic techniques used to detect amyloid deposits in tissues, genetic mutations of the
TTR gene, and variant TTR in serum. Mass spectrometric analyses can clearly detect TTR variants in the most of FAP cases andcan be used for screening and double checking TTR variants with the genetic testing. Using protein chip system, presence orabsence of variant TTR in serum can be screened within 3 hours. We introduce recent research progress in ATTR amyloidosis andour diagnostic system.

Oral Session
Tuesday, 18th SeptemberRoom D

15:00 － 17:00

Session 19: Mass Spectrometric Diagnosis
Chair:

Toyofumi Nakanishi（Osaka Medical College, Japan）

S19-1620
16:20 － 16:40

Cerebrospinal fluid proteomics: A new window for understanding humanneurological disorders

Masaya Ikegawa

Kyoto Prefectural University of Medicine

Keywords:
MALDI-TOF MS, Cerebrospinal fluid, Proteomic pattern analysis, multiple sclerosis, Parkinson’s diseases
Novel aspects:
CSF proteomic pattern analysis is an effective new technology for identifying biomarker signatures of human neurologicaldisorders such as MS and PD and their related disorders.
Abstract:
The rapid evolution of research technologies has provided novel, high-throughput tools for the investigation of intractableneurological disorders. The ability to perform in-depth analyses of genomes, transcriptomes, proteomes, and metabolomes has
fueled efforts to uncover these neurological disease related targets and biomarkers. Initial evaluations of serum and cerebrospinal
fluid（CSF）from case and control subjects have led to the identification of a diverse group of candidate disease associatedproteins. However, being study design, sample treatment, and data complexity contributed to the significant inter study variability,
the urgent demand for diagnostic, therapeutic, and prognostic biomarkers in the future care of individuals with neurologicaldisorders has spurred novel efforts to focus these technologies on defined clinical questions. Recently, two papers1,2 appeared
from our group employing a novel proteomic strategy uncover potential diagnostic biomarkers among neurodegenerative
disorders as well as demyelinating disorders. First we have succeeded in discriminating multiple sclerosis（MS）and neuromyelitis
optica（NMO）by a CSF proteomic pattern analysis by using magnetic bead-based enrichment of peptides and proteins followedby matrix-assisted laser desorption / ionisation time-of-flight（MALDI）mass spectrometry1. In this study, the proteomic patterns ofacute NMO patients, both aquaporin- 4 （AQP 4 ）seropositive and seronegative, were easily distinguished from relapsingremitting
MS（RRMS）and primary progressive MS（PPMS）. These distinctions were less clear, however, when CSF was obtained during
disease quiescence. This finding suggests that the CSF proteome may be a sensitive barometer of CNS disease activity andtherapeutic response. A smaller but measurable variability was also observed between CSF proteomes in RRMS and PPMS
patients. The additional observation that the PPMS proteome may be more closely aligned with amyotrophic lateral sclerosis（ALS）than RRMS was quite intriguing and may be consistent with enhanced neurodegeneration in PPMS patients. In anotherstudy, we have succeeded in discrimination between Parkinson’s diseases（PD）and multiple systemic atrophy（MSA）with thesame strategy2. Our findings suggest that CSF proteomic pattern analysis can increase the accuracy of disease diagnosis of MSand PD-related disorders and will aid physicians in appropriate therapeutic decision-making even for expanding neurologicaldisease entities.
1. Komori et al., Annals of Neurology 2012； 71： 614623.
2. Ishigami et al., Movement Disorders 2012； 9 Mar accepted.

Oral Session
Tuesday, 18th SeptemberRoom D

15:00 － 17:00

Session 19: Mass Spectrometric Diagnosis
Chair:

Toyofumi Nakanishi（Osaka Medical College, Japan）

S19-1640
16:40 － 17:00

Digging deeper into Small Cell Lung Cancer. Can targeted LC-MS reveal
tomorrows diagnostic level?

Silje B Torsetnes1, Marianne S Nordlund2, Elisabeth Paus2, Trine G Halvorsen1, Leon Reubsaet1
1

University of Oslo, Oslo, Norway, 2Central Laboratory - Radiumhospitalet, Oslo, Norway

Keywords:
SRM, Triple quadrupole, SCLC, ProGRP
Novel aspects:
The combined immunocapture with the LC-MS/MS SRM detection allows determination of isoforms of the tumor marker ProGRP
revealing a potential new diagnostic level in Small Cell Lung Cancer
Abstract:
The overall survival rate for Small Cell Lung Cancer（SCLC）patients is very low, less than 5 % at 5 years, despite the sensitivity ofthe tumors to chemotherapy and radiotherapy. The difficulty, however, is early diagnosis and monitoring of the treatment. This
hampers the improvement of the disease prognosis.
Out of several possible biomarkers for SCLC, ProGRP is considered very promising, with reported qualities of high disease
specificity and sensitivity. On a routinely basis, so far only the total amount of ProGRP has been measured in correlation withdisease, using conventional immunometric analysis. Challenging is the very low serum concentration reported for this tumor
marker（ref. level < 60 pg/mL levels）.
ProGRP comprise three known isoforms, known to be expressed on the mRNA level, but their individual prevalence has until now
not been shown in serum. From a diagnostic point of view, it might be of great interest to confirm the protein expression of these
isoforms as well as their presence in serum to see if ratios between them vary, e.g. between disease states or -types.
A method for total ProGRP, developed by Winther, B., et al.（ 1 ）, was used as a starting point for an improved immuno-MS
method. A monoclonal antibody, E146（ 2 ）, immobilized on magnetic beads, was used for the extraction of ProGRP-isoforms
spiked to human serum. The captured ProGRP-isoforms were subjected to tryptic digestion, generating signature peptides thatwere further separated by liquid chromatography and detected by selected reaction monitoring（SRM）on a triple quadrupole（QqQ）mass spectrometer（MS）. Orthogonal selectivity, conferred by the combination of immunoaffinity extraction and LC-MS/
MS analysis, enables development of isoform-differentiating methods.
Highly specific and accurate measurements of tumor marker concentrations, aided by LC-MS/MS, may to a larger extent thancurrent methods give valuable clinically relevant information in diseases such as SCLC. In this presentation it will be shown thatthrough combined benefits of three complementary separation methods, a validated method for quantifying clinically relevant
concentrations of ProGRP-isoforms has been developed. Evidence of applicability on SCLC patient samples will be presented andthe potential relevance of isoform differentiation will be discussed.
1. Winther, B., Nordlund, M., Paus, E., Reubsaet, L., and Halvorsen, T. G.（2009）Immuno-capture as ultimate sample cleanup in LCMS/MS determination of the early stage biomarker ProGRP, J Sep Sci 32, 2937-2943.
2. Nordlund, M. S., Fermer, C., Nilsson, O., Warren, D. J., and Paus, E.（2007）Production and Characterization of Monoclonal
Antibodies for Immunoassay of the Lung Cancer Marker proGRP, Tumor Biol 28, 100-110.

Oral Session
Tuesday, 18th SeptemberRoom E

15:00 － 17:00

Session 20: The Ion formation and Dissociation Mechanisms in MALDI
Chair:

Myung Soo Kim（Seoul National University, Korea）

S20-1500
15:00 － 15:40[Keynote Lecture] MALDI and Related Desorption / Ablation / Ionization
Methods:A Solved Problem or Still a Mystery?

Richard Knochenmuss

Tofwerk, Thun, Switzerland

Keywords:
MALDI Ionization Ablation
Novel aspects:
Keynote on MALDI ionization mechanisms
Abstract:
MALDI is one of the key technologies of modern mass spectrometry. The ablation and especially ionization mechanisms underlying
it have therefore been the object of much speculation and many studies. With the recent development of variants and alternatives
to MALDI and electrospray, more mechanistic questions have arisen, as well as questions of similarities between the methods.
A brief, critical overview will be presented of the development of MALDI mechanisms. A recurring theme is the hope that a simple
basic mechanism largely explains MALDI, followed by disappointment as reality is found to be more complicated. This will befollowed by a summary of the current status of MALDI models, both qualitative and quantitative. The author’s models will be
discussed in deeper detail. Some related recent methods will be compared, and parallels discussed. A common aspect is a rapid,
nonequilibrium phase change, during which multiple interconnected ion-molecule reactions take place. Some opinions on what issolved and what remains mysterious may be offered.

Oral Session
Tuesday, 18th SeptemberRoom E

15:00 － 17:00

Session 20: The Ion formation and Dissociation Mechanisms in MALDI
Chair:

Myung Soo Kim（Seoul National University, Korea）

S20-1540
15:40 － 16:00

Ion yield and laser shot number-dependent variation of mass spectral
pattern in MALDI of peptides; a thermal model for MALDI

Yong Jin Bae, Kyung Man Park, Sung Hee Ahn, Myung Soo Kim
Seoul National University, Seoul, Korea

Keywords:
MALDI, Ion yield, Thermal model
Novel aspects:
MALDI-TOF spectra for peptides became highly reproducible when a homogenous sample prepared by vacuum-drying was used
and when the spectra displaying the same ’early’ plume temperature were compared.
Abstract:
The mechanism for the formation of gas-phase peptide ions in matrix-assisted laser desorption ionization（MALDI）and the factors
determining mass spectral patterns in MALDI-TOF of peptides were investigated. α-Cyano- 4 -hydroxycinnanmic acid（CHCA）wasused as the matrix. The main tools for the study were the measurements of the ion yields（IY, number of ions formed ÷ number ofmolecules in a sample）and the laser shot number-dependence of the spectral pattern. A rather homogeneous sample wasprepared by vacuum-drying of water/acetonitrile solution of a peptide/CHCA mixture, rather than the usual air-drying.
IYs were low 10-  4 for peptides and 10-  7 for CHCA. Both of these were smaller than those speculated by Hillenkamp and Karas by
two orders of magnitude. Total number of ions in a spectrum, i.e. the sum of peptide- and matrix-derived ions, was the same
regardless of peptides and their concentrations. Namely, the number of ions generated from a pure matrix was the upper limit to
the number of peptide-derived ions. The total number of ions was unaffected by the laser pulse energy, invalidating laser-induced
ionization of matrix molecules as the mechanism for the primary ion formation. Instead, the excitation of matrix by laser seems to
be a way of supplying thermal energy to the sample. Accepting strong Coulomb attraction felt by cations in a solid sample, we
propose three hypotheses for gas-phase peptide ion formation. In Hypothesis 1, they originate from the dielectrically screened
peptide ions in the sample. In Hypothesis 2, the pre-formed peptide ions are released as part of neutral ion pairs, which generate
gas-phase peptide ions via reaction with matrix-derived cations. In Hypothesis 3, neutral peptides released by ablation get
protonated via reaction with matrix-derived cations.
By repeatedly irradiating a spot on a sample with laser, a set of mass spectra was collected. For each spectrum, the temperaturein the early plume（T early ）was estimated by kinetic analysis of the peptide ion dissociation yield. Tearly decreased as the shot
continued because thermal conduction at the irradiated spot became more efficient as it got thinner. At a given Tearly, the peptideto-matrix ion abundance ratio was proportional to the peptide-to-matrix ratio in the solid sample. It increased as the shot
continued. These indicate that the exothermic matrix-to-peptide proton transfer was in quasi-thermal equilibrium in the earlymatrix plume. For samples with the same peptide-to-matrix ratio, the overall spectral pattern was the same regardless of theexperimental condition when the spectra with the same T early were compared, suggesting thermal determination of MALDI spectral
pattern.

Oral Session
Tuesday, 18th SeptemberRoom E

15:00 － 17:00

Session 20: The Ion formation and Dissociation Mechanisms in MALDI
Chair:

Myung Soo Kim（Seoul National University, Korea）

S20-1600
16:00 － 16:20

Plume Expansion Dynamics of Ionic Liquid Matrices for Matrix-assisted
Laser Desorption/Ionization

I-Chung Lu, Sheng Lee, Yuan T Lee, Chi-Kung Ni
Academia Sinica, Taipei, Taiwan

Keywords:
Ionic liquid, MALDI, matrix,
Novel aspects:
High-resolution angular and velocity distribution of ionic liquid and solid matrices are obtained to describe expansion dynamics of
MALDI
Abstract:
Ionic liquid has been used as MALDI matrix in recent years. Comparing to conventional solid matrices, it shows homogeneous
sample preparation, higher analyte signal intensity, and wider detection mass range. In this study, we used modified crossedmolecular beam apparatus to measure the angular, velocity, and mass distributions of desorbed neutral products from theirradiation of 355 nm on ionic liquid matrix. We investigate the effect of amine（n-Butylamine, IMTBA）as additive base for thecommonly used solid matrix α-Cyano- 4 -hydroxycinnamic acid（CHCA）. The results show that the desorbed neutral moleculesfrom ionic liquid matrix are more than ten times larger than that from conventional solid matrix. The angular, velocity, and mass
distribution measurements provide the clues to understand the MALDI mechanism of ionic liquid.

Oral Session
Tuesday, 18th SeptemberRoom E

15:00 － 17:00

Session 20: The Ion formation and Dissociation Mechanisms in MALDI
Chair:

Myung Soo Kim（Seoul National University, Korea）

S20-1620
16:20 － 16:40

New Insights in MALDI In-Source Decay Process

Daiki Asakawa1,2, Nicolas Smargiasso1, Edwin De Pauw1
1

University of Liege, Liege, Belgium, 2Yokohama City University, Yokohama, Japan

Keywords:
In-source decay, Initial velocity, Internal energy, Hydrogen transfer
Novel aspects:
Consider the ions initial velocity and the presence of O-centered radicals in MALDI plume allows better understanding the MALDIISD process.
Abstract:
Introduction： Matrix-assisted laser desorption/ionization in-source decay（MALDI-ISD）has been utilized as a tool for top-down
protein identification. The major MALDI-ISD fragments are the c’and z’ ions which are originated from the N-Cα bond cleavage via
hydrogen transfer from matrix to peptide.［ 1 ］In contrast, unexpected fragments, such as a’, y’ and w ions, are also observed.
Those fragments cannot be explained by the N-C α bond cleavage alone and their origin is studied in this study using threedifferent matrices, 2,5-dihydroxybenzoic acid（2,5-DHB）,2-aminobenzoic acid（ 2 -AA）and 1,5-diaminonapthalene（1,5-DAN）. The
influence of the initial velocity and the internal energy of analyte ions on fragment ion yield in MALDI-ISD processes is described.
Experimental： Analyte peptides were dissolved in water at concentration of 20 pmol/μl. Matrices were dissolved in water/acetonitrile（ 1 ；1, v/v）with 0.1 % formic acid. The each 0.5 μl of matrix and analyte solutions were deposited on the MALDI plate
and allowed to dry at room temperature. MALDI-ISD mass spectra were recorded using a time-of-flight mass spectrometer,
UltraFlex II（Bruker Daltonics, Germany）equipped with freauency-tripled Nd：YAG laser（355 nm）.
Results： MALDI-ISD generates c’ and z’ ions by the radical-induced cleavage at the N-Cα bonds. The［z+matrix］fragments werealso generated, whereas the matrix adducts bound to c’ ions were not observed. Therefore, MALDI-ISD processes may form the c’/z· fragment pair, and the z· radical fragments then gain a hydrogen atom or react with the matrix. The z’ and w ions originate
from z· radical. These reactions competitively occur during MALDI-ISD processes. The hydrogen attachment reactions of z· radicals
must occur by the collision between z· radicals and matrix molecules in the MALDI plume. Therefore, it is suggested that the ionyield of z’ depends on the collision rate of analyte molecules in the MALDI plume. According to desorption model described by
Spengler and Kirsch, the collision rate is proportional initial velocity of analytes.［ 2 ］It is known that the initial velocity of analytes,
vin, is dependent on the matrix.［ 3 ］The order of initial velocity is 2,5-DHB > 2 -AA > 1,5-DAN. The use of 1,5-DAN gave high ion
yields of w ions, while the use of 2,5-DHB gave low ion yields of wions. The order of ion yield of w ions is 1,5-DAN > 2 -AA > 2,5DHB, which is inversely proportional initial velocity of analyte ions. When the 1,5-DAN is used, the high abundance of w ionscompared when using 2,5-DHB can be understood as resulting from low collision rate in the MALDI plume allowing unimoleculardissociation. We found that MALDI-ISD with 1,5-DAN can be a useful method for the discrimination of Leu and Ile residues.
MALDI-ISD also generated a·/y’ ions. Their intensity increases with the internal energy of analyte ions. As they cannot be explainedby the N-Cα bond cleavage, it is suggested that the formation of a·/y’ fragment pair is initiated by thermal activation of carboncentered radical, leading to an oxygen-centered radical. Subsequent radical-induced fragmentation leads to a·/y’ fragment pair.
The relative importance of the a·/y’ formation increases with decreasing proton affinity of matrix.
References
1. Takayama M., J. Am. Soc. Mass Spectrom., 2001； 12： 1044-1049.
2. Spengler, B., Kirsch, D., Int. J. Mass Spectrom.2003, 226, 71-83.
3. GluckmannM., Karas M., J. Mass Spectrom., 1999； 34： 467-477.

Oral Session
Tuesday, 18th SeptemberRoom E

15:00 － 17:00

Session 20: The Ion formation and Dissociation Mechanisms in MALDI
Chair:

Myung Soo Kim（Seoul National University, Korea）

S20-1640
16:40 － 17:00

CO2-Laser Atmospheric Pressure Ionization of Acoustically LevitatedDropletsArne Stindt1, Merwe Albrecht1, Ulrich Panne1,2, Jens Riedel1

1
BAM Federal Institute for Materials Research and Testing, Berlin, Germany, 2Humboldt-Universitaet zu Berlin,
Department of Chemistry, Berlin, Germany

Keywords:
Ultrasonic Levitation, AP-Ionization, Lucky Survivor
Novel aspects:
A simplified ionization scheme for levitated droplets was established. Besides its direct practical applicability it helps
understanding classical ionization mechanisms.
Abstract:
Once designed for zero gravity experiments, in recent years levitation devices have experienced a renaissance in microfluidics
where they serve as wallless reactors and for contactless sample handling. Until now many optical analytical techniques havebeen employed to interrogate the contents of levitated droplets, however only one approach towards a coupling to mass
spectrometry has been successfully conducted utilizing a combination of corona charging, pulsed high voltage electrodes, MALDI
matrix addition to the droplet and desorption with an UV-laser［ 1 ］.
In the presented work we used a single CO 2 laser pulse for vaporization and ionization out of previously uncharged droplets
following the approach of CO 2 MALDI proposed by Hillenkamp 20 years ago［ 2 ］. We used water as solvent, glycerol as IR
chromophore and amino acids and small peptides as analytes. The resulting spectra resemble results expected for MALDI
conditions and show no fractionation products. This soft ionization is attributed to the low energy coupled to the system byindividual photons dictating mere dynamics in the electrical ground state. The observed ions therefore corroborate the lucky
survivor mechanism postulated by Karas et al.［ 3 ］. In order to visualize the vaporization and plume formation, the experiments
were accompanied by high repetition rate shadowgraphy.
References［ 1 ］M. S. Westphall, K. Jorabchi, L. M. Smith, Anal. Chem., 2008, 80, 5847-5853.
［ 2 ］A. Overberg, M. Karas, F. Hillenkamp, Rapid Commun. Mass Spectrom., 1991, 5, 128-131.
［ 3 ］M. Karas, M. Glueckmann, J. Schaefer, J. Mass Spectrom., 2000, 35, 1 -12.

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 11: Glycomics: From Disease Markers to Therapeutic Antibody Products

PTu-001

11:10 － 12:20

Identification of Native N-linked Glycan Structures from Human Cancer
Cell Using HPLC on a Microfluidic Chip and Time-of-Flight Mass
SpectrometryJong-Moon Park1, Kyeong-seob Lee4, Na-Young Han1, Eugene C Yi3,4, Hookeun Lee1,2

1
Lee Gil Ya Cancer and Diabetes Institute, Incheon, Republic of Korea, 2College of Pharmacy, Gachon University,
Republic of Korea, 3Dept of Molecular Medicine and Biopharmaceutical Sciences Seoul National University, College ofMedicine, Republic of Korea, 4Dept.of Molecular Medicine and Biopharmaceutical Sciences Graduate school ofConvergence Science and Technology Seoul National University, Republic of Korea

Keywords:
Glycan, Q-TOF
Novel aspects:
N-linked glycan analysis
Abstract:
Glycan play key roles in protein folding, cell-cell recognition, cancer metastasis, and the immune system. In cell, glycosylation
comprise that many kinds of glycan structures associated with a specific glycoprotein have different functions. So, the biologicalmeaning of glycosylation has made them a prime target for discovering biomarker in diseases. Identifying the numerous glycan,
we have used new method to assess the diversity of the N-linked oligosaccharides without derivatization has been developed
using on-line nano-liquid chromatography（nanoLC）and high resolution time-of-flight mass spectrometry. In three times MS/MS
results, chips offered good sensitivity and reproducibility in separating a intact protein. In tandem mass spectra, glycan fragment
ions were identified asN-glycan sequences for mining glycoproteome. As a result, we have identified twelve N-glycan structuresfrom murine myeloma recombinant Human epidermal growth factor receptor（EGFR）.

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 11: Glycomics: From Disease Markers to Therapeutic Antibody Products

PTu-002

13:30 － 14:40

Development of Analytical Method of Keratan Sulfate Structure

Shigeyasu Ito, Hiroshi Fujita, Toshikazu Minamisawa
SEIKAGAKU CORPORATION, Tokyo, Japan

Keywords:
keratan sulfate, oligosaccharide, marker, diagnosis, LC/MS
Novel aspects:
We have successed in developing an advanced profiling method for oligosaccharides of keratan sulfate from different biosources.
Abstract:
Keratan Sulfate（KS）is known as a linear polymer of N-acetyllactosamine, 3 Galβ 1 - 4 GlcNAcβ1. The majority of the GlcNAc anda significant proportion of the Gal are sulfated at the C 6 position, and it was also reported that fucosylation and/or sialic acidend-capping can occurred. This polymer is attached to cartilage proteoglycans such as aggrecan and fibromodulin. Several studies
have been reported that structural changes, such as sulfation degree, sialylation and fucosylation was occurred on KS in cartilage
during maturation1)-3). Besides, it was confirmed that the serum concentration of KS correlated with the severity of the damage ofarticular cartilage 4) . However, there are few reports that studied about the structural changes of cartilage and/or serum KS
molecules in such diseases as osteoarthritis and intervertebral disk degeneration. In order to investigate this subject, weconstructed a high sensitivity evaluation system, which enables to detect a small amount of KS, and attempted to analyze whether
the difference between two KSs derived from different origins becomes clear or not, using the system.
KS isolated from bovine cornea（BKS）and from shark cartilage（KPS）were used as model samples. These samples were
thoroughly digested with Keratanase II. Generated oligosaccharides were separated on a Superdex 75 10/300 GL（GE healthcare）
with 50% ethanol as a running solution. Structural and quantitative analysis of these oligosaccharides were performed using LCMSIT-TOF system（Shimadzu）equipped with nanoelectrospray ionization source with negative ion mode.
KS degradation products were detected in trace amounts which were assumed to be several ten micrograms. It was revealed
that more than 70 % of the generated compounds were disaccharides in each KS sample. Other oligosaccharide structures,presumed to be sialylated or de-acetylated, could also be observed, and the amount varied between BKS and KPS. In this way,
analysis of KS oligosaccharide structures and the amounts by this sensitive system was considered promising to provide
information on sulfation, sialylation and de-acethylation pattern of KS which change could reflect the cartilage disease.
References
1 ）Lauder, R. M. et al., Biochem. J. 330, 753-757（1998）.
2 ）Brown, G. M., et al., J. Biol. Chem. 273, 26408-26414（1998）.
3 ）Pratta, M. A., et al., J. Biol. Chem. 275, 39096-39102（2000）.
4 ）Wakitani, S., et al., Rheumatology 46, 1652-1656（2007）.

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 11: Glycomics: From Disease Markers to Therapeutic Antibody Products

PTu-003

11:10 － 12:20

Detection of the Heterogeneous O-Glycosylation Profile of MT1-MMP
Expressed in Cancer Cells by a Simple MALDI-MS Method

Takuya Shuo1, Naohiko Koshikawa1, Daisuke Hoshino1, Tomoko Minegishi1, Hiroko Ao-Kondo2,
Masaaki Oyama2, Sadanori Sekiya3, Shinichi Iwamoto3, Koichi Tanaka3, Motoharu Seiki1

Division of Cancer Cell Research, Institute of Medical Science, University of Tokyo, Tokyo, Japan, 2Medical Proteomics
Laboratory, Institute of Medical Science, University of Tokyo, Tokyo, Japan, 3Koichi Tanaka Mass Spectrom. Res. Lab.,
Shimadzu Corp., Kyoto, Japan

1

Keywords:
MALDI-MS, Liquid matrix, On-target separation, Glycoprotein, Cancer cell
Novel aspects:
A single droplet of a whole protein digest applied to a liquid matrix 3 AQ/CHCA on a MALDI target plate was sufficient to identify
glycosylated and nonglycosylated peptides.
Abstract:
Glycosylation is an important and universal post-translational modification for many proteins, and regulates protein functions. In
particular, specific changes in protein O-glycosylation have been implicated in malignant tumors and therefore development of asimple method to determine O-glycosylation of limited amounts of individual proteins has been desired. MALDI-MS is anindispensable analytical tool for elucidating both the peptide and glycan moieties of glycopeptides. However, it has been difficult
to analyze glycosylated and nonglycosylated peptides within a proteolytic digest simultaneously by MS because the ionization
efficiency of glycosylated and nonglycosylated peptides is so different. Although several methods have been developed for
isolation of O-glycopeptides, they are time-consuming and give rise to poor yields. A new on-target separation technique toanalyze glycopeptides in a highly sensitive manner by MALDI-MS using the liquid matrix 3 AQ/CHCA, which is composed of
3 -aminoquinoline and alpha-cyano- 4 -hydroxycinnamic acid, was developed recently［Sekiya et al.（2012）Rapid Commun. Mass
Spectrom. 26, 693-700］. In this method, a single droplet of proteolytic digest of a protein sample containing both glycosylated andnonglycosylated peptides is spotted directly onto the surface of the liquid matrix 3 AQ/CHCA on a MALDI target plate. During
evaporation of solvents of the digest on the matrix, hydrophilic constituents such as glycopeptides are concentrated within thecentral area. In contrast, hydrophobic peptides tend to be excluded from the evaporating solution and are left at the periphery of
the liquid matrix.
Here we optimized the technique to analyze a small amount of transmembrane protein separated by sodium dodecyl sulfatepolyacrylamide gel electrophoresis（SDS-PAGE）, which is a common procedure for the small-scale separation of proteins. Then
we applied the MALDI-MS method to evaluate the O-glycosylation profile of membrane-type matrix metalloproteinase MT 1 -MMP
expressed in cancer cells at a low level and tried to detect the heterogeneous modification status. MT 1 -MMP is a potent invasion
promoting protease expressed on the surface of aggressive cancer cells. O-glycosylation of MT 1 -MMP is reported to modulate its
protease activity and thereby to affect cancer cell invasion. MT 1 -MMP expressed in human fibrosarcoma HT1080 cells was
immunoprecipitated and resolved by SDS-PAGE. After in-gel tryptic digestion of the protein, a single droplet of the digest wasapplied directly to the liquid matrix 3 AQ/CHCA on a target plate and analyzed by a digital ion trap-type MALDI-MS. The tryptic
MT 1 -MMP digest was allowed to evaporate and the central portion of the sample load area was irradiated using a laser beam.
The MS spectra generated comprised several protonated ion［M + H］+ peaks and the distances between the peaks corresponded
precisely to the masses of typical monosaccharides： 162 and 203 Da for hexose and N-acetylhexosamine, respectively. The
collision-induced dissociation of these peptide peaks via MS2 analysis indicated that the fragment ions correspond to a series oflosses of monosaccharides from a single peptide glycosylated differentially. The glycoforms of this peptide contained 2 to 5
hexose and N-acetylhexosamine. Fragment ion spectra were also obtained from another glycopeptide as a sodiated ion［M +Na］+ peak. On the other hand, many unmodified peptides derived from the MT 1 -MMP digest were detected in the periphery of
the liquid matrix. Overall, we achieved approximately 50 % sequence coverage of MT 1 -MMP using this method. Thus, a wholeprotein analysis of glycosylated and nonglycosylated peptides was accomplished following conventional SDS-PAGE, in-gel
digestion, and direct application of a single droplet of a sample digest onto the liquid matrix 3 AQ/CHCA on the MALDI target
plate.

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 11: Glycomics: From Disease Markers to Therapeutic Antibody Products

PTu-004

13:30 － 14:40

Age-related Changes in the Composition and Structure of Glycan released
from Mouse Skin Tissues

Bum Jin Kim1, Inae Kang1, Su Hee Kim1, Sureyya Ozcan2, Lauren Dimapasoc2, Ik-Soon Jang3,
Jong-Soon Choi3, Hyun Joo An1

1
Chungnam National University, Daejeon, Republic of Korea, 2Department of Biochemistry, University of California
Davis, Davis, CA95616, 3Korea Basic Science Institute, 113 Gwahangno, Yuseong-gu, Daejeon, Korea

Keywords:
Aging, N-glycan, O-glycan, Aging biomarker, Mass spectrometry
Novel aspects:
Rapid, sensitive, and quantitative monitoring for the change of glycans during aging by mass spectrometry
Abstract:
Glycosylation is highly sensitive to the biochemical environment and glycans on a protein play an important role in cell
development. Aging changes have been found in all of the body’s cells, tissues, and organs. These changes affect the functioningof all body systems. Thus, monitoring of the change of glycans during aging may be new approach for aging biomarker discovery.
Mass spectrometry was employed for this study because it is particularly fit for quantitative and qualitative analysis of glycans due
to its intrinsic speed and sensitivity. Here, we have monitored the alteration of glycans released from mouse skin tissues withchronological aging by high performance mass spectrometry.
Mouse skin tissues（three mice sets of 3, 6, 9, 12, 15, 18, 21 and 24 months）were cut about 20mm diameter and ground into small
dices with 70 % ethanol. Ground tissues were dialyzed against water for 24 hours. Supernatant containing glycans were readilycollected. N-glycan were enzymatically released using PNGase F for overnight in a water bath at 37&#8451；. O-glycans werechemically released by β-elimination with sodium borohydride（NaBH 4 ）. Released glycans were purified and fractionated bysolid phase extraction using a porous graphitized carbon. Enriched glycans were profiled by MALDI-TOF/TOF mass spectrometry.
Quantitative analysis and structure elucidation of glycans were further performed by Nano LC-chip Q-TOF mass spectrometry.
PCA analysis of glycans was also performed to examine the alteration of glycosylation during natural aging.
We have profiled several N-glycans including high mannose and neutral-complex type by MALDI-TOF/TOF mass spectrometry.
Fucosylated glycans consisting of only Hex, HexNAc, and Fuc were highly abundant in both young（ 6 months）and old（21months）mouse skin tissues. Tandem mass spectrometry were further performed to assign the structure of glycans. O-glycans
released from skin tissues of mouse aged from 3 months to 24 months were analyzed by mass spectrometry. Approximately 40
glycans（composition only）were identified in mouse skin tissues. Of them, 12 O-glycans having O-glycan cores were reselected forfurther analysis. Interestingly, we observed two distinct O-glycans： m/z 693.2586（Hex 1 HexNAc 1 NeuGc 1 ）and m/z 751.3024（Hex2HexNAc2）. The abundance of two glycans gradually were changed during natural aging. At first, the abundance of ion atm/z 693.2586 was readily increased from 3 months to 12 months and decreased since 15 months. The abundance of another ion atm/z 751.3024 also shows very similar change pattern like m/z 693.2586, which suggests two glycans follow same glycosylation
biosynthesis pathway. Two selected O-glycans were further explored to obtain isomer specific structure information using Nano
LC-chip Q-TOF mass spectrometry. The first（Hex1HexNAc1NeuGc1）has three isomers（RT： 8.363 min, 8.799 min, and 8.995 min）,
while the latter（Hex 2 HexNAc 2 ）has two isomers（RT： 10.293 min and 10.391 min）. In addition, we could identify linkage andstructure isomers by tandem mass spectrometry. Glycans at m/z 693.2586（Hex1HexNAc1NeuGc1）are linkage isomers which havesame structures with a different linkage between monosaccharide, thus, they have same fragments. Structure isomers can beeasily identified by tandem mass spectrometry. Glycans at m/z 751.3024（Hex2HexNAc2）are structure isomers containing unique ’finger printing’ fragments. We will explore more glycans released from mouse skin tissues to examine the change during natural
aging.

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 11: Glycomics: From Disease Markers to Therapeutic Antibody Products

PTu-006

13:30 － 14:40

An MSn platform for detailed characterisation of both the peptide and theglycan moieties and the peptide/glycan linkage in glycoproteins.

Helen V Montgomery1, Matthew E Openshaw2, Omar Belgacem2, Masaki Murase3, Koichi Tanaka3

Shimadzu Koichi Tanaka Mass Spectrometry Research Laboratory, Manchester, UK, 2Kratos Analytical Ltd, Manchester,UK, 3Shimadzu Corp. , Kyoto Japan

1

Keywords:
glyoprotein, glycopeptide, MALDI, MSn, characterisation
Novel aspects:
Complete MSn characterisation of glycoproteins, including peptide sequence, glycan isomeric structure and peptide/glycan
linkage using a MALDI-QIT-TOF Mass Spectrometer and novel software solutions.
Abstract:
The analysis of glycoproteins has become essential in light of the recent developments in the biosimilars /follow-on biologics
market. As increasing numbers of pharmaceutical companies have begun research in the field of therapeutic proteins andpeptides, competition has increased in terms of creating and manufacturing recombinant versions of biologically active proteinsusing E Coli, yeast, human or CHO cell expression models. EU and FDA regulation regarding what may be classified as a biosimilar
is very strict and requires extensive characterisation of the recombinant protein, in particular the glycosylation sites, the glycan
structure and the protein sequence.
The platform presented here combines MS n acquired on a MALDI-QIT-TOF mass spectrometer and dedicated software tools：
SIMSE1（Shimadzu Corp., Japan）for glycopeptides characterisation and localisation of the peptide glycan linkage and Accurate
Glycan Analyser3 - AGA（Shimadzu Corp, Japan）for glycan structural identification.
CID of N-linked glycopeptides exhibits a characteristic cleavage pattern（triplet of peaks）that corresponds to a single HexNAc（peptide + 83Da）and themolecule bound to the peptide（peptide + 203 Da）, the peptide linked to a ring cleaved HexNAc（0,2X）
peptide with no glycan2. This triplet of peaks is used by SIMSE to determine the respective molecular weights of the glycan andthe peptide moieties. Furthermore software guided de-novo sequencing within SIMSE provides the peptide sequence. However,SIMSE can only provide the general composition of the glycan, not the precise structure and linkages. For structural isomercharacterisation of the glycan moiety, AGA software uses MS to MS 4 analyses and a populated database of real MS and MS n
glycan spectra. Determination of the correct glycan structure is achieved using a combination of the MS and MS n data, relative
intensities of the fragment ions and a probabilistic scoring scheme to differentiate isomeric glycan structures.
Glycoproteins were reduced and alkylated prior to tryptic digestion and glycopeptide enrichment. The enriched sample wasanalysed by MS2 and MS3 and these data were investigated using SIMSE software. In parallel, the enriched sample was treatedwith PNGase F and the resulting glycans isolated using a combination of C18 and graphite materials and labelled with aminopyridine. These purified glycans were analysed using AGA software-directed multi-stage MALDI mass spectrometry combining
upto MS4 spectra to unambiguously identify the specific glycan isomeric structure.
Preliminary MS, MS2 and MS3 data using the tryptic digest of the standard glycoprotein Human Transferrin provided the complete
peptide sequence of the glycopeptides GP 2 （QQQHLFGSNVTDCSGNFCLFR； m/z 4137）. SIMSE identified the characterisitic
triplet peak for the localisation of the peptide/glycan bond（Asn 630）, as well as the general composition of the glycan（Hex 5,
HexNAC 4 ）deduced from the glycan moiety molecular weight and fragmentation of the whole glycopeptide. A combination of
MS and MS2 of the PNGaseF cleaved glycan using AGA provided the precise bi-antennary glycan structure for transferrin GP2. Thismethod was applied to the characterisation of other biologically relevant glycoproteins.
1
M. Murase et al., Integrated MALDI MS approach for structural characterisation of intact N-Linked glycopeptides using De Novo
sequencing and protein database search, ASMS TP148（2010）
2
S. Sekiya, T. Iida, Trends in glycosci. and glycotech. 20, 51-65（2008）
3
Kameyama et.al. Anal. Chem., 2005, 77, 4719-4725

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 11: Glycomics: From Disease Markers to Therapeutic Antibody Products

PTu-007

11:10 － 12:20

Comparative Study of Protein Concentration, Glycoform Profile and Site
Occupancy of a Glycoprotein with Multiple Glycosylation Sites andComplex Glycans

Yi-Chun Ma, Guor-Rong Her

National Taiwan University, Taipei, Taiwan

Keywords:
Glycoprotein quantitation； glycosylation site occupancy； glycan profile； mass spectrometry； isotope labeling
Novel aspects:
This is the first strategy capable of differentiating the variation of a glycoprotein which has multiple glycosylation sites bearing
complex type glycans.
Abstract:
Over half of human proteins are glycoproteins. The glycoprotein concentration, glycosylation site occupancy and glycan profile
may vary with regulatory states, types of diseases and even with disease progression; therefore, comparative analysis may
provide useful information. A strategy was proposed for comparative analysis of glycoproteins in which the change of protein
concentration, the change of glycosylation site occupancy and the change of site-specific glycoform profile could be
differentiated and determined. To demonstrate the utility of the strategy, the glycoprotein asialofetuin which has multiple
glycosylation sites bearing complex type glycans was analyzed. Comparative analysis was performed using stable isotope
labeling of glycopeptides and peptides by formaldehyde-D 0 and formaldehyde- 13 C, D 2 and analyzed by RPLC-ESI-MS.
Glycopeptide signals were identified by the largely present glycosidic fragments in the MS/MS spectra. Dimethyl labelingreaction of glycopeptides was examined to be complete. The relative intensity of nonglycosylated peptides provided
information about protein concentration variation. Change of site-specific glycoform profile was obtained by the relative
glycoform profile of D0-dimethyl and 13C, D2-dimethyl labeled glycopeptides. By knowing the variation of protein concentrationand the variation of site-specific glycoform profile, the glycosylation site occupancy could be obtained.

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 11: Glycomics: From Disease Markers to Therapeutic Antibody Products

PTu-008

13:30 － 14:40

Automated intact mass analysis for routine identity testing and quality
tracking of recombinant monoclonal antibodies

Yoshihiko Morishita1, Jette Wagtberg2, Christian Albers3, Laura Main3, Wolfgang Jabs3
1

Bruker Daltonics K.K., 2Symphogen A/S, Lyngby, Denmark, 3Bruker Daltonik GmbH, Bremen, Germany

Keywords:
Antibody, protein, QC, glycosylation
Novel aspects:
Fully automated solution for the characterization of antibodies. Automated platform for identity testing
Abstract:
Symphogen is developing recombinant antibody mixtures, a new class of antibody therapeutics for the treatment of serious
human diseases. Development of such products presents a number of challenges, including the development of effective methods
for manufacturing and release of a vast number of recombinant antibodies as well as mixtures. Characterization methods andidentity tests represent key analytical methods that need to be established during development.
To ensure the correct sequence and identity of batches throughout development from discovery to tech transfer for GMP
production, an intact mass analysis has been implemented as identity test. The same analysis is used for characterization toevaluate fragmentation and glycosylation pattern. Initially, this method was time consuming due to the high number of samples
and the need for hands-on data processing. In addition, a system was needed for tracking data on older batches and forcomparative analysis. However, with the implementation of newly developed software, we are able to run intact mass both as an
identity assay and for characterization, in a fast and automated manner, including all steps from sample injection to reporting
results in one seamless workflow.
The glycosylation pattern of antibodies is an important quality attribute, as it potentially affects antibody potency and clearance.
The overall glycosylation pattern is influenced by changes in the upstream and downstream process, as well as individual
production clones gives rise to glycosylation variations. Therefore, a workflow for fast and cost-effective evaluation of change inglycosylation is crucial during process development and clone selection. We demonstrate data in visually easy to understandviews and reports that show how our new method enabled us to guide process development and clone selection.
Besides quality assessment of glycosylation patterns, we use the new methodology in high throughput identity assays, where aminimum of 75 samples can be analyzed in a day including automated data processing and data evaluation. We show here howwith a searchable database of processed results, it is easy to access old data, and/or track quality over time for a specificantibody, process or clone during preclinical development.

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 11: Glycomics: From Disease Markers to Therapeutic Antibody Products

PTu-009

11:10 － 12:20

Comprehensive Glycan Profiling of Exosomes Derived from Human Body
Fluids for Disease Biomarker Discovery

Seunghyup Jeong1, Do-Young Choi2, Pyong-Gon Moon3, Kwang Pyo Kim2, Moon Chang Back3,
Hyun Joo An1

Chungnam National University, Daejeon, South Korea, 2Konkuk University, Seoul, South Korea, 3Kyungbuk National
University, Daegu, South Korea

1

Keywords:
Exosomes, microvesicles, glycan, glycosylation, biomarker
Novel aspects:
The first study of comprehensive glycan profiling of exosomes using mass spectrometry.
Abstract:
Exosomes（or microvesicles）are secreted by various cell types including tumor cells. They are composed of membrane, cellular
proteins and RNA derived from their parent cells. They play an important role as mediators in extracellular communication. It isalso known that exosomes are involved in tumor metastasis, angiogenesis, and antitumor immunity in cancer cells. However, theglycosylation of exosomes has not yet been studied. In this study, we have analyzed N-glycans derived from two types of
exosomes, namely cancer cells and urine. First, we developed the method to profile exosome glycans by mass spectrometry.
Using newly developed analytical method, glycans of exosomes derived from several lung cancer cell lines（PC9, PC 9 /R, A549）
were profiled. Then we compared exosome glycans with glycans on their parent cell membranes to examine glycosylation
correlation between origin cells and their exosomes. Furthermore, we have profiled glycans released from urinary exosomes ofhematuria patients and control group（n=18）to examine the difference of glycosylation between two sample groups for biomarker
discovery. All exosomes（about 70ug each）were treated with PNGase F to release glycans without any further sample
preparation. Released glycans were then enriched by solid phase extraction（SPE）using a porous graphitized carbon（PGC）
cartridge and solvents of varying polarity（different concentrations of aqueous acetonitrile）to partition them into highly anionicand neutral components. The fractions were then analyzed by two types of mass spectrometry. Ultraflextreme MALDI-TOF/TOF
MS（Bruker daltonics）and Nano LC-chip Q-TOF MS（Agilent technologies）were used for overall glycans profiling and quantitation,
respectively.
Interestingly, we found that glycans of exosomes derived from cancer cell lines contain over 75 % of high-mannose glycans
consisting of trimannosyl core and［Man］5-9. The most abundant glycan is Man 8 （m/z 1743.581, Hex8 HexNAc2）. While complex /hybrid type glycans with at least one fucose account for 25%.
In order to study the differences of glycosylation between wild type exosomes and anticancer drug Gefitinib（Iressa, EGFR
antagonist）resistance type, we compared exosome glycans between PC 9 and PC 9 /R. Total abundance of glycans on PC 9 isslightly higher than that of PC 9 /R although the number of glycans analyzed by LC/MS is quite similar. We performed bio- andanalytical replica to validate our preliminary data. In addition, we further analyzed N-glycans from more exosomes derived fromvarious cancer cell lines and their origin cancer cells, respectively to examine the correlation if cancer cell lines and their
exosomes have similar glycan profile.
In hematuria patient, IgAN（IgA nephropathy）develops end-stage renal disease and TBMN（Thin basement membrane
nephropathy）excellent outcome which cannot be considered a disease. These two diseases have same symptom and are only
diagnosed through a biopsy. This traditional way is invasive. Here, we have studied to investigate non-invasive diagnosis tool
focused on patient’s urine. Urine sample can be easily obtained. Using newly developed method glycans have been released fromexosomes prepared from 18 clinical samples consisting of one control group（n= 6 ）and two hematuria patient groups（IgAN（n= 6 ）, TBMN（n= 6 ））. Glycans between groups will be further examined for biomarker discovery.

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 11: Glycomics: From Disease Markers to Therapeutic Antibody Products

PTu-010

13:30 － 14:40

Determination of core fucosylation in glycopeptides of monoclonal
antibodies

Toshiji Kudo1, Anja Resemann2, Ulrike Schweiger-Hufnagel2, Detlev Suckau2, Daniel Kolarich3

1
Bruker Daltonics K.K., 2Bruker Daltonik GmbH, Bremen, Germany, 3Max-Planck-Institut for Kolloid- undGrenzflaechenforschung, Berlin, Germany

Keywords:
Glycosylation, glycopeptide, ion trap, MALDI
Novel aspects:
Automatic fragmentation pattern based detection of core fucosylation of N-linked glycopeptides in LC-MS/MS datasets
Abstract:
Fucosylation is a common modification of glycans known to affect glycoprotein confirmation and function. alpha-1,6 fucosylation of
the chitobiose core commonly found on N-linked glycans plays an important role in many physiological processes such as
signaling pathways and immune response. Involvement of core fucosylation has also been shown in pathological processes likecancer and has been described as biomarker for liver cancer malignity. Antibody-dependent cell-mediated cytotoxicity of IgG has
been shown to be crucially mediated by different grades of core fucosylation. Here we describe a fast and reliable approach tounambiguously determine core fucosylation directly on the glycopeptides in tryptic digests by MALDI-TOF/TOF and ESI-IT.
Standard glycoproteins like asialofetuin, horseradish peroxidase and a panel of therapeutic and standard monoclonal antibodies（e.g., MOPC21 from Sigma）were reduced, alkylated and trypsinized. Tryptic peptides and glycopeptides were separated by RPLC and fractions were analyzed by ESI-IT-MS and MALDI-TOF/TOF-MS in positive ion mode. A dedicated software algorithm for
N-linked glycopeptide analysis automatically classified MS/MS spectra taking the specific fragmentation patterns of MALDI and ESI
CID into account, clearly distinguishing glycopeptides with or without core fucose. Subsequent protein and glycan databasesearches provided glycan structures and peptide sequences of the respective glycopeptides.
The typical fragmentation patterns derived from N-linked glycopeptide fragmentation by MALDI and ESI analysis was used to filter
glycopeptide spectra from a large number of MS/MS spectra. This allowed determination of both, peptide and the glycan masses
of individual glycopeptides and enabled detection of fucosylation at the chitobiose core.
In MALDI-TOF/TOF-MS, the N-acetyl glucosamine（GlucNAc）attached to the peptide undergoes a 0,2X cross ring fragmentation
resulting in the specific fragmentation pattern（Mpeptide+H）/ +83/ +120 Da（Wuhrer et al., 2007）. In core fucosylated glycopeptides
an additional fucose distance of Dm/z=146 is found adjacent to the fragment peak from the Y type cleavage（Domon and Costello
nomenclature, 1988）.
In ESI-IT, the fragment mass of the peptide including the first GlucNAc of the chitobiose is detected in the majority of cases ofN-glycopeptides. In addition to this YY or Y fragment（for core fucosylated or non-fucosylated N-glycans）, the ZZ fragment of thepeptide & GlucNAc（mass difference -36）, the Z fragment of peptide & GlucNAc & fucose（+128）and the Y fragment of thepeptide & 2 GlucNAc & fucose （+ additional 203） are observed and were used for the detection of core fucosylated
N-glycopeptides including determination of the peptide mass.
This information was used to adapt the glycoprotein analysis software by extending lists of fragmentation patterns to distinguishdifferent glycan structures. That capacity enabled us to identify the high glycan micro heterogeneity at HC-Asn294 in murine IgG 1
MOPC21. Core fucosylation was found to be a common modification in more than 50 % of the identified N-glycans. The main
glycan in MOPC21 is G 0 F 1 （GlucNAc 4 Man 3 Fuc 1 ）, followed by the same structure lacking the core fucose（G 0 ）.
Subsequently, core fucosylation classification results were verified by glycan database searches and spectrum matches.

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 11: Glycomics: From Disease Markers to Therapeutic Antibody Products

PTu-011

11:10 － 12:20

Identification and structural analysis by chip-based nanoelectrospraymass spectrometry of novel brain-associated chondroitin/dermatansulfate motifs

ALINA D ZAMFIR1, CORINA FLANGEA1, ROXANA M GHIULAI2, DANIELA G SEIDLER3
1
3

Aurel Vlaicu University, Arad, Romania, 2Victor Babes University of Medicine and Pharmacy, Timisoara, Romania,
University of Muenster, Germany

Keywords:
glycosaminglycans；chondroitin/dermatan sulfate； chip-based nanoESI；brain
Novel aspects:
Novel oversulfated CS/DS species from brain were for the first time discovered and characterized using fully automated chipbased nanoESI MS and CID multistage MS.
Abstract:
Brain chondroitin/dermatan sulfate（CS/DS）represent a class of glycosaminglycans involved in many biological processes, such ascell proliferation and recognition, cell adhesion and migration, neurite outgrowth and wound repair. CS chain backbone consistsof repetitive disaccharide units containing D-glucuronic acid（GlcA）and N-acetylgalactosamine（GalNAc）residues, whereas DS is
the stereoisomeric variant of CS with varying proportions of L-iduronic acid（IdoA）. Recent studies have shown that proportionsof CS/DS disaccharide units in brain change with development/affliction suggesting that CS/DS differing in the degree and profile
of sulfation may be involved in the functional diversity of neurons［1,2］. In the last few years, due to the technical advancements
in MS instrumentation and the development of efficient sequencing protocols［ 3 - 5 ］, the determination of CS/DS structure interms of chain length, epimerization, sulfation content, pattern and site（s）became feasible.
In this study, CS/DS chains extracted from 4 weeks old normal mouse brain were released by beta-elimination and digested bychondroitin lyases. The enzymatic hydrolysis mixtures of hybrid CS/DS chains were separated by size exclusion chromatography.
Tetra- and hexasaccharide pools were submitted to fully automated chip-nanoelectrospray ionization （nanoESI） mass
spectrometry（MS）in negative ion mode. Fully automated chip-based MS was performed on a high capacity ion trap（HCT）mass
spectrometer couplet to a NanoMate 400 robot. Multistage MS（MSn）was carried out by collision-induced dissociation（CID）at
variable low energies using helium as the collision gas.
For this assay, to discriminate between domains containing GlcA or IdoA, the whole CS/DS chain was digested with chondroitin
AC I lyase and in parallel with chondroitin B lyase. Chondroitin B lyase cleaves the linkage between GalNAc and L-IdoA, whilechondroitin AC I lyase cleaves the linkage between GalNAc and D-GlcA irrespective of the sulfation content and distribution along
the chain or within the monomer. Screening in the MS1 mode of tetra- and hexasaccharides resulted after depolymerization withAC I lyase revealed molecular ions assigned to regularly and oversulfated species. CID MS n applied on tetra- and pentasulfatedtetra- and hexasaccharides gave rise to a fragmentation pattern characteristic for 4,5-delta-［GlcA（S）-GalNAc（S）-IdoA（S）-GalNAc（S）］and 4,5-delta-［GlcA（S）-GalNAc（S）-IdoA（S）-GalNAc（S）-IdoA-GalNAc（S）］structures. Chip ESI MS profilingand CID MS n analysis of the two oligosaccharide fractions obtained by depolymerization of the same CS/DS chain using
chondroitin B lyase provided evidence upon the occurrence of mono- and bisulfated dissacharide sequences. CID MS nexperiments provided a fragmentation pattern consistent with an atypical, previously unidentified 4,5-delta-［IdoA（S）-GalNAc（S）-GlcA-GalNAc（S）］and 4,5-delta-［IdoA（S）-GalNAc（S）-GlcA-GalNAc（S）-GlcA-GalNAc（S）］structural motifs. Consequently,
due to the high sensitivity and reproducibility of the employed MS method, novel brain-associated CS/DS species exhibiting an
unusual location of the sulfate groups at GlcA and IdoA were for the first time discovered and characterized. Obviously, thisanalytical platform opens new perspectives in glycomics of brain glycosaminoglycans for identification of brain CS/DS domains ofirregular sulfation pattern and elucidation of their role in development, maturation, aging and degeneration of the central nervous
system.
［ 1 ］A. Purushothaman, J. Fukuda, S. Mizumoto, G. B. ten Dam, T. H. van Kuppevelt, H. Kitagawa, T. Mikami, K. Sugahara, J. Biol.
Chem., 282, 1944219452（2007）.
［ 2 ］E. Sisu, C. Flangea, A. Serb, A. D. Zamfir, Amino Acids, 41, 235-256（2011）.
［ 3 ］C. Flangea, C. Schiopu, E. Sisu, A.Serb, M. Przybylski, D. G. Seidler, A. D. Zamfir, Anal. Bioanal. Chem., 395, 2489-2498（2009）.
［ 4 ］C. Flangea, E. Sisu, D. Seidler, A. D. Zamfir, Anal. Biochem. 420, 155-162（2012）.
［ 5 ］A. D. Zamfir, C. Flangea, E. Sisu, D. G. Seidler, J. Peter-Katalinic, Electrophoresis, 32, 1639-1646（2011）.

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 11: Glycomics: From Disease Markers to Therapeutic Antibody Products

PTu-012

13:30 － 14:40

Glycobiomarkers for atherothrombosis

Oliver Ozohanics1, Lilla Turiak1, Anita Jeko1, Angel Puerta2, Jose Carlos Diez-Masa2,
Mercedes de Frutos2, Karoly Vekey1, Laszlo Drahos1

Research Centre for Natural Sciences Hungarian Academy of Sciences, Budapest, Hungary, 2Institute of Organic
Chemistry (CSIC), Juan de la Cierva 3 Madrid, 28006, Spain

1

Keywords:
Alpha- 1 -acid-glycoprotein AGP Glycosylation atherothrombosis atheroslerosis
Novel aspects:
Early diagnosis tool for atherothrombosis by analysing AGP glycoforms（78 % sensitivity and 95 % specificity）
Abstract:
Cardiovascular diseases, like atherothrombosis, are the leading cause of death in the western world. Currently there is no mass
diagnostic procedure of atherotrombosis, as Computed Tomography（CT）is costly and not suitable for high throughput, andangiography is invasive and not suited for routine diagnosis.
Proteomics is one of the most dynamically improving research field recently focusing on post-translational modifications（eg.
phosphorylation, glycosylation）. Glycosylation is immensely important； two typical examples are biopharmaceuticals and FDA
accepted biomarkers, most of which are glycoproteins. There is a lack of adequate techniques for determining glycosylation
patterns, suitable high-throughput analytical methodologies are just starting to appear. In this presentation we will show a novel
high-throughput approach to determine site-specific glycosylation patterns.
The developed workflow is based on LC-MS quantitation of glycopeptides and LC-MS/MS based glycopeptide identification. The
key, novel aspect of the workflow presented is automatic spectra evaluation. To identify glycopeptide structures based on tandem
mass spectra we have developed the algorithm and software named GlycoMiner. This works automatically（does not require
manual control）and has a very low false positive and false negative rate（both 0.1 %）. To analyze glycosylation patterns
automatically, we have developed another software（GlycoPattern）. This works in conjunction with GlycoMiner. Besides
quantitation, it is capable of identifying minor glycoforms.
We have applied the developed workflow to analyze the glycosylation pattern of α- 1 -acid glycoprotein（AGP）samples, isolated
from the plasma of healthy individuals and patients suffering from 2 different forms of atherothrombosis（carotid atherosclerosis
and abdominal aortic aneurism）. Statistical analysis showed significant differences among all three groups （healthy,atherosclerosis and aneurism）based on the glycosylation pattern. Cross-validation showed 78 % sensitivity and 95 % specificity for
identifying atherothrombosis（comparing sick vs. healthy groups）. The glycosylation pattern of AGP is therefore a very promisingbiomarker candidate.
Acknowledgement:This work has been supported by the Hungarian Scientific Research fund（grant No. OTKA NK83857）, National Office for ResearchTechnology（KMOP-1.1.2-07/ 1 -2008-0002）and the Spanish Ministry of Science and Innovation for grant CTQ2009-09399. Angel Puerta
acknowledges the CSIC for a contract in the JAE doc program. Dr. Egido and Dr. Tuñon groups from IIS-Fundacion Jimenez Diaz
（Madrid, Spain）are acknowledged for the plasma samples.

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 11: Glycomics: From Disease Markers to Therapeutic Antibody Products

PTu-013

11:10 － 12:20

Top-Down Mass Spectrometric Analysis of Glycosylated andPhosphorylated Proteins

Fujio Nishida1, Anja Resemann2, Lars Vorwerg2, Paul Kowalski3, Deltev Suckau2

1
Bruker Daltonics K.K., Yokohama, Japan, 2Bruker Daltonik GmbH, Bremen, Germany, 3Bruker Daltonics, Inc., Billerica,
MA, USA

Keywords:
Top-down, N-linked glycosylation site, phosphorylation site, new sample preparation
Novel aspects:
Intact N-linked glycans were detected by positive mode MALDI-ISD and phosphorylation sites were analyzed using negative mode
MALDI-ISD.
Abstract:
Introduction：
Top-down approaches allow for direct analysis of intact proteins using their mass and their fragments for identification andcharacterization. In contrast to bottom-up techniques, labile PTM’s like phosphorylation remain stable in top-down analysis andenable direct modification site determination. MALDI-In-Source-Decay（MALDI-ISD）is a powerful Top-down-Sequencing（TDS）
tool and typically provides long N- and C-terminal sequence tags of up to 80 amino acids and more, allowing for the detection oflarge PTM’s like N-linked glycans even at central sites of proteins up to 15 kDa. TDS were applied to standard glycoproteins such
as RNAseB, horseradish peroxidase, alpha- and beta-caseins for positive as well as negative ionization mode MALDI-ISD was
explored.
Methods：
Proteins（Sigma）were dissolved in water/ 0.1 % TFA at 10-100 pmol/ul, applied to a MALDI sample plate carrying 2 mmhydrophilic anchor sample spots, allowed to dry and rinsed. Matrices for MALDI-TDS were 1,5-diaminonaphtalene（DAN）and
super-DHB. They were dissolved in 50 % acetonitrile/water/0.1 % TFA and 1 ul was applied to the rinsed sample spots. Spectra
were acquired manually or automatically in positive and negative ion mode. Proteins were identified using ISD signals as virtual
precursors and as fragments for MASCOT（Matrix Science, Inc.）MS/MS ions searches. Protein sequences were matched to thespectra including proposed modifications and ranked by a proximity score reflecting how closely the sequences match theexperimental data.
Preliminary Data：
This presentation describes a new method for sample preparation, TDS spectrum acquisition and interpretation for the analysis ofglycosylated or phosphorylated proteins. The usage of the MALDI sample plates with hydrophilic sample areas allowed forwashing the dried, buffer containing samples before matrix application. This led to higher S/N values and extended mass range for
TDS analyses. ISD fragmentation series are typically terminated by disulfide bonds and limits the utility of TDS.
Glycosylation： Using DAN as matrix, reduction of the SS-bridges occurred and ISD fragmentation of RNaseB was extended toresidue 40 including the glycosylated Asn34. The most abundant high-mannose-type glycans Man 5 and Man 6 were detected.
Horseradish peroxidase was prepared the same way and the N-linked glycan structure typical for plants was identified at Asn13.
Additionally, the pyro-glutamate at the N-terminus of horseradish peroxidase was found. To our knowledge, this is the first time
TDS analysis included the assignment of N-linked glycans.
Phosphorylation： Negatively charged phosphate modifications in -casein limited the N-terminal ISD fragment detection in positive
ion mode, only 2 phosphorylation-sites were detected. Negative ion mode, however, enabled the detection of all 5 N-terminallyphosphorylated serine residues. In -S 1 -casein, Ser--41 was identified as non-phosphorylated in contrast to uniprot description, S46
and S48 were confirmed as being phosphorylated. -S 2 -Casein carries 5 phosphorylated serine residues near the N-terminus, all ofthem were confirmed using negative mode ISD. As a result, we propose to use negative ion mode ISD in cases where
phosphorylation or other negative charged residues effect the formation of fragment ions in TDS analyses.

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 12: On-site Mass Spectrometry -Miniaturized Instruments and Allied Technologies-

PTu-014

13:30 － 14:40

Design and Testing of a Miniature Magnetic Sector Mass Spectrometer

meiru guo, liang wang, yide zhao, yuhua xiao, taiguo li

Science and Technology on Vacuum & cryogenics Technology and Physics Laboratory, Lanzhou Institute of Physics,
Lanzhou, China

Keywords:
magnetic-sector； double trajectory； miniaturization
Novel aspects:
A small, lightweight, low power consumption instrument has been designed. Including the miniaturization of ion source, highvoltage technique, sweep high voltage subsystem, and micro-current amplifier subsystem.
Abstract:
A miniaturized magnetic-sector mass spectrometer has been developed and tested. The instrument was designed which consistof Nier-type thermionic ion source, a 90o magnetic sector-field analyzer with double trajectory, electronic unit and ion detectors.
Miniaturization（170 x 165 x 170 mm 3 ,  <4.3 kg）was accomplished by using high-energy-product magnet material（NdFeB alloy）
which has an magnetic of 0.65T. Two special trajectories are selected to cover the mass ranges 1 -12amu and 6 -50amu. The
electronic unit consists of sweep high voltage subsystem, and micro-current amplifier subsystem. The sweep high voltage
subsystem consist of the sweep high voltage supply and the digital sweep control circuit. The sweep high voltage power supply
develops the high voltage level on which the ion source rides. Digital control develops a sweep output and a pedestal output.
Together these two output control the sweep high voltage power supply. The instrument was able to attain a mass resolutionof7.7x10 -5 A/Pa, and a mass range up to 50 atomic mass units. Its power consumption was measured to be 15W. All thesecharacteristics make this miniature mass spectrometer suitable for space applications and chemical analysis and also for low-cost
analytical instrumentation.

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 12: On-site Mass Spectrometry -Miniaturized Instruments and Allied Technologies-

PTu-016

13:30 － 14:40

Real-Time Monitoring of a Suzuki Reaction using a Hood-Based Compact
Mass Spectrometer

Daniel Eikel, Jack Henion, Simon Prosser, Nigel Sousou, Collier Lee, Jamey Jones
Advion Inc., Ithaca, New York, USA

Keywords:
reaction monitoring, on-site mass spectrometry
Novel aspects:
Real-time reaction monitoring of a Suzuki reaction is described with a hood-based chemical synthesis- compact LC/MS system.
Abstract:
Medicinal chemists are routinely faced with personally synthesizing over a hundred new chemical entities（NCE’s）each year for
testing as future pharmaceutical drug candidates. The goal is to prepare a high yield, relatively pure product via an optimized
synthetic route. TLC and LC/UV/MS are routinely used to monitor and guide these reaction outcomes. In most cases the LC/UV/MS technology is only available through a central core facility or via shared open access of systems. A preferred alternative is apersonal small LC/MS system located in a hood adjacent to the synthetic reaction apparatus. This presentation will describe suchan LC/MS system used to monitor and optimize a Suzuki coupling reaction.
A compact single quadrupole mass spectrometer（expression, Advion, Inc.）with dimensions of 11 X 21 X 26 inches was installed ina standard chemical fume hood adjacent to a round-bottom flask chemical synthesis setup. The mass spectrometer was equippedwith an atmospheric pressure ionization（API）source coupled with electrospray ionization. A Shimadzu 20 AD binary solvent
delivery system employing a 2.1 mm i.d. x 5.0 cm C-18 reversed-phase HPLC column operated in the isocratic mode with 40/60
aqueous acetonitrile containing 0.1% formic acid and maintained at 200 microliters/min. The LC/MS system was used to monitor
product formation from a conventional Suzuki coupling reaction to prepare p-aminobiphenyl. Direct injection of diluted crude
reaction product mixture for LC/MS analysis was used.
The Suzuki reaction selected in this study consisted of coupling p-aminobromobenzene with phenylboronic acid at room
temperature stirred with palladium dichloride and NaOH in ethanol. This reaction prepares p-aminobiphenyl at the 5 mmol level
which makes nearly a gram of product in 5 mL of a heterogeneous solution if it goes to completion with a quantitative yield. The
para amino group retards this coupling reaction thus providing the opportunity to improve the yield with the application of heator longer reaction time. The described compact LC/MS system thus may be used to monitor the starting material disappearance
vs. the production of the desired product during the course of the reaction. A series of reactions were carried out while
systematically varying the reaction temperature and reaction time. At regular 10 min. interval five microliter aliquots were manually
removed from the reaction vessel, diluted and mixed in a 5 mL injection solvent mixture of 80/20 water/0.1% acetonitrile and then
5 microliters of this solution injected into the LC/MS system under isocratic conditions. The latter 40/60 aqueous acetonitrile
containing 0.1% formic acid mobile phase had been previously optimized to provide a run cycle time of only 2 min. with good
separation of each targeted compound. A post-column split of 200/ 1 was provided by RMA valve providing reduced exposure ofhigh concentration compounds direct to the compact mass spectrometer. The typical experimental sequence provided real-time
LC/MS results of the reaction progress within one-hour reaction intervals. This allows immediate changes in reaction conditions forthe next synthetic run allowing the optimization of this reaction to provide a 90% yield within a half-day period.

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 12: On-site Mass Spectrometry -Miniaturized Instruments and Allied Technologies-

PTu-017

11:10 － 12:20

Explosives Trace Detection by Mass Spectrometry: An Automated Particle
Sampler for Collecting Explosives Particles Adhering to Passenger’s
Baggage

Hisashi Nagano, Yuichiro Hashimoto, Yasuaki Takada, Yasutaka Suzuki, Hideo Kashima,
Masakazu Sugaya, Yasunori Doi, Koichi Terada, Minoru Sakairi
Hitachi,Ltd.,Central Research Laboratory,Tokyo,Japan

Keywords:
Explosives_Trace_Detection, Mass_Spectrometry, Linear_Ion_Trap, Cyclone_Particle_Concentrator
Novel aspects:
Explosives Trace Detection by Mass Spectrometry： An Automated Particle Sampler for Collecting Explosives Particles Adhering toPassenger’s Baggage.
Abstract:
Introduction
The threat of terrorism or criminal bombings has become a serious problem for all countries. To maintain a safe society, detection
technologies for hidden explosive devices are in high demand. Two methods are primarily used to detect hidden explosives； bulk
detection, which determines the existence of suspicious objects such as knives, firearms, and explosive devices from their shapes,
and trace detection, which detects the presence of explosive contaminants by chemical analysis of vapor or particles fromobjects. The bulk and trace detection methods have different characteristics, and they complement each other’s weak points.
Therefore, the combined use of bulk detection and trace detection is effective to improve security at important facilities.
We have already developed explosives trace detectors（ETDs）by mass spectrometry（MS）for airport checkpoints. However,current ETDs require human operations for swab-sampling of contaminants from surface of baggage, which makes 100% inspection
high-cost. Therefore, an auto-sampling ETD will dramatically reduce human operations, which can be applied to all baggages withlower cost. In this paper, we report preliminary results of an automated particle baggage screener combined with the MS-based
ETD.
Methods
The automated particle sampler consists of a nozzle, a sampling port, and a cyclone-type particle concentrator. The compressed
air is emitted from the nozzle to the surface of baggage to be checked. Explosives particles adhering to the baggage are detachedby the air jet and introduced into the concentrator through the sampling port.
The explosives particles collected by the sampler are then vaporized by a heating unit that is located below the concentrator. The
vaporized explosives molecules are introduced into an atmospheric pressure chemical ionization（APCI）ion source and the ionsproduced by the ion source are analyzed by a linear ion trap mass spectrometer with wire electrodes（wire-LIT）［1,2］.
Preliminary Data
We tested the prototype of the automated particle sampler using NIST SRM2905 which is trace particulate explosive stimulants
developed by NIST［ 3 ］. The mg -level of the particle explosive simulants, which included ng-level of 2,4,6-trinitrotoluene（TNT）,
were adhered to a baggage and we observed the M- ion（m/z 227）to detect TNT in a negative APCI mode. The signal of TNT wasclearly detected within 5 seconds after the emission of the air jet from the nozzle to the baggage. The collection efficiency of theautomated particle sampler, which are defined as the ratio of the amount of the particles collected by the sampler to the amountof the particles initially adhered to the baggage, were 0.4- 1 %.
The sensitivity and throughput of the ETD system is high enough to use it with X-ray bulk detection systems at airport checkpoints.
Therefore, we concluded that our newly developed automated particle baggage screener combined by the MS-based ETD would
be useful to improve security at important facilities.
This work was supported by“R&D Program for Implementation of Anti-Crime and Anti-Terrorism Technologies for a Safe andSecure Society“, Strategic Funds for the Promotion of Science and Technology of the Ministry of Education, Culture, Sports,
Science and Technology, Japan.
References［ 1 ］Y. Takada et al., Rapid Commun. Mass Spectrom. 2011； 25（17）： 2448.
［ 2 ］Y. Hashimoto et al, Trace Explosives Detection Workshop, April, 2011, Portland.
［ 3 ］W. A. MacCreham, Anal. Chem. 2009；81： 7189.

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 12: On-site Mass Spectrometry -Miniaturized Instruments and Allied Technologies-

PTu-019

11:10 － 12:20

Radio Frequency Mass Reflectrons with plane Discrete Electrodes

Alexander A Dyagilev, Eugeny Y Grachev, Vladimir V Zhuravlev, Eugeny V Mamontov
Ryazan State Radio Engineering University, Ryazan, Russia

Keywords:
discrete planar electrodes, quadrupole fields
Novel aspects:
New method of formation of 2 D linear electric fields with discrete planar equipotential electrodes is investigated. This method isbased on a linear discrete distribution of the averaged potential.
Abstract:
Modern technologies employ mass spectrometers as an effective means of accurate determination of substance structure. The
highest analysis speed belongs to time-of-flight mass spectrometers（TOF MS）. Usually, time-of-flight mass analysis occurs instatic fields, which requires high energy homogeneity of injected ions. To overcome this problem time-of-flight mass spectrometerswith quadrupole RF fields can be used. Implementation of such devices for time-of-flight sorting requires creation of ion optics
with quadrupole RF field extended along the axis of the flight. Hyperbolic electrode systems are not effective here because oflarge size of electrodes required. Most effectively quadrupole RF fields can be formed by a system of plane electrodes with linear
one-dimensional distribution of RF potential. A continuous linear potential distribution, which is difficult to create, is replaced by adiscrete linear distribution. Numerical methods were used to study the potential distribution in a flight region. Relative departureof potential distribution was investigated as a function of plane electrodes discrete step size dy.
It was found that the value of the departures is proportional to the fifth order of electrode discretion step. The relative departureof potential within the flight region does not exceed 10-4 for dy/L < 0.014, where L is the length of the analyzer along the axis of theflight. Ion reciprocating oscillations were simulated with a spread in initial energies dW/W=100%, initial coordinates dx/x=0.1, andinjection angles ± 5 °for the stability parameter 0.01. The relative time defocusing did not exceed 10-5.
Ion-optical systems with a discrete linear potential distribution can be created using inductive and capacitive dividers of RF
voltage. Another approach is based on plane discrete equipotential electrodes. Analyzers can be created by a system of plane
discrete electrodes with discrete linear distribution of mean potential along one of axis.
Two types of plane discrete electrodes were examined： with different electrical transparency along the Y axis and with different
distribution of elements along the Y axis. In the first case two-dimensional linear electric fields are formed by a system, which
consists of paired electrodes, one of which is solid, while the other one, half-transparent, is grounded. The plane half-transparent
electrodes are placed along the Y axis uniformly with step dy. Their width l i is selected so that the average potential of theelement is proportional to its number. In the second case the two-dimensional linear field is formed by a system of paired
electrodes, one of which is solid and grounded. The other electrodes represent a set of identical equipotential elements. Fine
metallic wires, parallel to the Z axis, can be used as elements of these discrete electrodes. The linear distribution of the meanpotential within the plane of each discrete electrode is provided by a change in spacing between the wires.
An RF mass reflectron with plane discrete electrodes with variable band width was investigated numerically. Electrode system
consists of 40 elements with the following dimensions： x 0=20 mm, y0=200 mm, dy= 5 mm. Trajectories were simulated for ionsinjected with a spread in the initial energies Wmax/Wmin=3, injection angles +/- 1 o, initial coordinates xmax/xmin=1.5. The obtained
accuracy of potential distribution within mass reflectron with plane discrete electrodes corresponds to resolution 1000. Further
optimization of electrode system geometry could yield two or three times greater resolution.

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 12: On-site Mass Spectrometry -Miniaturized Instruments and Allied Technologies-

PTu-020

13:30 － 14:40

Development of Low Pressure Dielectric Barrier Discharge Ionization
Source and Vacuumed Headspace Method for Portable Mass
Spectrometer

Shun Kumano1, Masuyuki Sugiyama1, Masuyoshi Yamada1, Hidetoshi Morokuma2, Kazushige Nishimura1,
Yuichiro Hashimoto1, Hiroyuki Inoue3
Hitachi, Ltd., Tokyo, Japan, 2Hitachi High-Technologies Corp., Hitachinaka, Japan, 3National Research Institute of Police
Science, Kashiwa, Japan

1

Keywords:
Ionization_source, Headspace_sampling, Portable_mass_spectrometer, Illicit_drug_screening
Novel aspects:
High sensitive new ionization source and efficient sample introduction method were developed for portable mass spectrometer.
Abstract:
The abuse of illicit drugs has been a global problem. To prevent the abuse of the illicit drugs, accurate and reliable screenings ofthose drugs are required. Compared with the conventional immunochemical assay which has been widely used for the onsite
screening, mass spectrometers（MSs）are known to produce much more accurate results. The reason why MSs have been not
used for the onsite screening despite their potential is that the MSs are not portable due to their size and weight. Therefore, toapply MSs for the screening, we have attempted to develop a portable MS applicable to the on-site initial test. To downsize MS, asmall pump must be chosen, which limits the volume of sample gas from the sample introduction part through the ionization
source into the mass analyzer, leading to the decrease in the sensitivity. Thus, the challenge in our project was to increase thesensitivity of the portable MS
For the enhancement of the sensitivity, we have developed two new technologies. One was the low pressure dielectric barrierdischarge ionization source. Our setup consisted of a glass tube, two electrodes placed outside and inside of such tube and AC
source. By applying high AC voltage on electrodes, dielectric barrier discharge was generated inside the glass tube, leading to the
low temperature plasma. Sample molecules were ionized during passing through such plasma. We confirmed that our ionization
source worked well under 3000 Pa. Compared with the case using the ambient ionization source, it was therefore possible toenlarge the conductance between the ionization source and the mass analyzer, enhancing the efficiency of the ion transfer to thatanalyzer. Compared with one of ambient ionization, atmospheric pressure chemical ionization, by using our new ionization source,
the sensitivity of the portable MS increased more than 10 times.
The other new technology was the vacuumed headspace method. As the headspace of the sample vial is usually connected witha gas chromatograph column or an ambient ionization source, such space is pressurized to transport sample gas. On the other
hand, our ionization source was in about 3000 Pa. Thus, it was possible to decrease the pressure of the headspace. The decreasein the pressure of the headspace resulted in the increase in the ratio of the amount of sample gas to that of whole gas in theheadspace, because the saturated vapor pressure of sample did not depend on the vial pressure. This indicated that when theflow volume into the ionization source from the sample vial was unchanged between the case with decreasing the vial pressure
and that without it, the amount of the sample gas introduced into that source in the former case was larger. When the headspacepressure was decreased to 7000 Pa, the sensitivity of the portable MS increased about 10 times.
In our portable MS, two new technologies described above were incorporated with a miniaturized linear ion trap. For thesensitivity evaluation, we measured one of illicit drugs, methamphetamine, in urine. In the obtained mass spectrum, m/z 150 which
was assigned to［M+H］+ ion of methamphetamine was detected. The detection limit of our portable MS for methamphetamine
was 0.1 ppm in urine, which would be sufficient for its use in the illicit drug screening.
This work was partially supported by“R&D Program for Implementation of Anti-Crime and Anti-Terrorism Technologies for a Safeand Secure Society“, Strategic Funds for the Promotion of Science and Technology of the Ministry of Education,Culture, Sports,
Science and Technology, the Japanese Government.

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 12: On-site Mass Spectrometry -Miniaturized Instruments and Allied Technologies-

PTu-021

11:10 － 12:20

Broad range detection in a low field FTICR instrument

Joel Lemaire1, Vincent Kromer1, Gwendoline Jamet1, Clotilde Le Vot1, Essyllt Louarn1,
Michel Heninger1,2, Helene Mestdagh1
1

LCP CNRS-Universite Paris Sud, Orsay, France, 2ALYXAN, Orsay, france

Keywords:
Permanent magnet FTICR, Real time, On site detection, Air monitoring
Novel aspects:
Broad range detection in a permanent magnet based FTICR instrument.Combination of different ionization modes.
Abstract:
FTICR instruments based on permanent magnet are well suited for on site measurements. They are easily moveable and provide
both a good resolution and a time response of a few seconds. They cannot be very highly miniaturized since a magnetic field
higher than about 1 Tesla is necessary for reaching good performances and the number of ions that can be stored is related to
the ICR cell dimensions. But compared to FTICR based on supraconducting magnets with weights above 3000 kg and impossible tomove while the magnet is on field permanent magnet FTICR with a weight close to 100 kg are small.
Most high field FTICRs are not able to detect low masses, because the cyclotron frequencies become too high for the detection
electronic and also because of the limited mass range of the ion guides often used for selection and transmission of the ions.
In our low magnetic field instruments it is possible to cover the range going from m/z 2 to 500 and therefore to detect all gases
from hydrogen to high mass volatile organic compounds with a resolution allowing to distinguish compounds having the same
nominal mass but different molecular formula.
Analysis of volatile organic compounds is done using chemical ionization. Most of them will be ionized by proton transfer from
H3O+ precursor ions with little fragmentation so that only one ion will be present on the mass spectrum for each neutral present
and the analysis will rely on precise mass identification. Alternative precursors such as O2 + or CF3 + or negative ions such as OH-,O- can be used to ionize alkanes, CFCs or HCFCs. For small molecules such as CO2, NO, NO2 and for main compounds of the air
matrix electron impact or charge transfer reactions from rare gases can be used.
We will show examples going from the detection of small masses（H2 +, HD+, H3 +, D2 +, He+）to large volatile organic compounds.
As the resolution is highest for small masses the different hydrogen isotopomers can be distinguished. And the high resolution（>10000 over the whole mass spectrum）allows for the monitoring of complex mixtures with many compounds simultaneouly
present.
The excitation conditions have to be adapted in order to be able to detect the ions over a wide frequency range.
We will give examples of reactions involving both hydrogen and volatile compounds of high mass, followed as a function of timein order to determine their kinetics.

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 12: On-site Mass Spectrometry -Miniaturized Instruments and Allied Technologies-

PTu-022

13:30 － 14:40

Digital Linear Ion Trap in a Portable Mass Spectrometer

Zhengxu Huang2, Hui Mu1, Xiaoyu Meng1, Junsheng Zhang1, Lin Tao1, Gongyu Jiang1, Li Ding1, Xu Wu2,
Zhen Zhou2, Wenjian Sun1, Zhong Fu2

1
Shimadzu Research Laboratory, Shanghai, China, 2Kunshan Hexin Mass Spectrometry Technology Co., Ltd, Kunshan,
China

Keywords:
digital linear ion trap, portable instrument, VOC detection
Novel aspects:
High performance such as resolution and sensitivity was expected to be obtained.
Abstract:
A digital driven ion trap based on ceramic plate structure was used for a portable mass spectrometer for the first time, and it wascurrently mainly targeted for detecting volatile organic compounds（VOCs）in the setting of environmental monitoring.
In this device, the VOC samples were introduced into the system through a heated PDMS membrane and ionized by photoionization source in the vacuum condition. The ions were focused by a set of lens into a linear ion trap（LIT）which was fabricatedusing silver printing process on the surface of ceramic plates. The LIT was driven by digital square wave in frequency scan modewith amplitudes of 100V or 250V（ 0 -p）according to the specific mass range. A diaphragm pump and a turbo pump were used formaintaining the pressure constant at 0.05Pa using helium as carrying gas, cooling gas, and collision gas.
According to the design, a mass range of 15-800 Th can be expected to cover the major environmental VOCs. For the scan speedof 10 to 50 Hz, a peak width of 0.4Th（FWHM）can be expected. MSn can be carried out in the LIT and detailed results will beshown.
By using the digital technology the electronics and its control can be relatively simple and flexible, whereas the ceramic plate
method eliminates the need for high precision machining of hyperbolic shape electrodes while maintaining good resolution andsensitivity. The results for testing various VOCs such as BTEX, formaldehyde, acetone at ppb level will be shown to verify theperformance of the device, and application data for using the device in a practical setting will also be presented.

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 12: On-site Mass Spectrometry -Miniaturized Instruments and Allied Technologies-

PTu-023

11:10 － 12:20

Photo ionisation time-of-flight mass spectrometry as a powerful tool forthe on-line analysis of tobacco and wood combustion and pyrolysis

Christian Busch1, Thorsten Streibel1,2, Chuan Liu3, Kevin G McAdam3, Christian Radischat1,
Ralf Zimmermann1,2

1
University of Rostock / Chair of Analytical Chemistry, Rostock, Germany, 2Helmholtz Zentrum Muenchen / German
Research Centre for Environmental Health, Neuherberg, Germany, 3British American Tobacco / Group Research &Development Centre, Southampton, United Kingdom

Keywords:
Photo ionisation time-of-flight mass spectrometry, biomass, combustion, pyrolysis, PAH
Novel aspects:
The first hyphenation of a PI-TOFMS and a cigarette smoking simulator as well as a stove was utilised to reveal formation
mechanisms and pathways of health relevant compounds during combustion.
Abstract:
First, a coupling between a cigarette smoking simulator and a time-of-flight mass spectrometer was built to enable the sampling
and analysis of fresh tobacco smoke under simulated burning conditions which are closely related to the conditions of a real
burning cigarette. However, the cigarette smoking simulator also allows cigarettes to be“smoked“ apart from normal burning
conditions by a flexible control of parameters such as smouldering and puff temperatures as well as combustion rate and puffing
volume in order to allow an investigation of the compounds’ formation mechanisms and pathways. The first study of the system
included the“smoking“ of reference cigarettes（here： 3 R 4 F）under nitrogen atmosphere to separate pyrolysis from combustion
processes.
The second part addresses a measurement campaign, which was carried out at the Technology and Support Centre（TFZ,
Straubing, Germany）, concerning wood combustion in a normal stove under several normal and malfunctioning conditions, which
includes the usage of spruce and beech wood as well as the usage of artificially dried and wet wood. The sampling point of themass spectrometer was located in the raw exhaust pipe. The objective of the study was the correlation of certain burning phaseswith the formation of several health relevant compounds such as polycyclic aromatic hydrocarbons（PAH）.
Both approaches enable the direct sampling and analysis of almost unaged smoke which is a complex and dynamic matrix.
Therefore, time-of-flight mass spectrometry together with photo ionisation（SPI = single photon ionisation； REMPI = resonanceenhanced multiphoton ionisation）was applied to analyse these mixtures on-line with a high time resolution. Both photo ionisation
techniques are unable to ionise prominent bulk compounds of combustion emissions such as nitrogen, carbon dioxide or water. In
addition, REMPI is highly selective and sensitive for the detection of phenols and PAH.
The two systems demonstrate clear distinctions between the different experimental conditions based on their corresponding
mass spectra and further statistical evaluations such as principal component analysis. In the first experimental setup the yield of
nearly all compounds decreased while changing the burning atmosphere from inert to oxidative. Other compounds such as
benzene and phenol were not significantly influenced by the type of burning atmosphere. The second setup reveals that the
maximum compounds’ yields can be measured over a few minutes directly after each new load of wood and during theexperiments involving the artificially dried and the wet fuel. Partially, a prompt change from pyrolysis to combustion could beobserved by monitoring their particular marker compounds.

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 12: On-site Mass Spectrometry -Miniaturized Instruments and Allied Technologies-

PTu-024

13:30 － 14:40

Development of a multi-turn time-of-flight mass spectrometer with anatmospheric ionization

Masanobu Nakazono1, Hiroki Andoh1, Shinichi Miki2, Michisato Toyoda1,2
1Osaka University, Osaka, Japan, 2MSI. TOKYO, Chofu, Japan

Keywords:
on site real-time analysis
Novel aspects:
API-MULTUM with the particular linear ion trap was developed.
Abstract:
In recent years, on site real-time analysis by a mass spectrometer is required. To achieve on site real-time analysis, the mass
spectrometer needs to have the following specialty. First, the instrument needs to be small enough to carry. Second, high mass
resolving power is desirable to analysis without any preparation. Last, an atmospheric pressure ionization（API）source such as adirect analysis in real time（DART）is recommended. The size of a miniature multi-turn time-of-flight（TOF）mass spectrometer（MULTUM-S II）is, including vacuum system and high voltage circuit unit, 50.4 cm x 58.4 cm x 27.3 cm and the resolution of it
achieved 31,600. Therefore an API-MULTUM might achieve on site real-time analysis.
We had developed the API-MULTUM, which had commercial API ion source. Various samples, for example cold medicine, wereanalyzed and their resolution achieved 10,000. However, detection limit of this equipment was not enough. It is because theamount of detectable ions is limited by the width of MULTUM’s electric sectors when ion packets are accelerated orthogonally. It
was estimated that the amount of detectable ions was about ten millions of ions inleted to acceleration region. As a result, it isclearly that traditional orthogonal acceleration method is not befitted for the interface of an API ion source and MULTUM.
Therefore we developed a new connection method and produced the equipment consisted of miniature differential pumping
chamber, the particular linear ion trap（LIT）, a potential lift and the MULTUM.
The ions generated by DART like ionization using dielectric barrier discharge were inhaled into the equipment. Through threesteps differential pumping room, the ions were transported to the LIT by an RF ion guide system. The length from sampling orificeto the entrance of the LIT was less than 150 mm. The LIT consists of plate electrodes that are inserted between the rod electrodes.
The shape of the rod electrodes of the LIT was hyperbolic, and their radius of inscribed circle is 5.307 mm. The length of them was
27 mm. While the ions were trapping, their kinetic energy were collisionally reduced by leaked He gas. When the ions wereejected from the LIT, the RF voltage was shut off, since the RF voltage can influence the TOF spectrum. Then, applying a pulse
voltage to the plate electrodes, the trapped ions were ejected. When a positive ion packet passed through the rod electrodes, it
was accelerated through the potential difference between the center of LIT and the potential-lift whose length was 100 mm.
During the passage of the ions through the potential-lift, the voltage on the potential-lift was switched from 2.0 kV to ground
potential（ 0 V）. Their velocities were conserved when the voltage of the potential-lift was changed. The accelerated ions were
spatially focused. As a result, they could be efficiently injected the sector electrodes of the MULTUM. The injected ions wereseparated at high resolution and detected by a micro channel plate.

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 12: On-site Mass Spectrometry -Miniaturized Instruments and Allied Technologies-

PTu-025

11:10 － 12:20

Sample Gas Concentration and Pulsed Injection System for a Portable Ion
Trap Mass Spectrometer

Hyun Sik Kim1, Seung Yong Kim1, Yong Hak Kim1, Mo Yang1, Minyoung Youn1, Yong-Moon Lee2
Korea Basic Science Institute, Ochang, Chungcheongbouk-do, 363-883 Korea, 2Chungbuk National
University,Cheongju, Chungcheongbouk-do, 361-763, Korea

1

Keywords:
Sample gas concentration, Pulsed injection, Ion trap mass spectrometer, CNT adsorbent, Field portable mass spectrometer
Novel aspects:
Sample gas concentration to enhance sensitivity up to 100 times, Pulsed injection of sample gas as small as 50 nL, Application inconnection with field portable mass spectrometer.
Abstract:
A field portable mass spectrometer［ 1 ］designed for detection of trace gases in the air is studied for improvements； an energy
saving feature to cope with the power consumption for vacuum pumping, and the improvements for detection sensitivity. Because
the trace gas components only amount as low as ppm level, they are outnumbered by background air molecules. As a possible
solution to the problems, a sample gas concentration system coupled with a pulsed sample injection device was considered. Sucha system can reduce power load caused by vacuum pumping, and it can removes background air molecules to enhance thesensitivity for trace gases. Based on a number of reports［ 2 ］［ 3 ］that described the adsorption/desorption characteristics ofcarbon nano-tube（CNT）, it is possible to enhance the sample gas concentration in the air up to 100 times. Present contribution
describes a study for sample gas concentration system that consists CNT adsorbent and a pulsed sample gas injection system.
The goals of this work are；（ 1 ）to develop a versatile concentration system for various trace gas species in the air,（ 2 ）to control
the memory effect under a repeated application for various samples,（ 3 ）to develop a pulsed injection of concentrated sample
gas for a portable ion trap mass spectrometer. A CNT-metal nano-composite is used as an adsorbent that is controlled by a small
vacuum pump and a heater for sample accumulation, evacuation, and desorption processes. As small as 50 nL of concentrated
sample gas is possibly injected to the tiny ion trap mass spectrometer［ 1 ］. Experimental results with the mass spectrometer are
discussed.
References［ 1 ］. Mo Yang, Tae-Young Kim, Hyun-Chul Hwang, Seok-Kyung Yi, and Do-Hoon Kim, J. Am. Soc. Mass Spectrom., 19（2008）14421448［ 2 ］Chaudhery Mustansar Hussain, Chutarat Saridara and Somenath Mitra, Songklanakarin J. Sci. Technol., 32（ 5 ）（2010）505-511［ 3 ］. Sandeep Agnihotri, Mark J. Rood, Massoud Rostam-Abadi, Carbon 43（2005）2379-2388

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 13: Accelerator Mass SpectrometryPTu-026

13:30 － 14:40

Various erosion rates of weathered soil surfaces of mountain ridges in
Tohoku District, Japan using in-situ cosmogenic nuclides depth profile.

Kazuyo Shiroya1, Junichi Itoh1, Yusuke Yokoyama2, Hiroyuki Matsuzaki2
1

Geological Survey of Japan, AIST, Tsukuba, Japan, 2University of Tokyo

Keywords:
In-situ cosmogenic nuclides, depth profile, weathered surface erosion rate, Tohoku District Japan
Novel aspects:
Quantitative determination of surface erosion rates using 10Be and 26Al depth profiles
Abstract:
Erosion rates of landforms are important information for quantitatively understanding geomorphological processes. However, it is
not easy to quantitatively determine erosion rates because eroded materials are removed from landform surfaces. Recently,
multiple-depth profiles of in-situ produced cosmogenic nuclides（e.g., 10 Be and 26 Al）constitutea powerful tool for directly,
accurately, and quantitatively determining erosion rates of weathered soil surfaces of mountain ridges in humid areas（e.g., Shiroya
et al., 2010）. In-situ cosmogenic nuclids are produced by interactions with secondary cosmic rays（muons and neutrons）near thesurface, which decreases with depth accompanied with attenuation of secondary cosmic rays（e.g., Gosse and Phillips, 2001）.
We present erosion rates of weathered soil surfaces of mountain ridges in Tohoku District（Abukuma and Kitakami areas）,
Japan using a depth profile of in-situ produced cosmogenic 10 Be and 26 Al. Tohoku is a well-known study area for fore-arc
tectonics. However, the relationship between landform evolution and factors such as tectonics, climate and weathering has notbeen quantitatively discussed. Measurements of 10Be and 26Al in quartz are performed by AMS at the University of Tokyo. Results
show that the study sites have significantly various erosion rates. This suggests that erosion rates in Tohoku District are notuniform. In this presentation, we will discuss causes of heterogeneous erosion rates in Tohoku District（Abukuma and Kitakami）.

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 13: Accelerator Mass SpectrometryPTu-027

11:10 － 12:20

Estimation of Isotopic ratio of radioactive iodine (I-129/I-131) released
from Fukushima Daiichi NPP accident

Yasuto Miyake1, Hiroyuki Matsuzaki1, Takeshi Fujiwara1, Takumi Saito1, Takeyasu Yamagata2,
Maki Honda2, Yasuyuki Muramatsu3
1

The University of Tokyo, Tokyo, Japan, 2Nihon Universityo, Tokyo, Japan, 3Gakushuin University, Tokyo, Japan

Keywords:
Fukushima, iodine, AMS, isotopic ratio, soil
Novel aspects:
The average isotopic ratio derived from Fukushima Daiichi nuclear power plant accident was estimated to be I-129/I-131 = 36.8±9.7
as of March 15, 2011.
Abstract:
On March 2011, an accident caused by a great earthquake occurred at Fukushima Daiichi nuclear power plant（NPP）and
radionuclides were discharged. Among them, Iodine-131, which has a short half-life of 8 days, is thought to be hardly detected
several months after. It is very important to research how leaked out Iodine-131 was diffused in order to estimate the healthimpact of radiation at the time of the accident. On the other hand, Iodine-129, which was leaked out and was thought to act
chemically-identically as Iodine-131, has an extremely long half-life of 1.57×107 years and it can be measured it equally after theaccident. By following the trail of Iodine-129, it is considered to be able to estimate the distribution of Iodine-131. For this, it isessential to measure the I-129/I-131 isotopic ratio of Iodine derived from the nuclear power plant.
In this study, Iodine-129 in surface soil within 60 kilometers from Fukushima Daiichi NPP was measured. Soils were collected and
Iodine-131 level in soil had already been determined by research team of Nuclear Professional School, School of Engineering, The
University of Tokyo. We discuss Iodine-129 derived from the Fukushima Daiichi NPP by considering the I-129/I-131 ratio. Since
Iodine-129, which had been leaked out from the nuclear fuel reprocessing plant in Europe, was already transferred to Japan by
way of the atmospheric transportation before the accident, it is important to distinguish between Iodine-129 from this accident
and from the reprocessing plant. Then, we want to obtain the I-129/I-131 ratio originating in the accident precisely.
As the way of experiment, iodine was extracted from soil and mixed with a carrier and I-129/I-127 ratio was determined by AMS（Accelerator Mass Spectrometry）at MALT（Micro Analysis Laboratory, Tandem accelerator, The University of Tokyo）.Iodine-127
was measured by ICP-MS.
As a result, certain apparent correlation between I-129 and I-131 from soil was observed. Surface deposition amount of I-129 ranges
between 11.77 mBq/m2 and 6057.6mBq/m2 within the area form 3.6 km to 59.0 km distant from Fukushima Daiichi NPP. Iodine-127
concentration was distributed from 0.21 to 17.41 ppm. The average isotopic ratio was estimated to be 129I/ 131I = 36.8±9.7 as ofMarch 15, 2011.

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 13: Accelerator Mass SpectrometryPTu-028

13:30 － 14:40

Depth profile and migration of 129I concentration in soil at Abukuma,
Fukushima

Maki Honda1, Hiroyuki Matsuzaki2, Takeyasu Yamagata1, Yoko Tuchiya2, Chuichiro Nakano2,
Yuki Matsushi3, Hisao Nagai1, Yuji Maejima41
4

Nihon university, Tokyo, Japan, 2The University of Tokyo, 3Disaster Prevention Research Institute Kyoto University,
National Institute for Agro-Environmental Sciences

Keywords:
AMS, 129I concentration, Depth profile, Migration, Fukushima
Novel aspects:
In this context, 129I accidentally released from the F 1 NPP accident is the 129I spike into the environment with precisely known
source position and time and ideal tracer for this study.
Abstract:
1. Introduction
The long-lived radionuclide 129I（T1/2=1.57×107 yr, Eβ, Max=0.194MeV）is widely distributed in environment. Natural production of 129I
occurs by cosmic-ray interactions with Xenon in the atmosphere and by spontaneous fission of 238 U in the crust, which inequilibrium with 127I and the 129I/127I was estimated to be about 1.5×10-12 before 1950（J.E. Moran et al, 1998）. As human began toutilize the nuclear power, enormously large amount of anthropogenic 129I has entered into the environmental iodine cycles andelevated 129I/127I by several orders of magnitude above the 1950 levels.
To evaluate the migration of iodine in the soil, long-lived radionuclide 129I is very useful tracer. The anthropogenic 129I has been
produced only recent years（after 1950’s）so that its distribution in the environment has not yet reach equilibrium at all. Thus we
can investigate the global dynamics of iodine by observing the distribution of anthropogenic 129I. In this context, 129I accidentally
released from the Fukushima Daiichi nuclear power plant（F 1 NPP）accident is the 129I spike into the environment with precisely
known source position and time and ideal tracer for this study.
This paper reports the analytical results for 129I/127I ratio depth profile in the soil collected from the area influenced by the F 1 NPP
accident.
2. Experimental
In this study, three 30cm-soil cores were analyzed, of which sample codes are ABK2, ABK 3 and ABK12. ABK 2 and ABK 3 werecollected in the Abukuma mountain region in Fukushima prefecture about 30km distant from F 1 NPP in the South West direction.
These were collected at almost the same position in a forest but before and after the accident so that we can observe theinfluence from the accident directly by comparing ABK 2 and ABK3. ABK12 was collected at the crop field 20km apart from theF 1 NPP after the accident.
The soil sample were first dried by oven. Then homogenized well. An amount of about 0.5g was mixed with V 2O5 in a ceramic
boat and placed in a quartz tube. The sample is then heated at 1000&#8451； under a flow of oxygen gas. The evaporated iodine iscollected with a trap solution（ 2 % TMAH solution）.（Here, an aliquot was separated for ICP-MS for the determination of thestable iodine concentration.）Iodine in the trap solution with 2 mg iodine carrier is purified by a sequential solvent extraction andback extraction and a finally extracted as AgI precipitation. Extracted AgI was dried well and pressed into a cathode of AMS
system. 129I/127I was measured at MALT-AMS system, The University of Tokyo.
3. Results and Discussion
At the depth of 1.5cm, 129I/127I ratio in ABK 3 （after accident）was five times higher than ABK 2 （before accident）, which should be
the accumulation of 129 I ejected by the F 1 NPP. At the other depths, almost identical 129 I/ 127 I profiles were observed. The
maximum 129I/127I ratio in ABK 3 （1.29×10 -7）was much higher than the surface ratios reported before the accident, at most 3 ×10 -8.
ABK12 was collected from a crop field and considered to be well mixed. This is supported by the observation that the depth
profile of 127I concentration was constant and lower than the surface layer of forest. 129I/127I decreases rapidly from the surface toat 9 cm depth and becomes constant below it. We considered this enhancement in the top 9 cm layer was the direct
accumulation from the F 1 NPP after the accident. Considering constant level at lower layer （ 129 I/ 127 I=8.24×10 -9 ） as a
background, 129I inventory was estimated as 3.41×1013 atoms m-2（47.7mBq m-2）.

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 13: Accelerator Mass SpectrometryPTu-029

11:10 － 12:20

Progresses in sample treatment for BAMS analysis

Minyoung Youn1, Seung Yong Kim1, Yong Hak Kim Kim1, Hyun Sik Kim1, Jae-Gook Shin2, Ji Hong Son2,
Soo Kyung Bae2, Hyun Sic Chae3
Korea Basic Science Institute, Ochang, Chungcheongbouk-do, 363-883 Korea, 2Inje University College of Medicine,
Busan, 614-735 Korea, 3Sans Frontier Technology, Daejeon, 305-510 Korea

1

Keywords:
BAMS, Sample treatment
Novel aspects:
Improved efficiency in the treatment time. Measures elemental density of carbon component in original sample.
Abstract:
Biomedical application of AMS（BAMS）is widely used in new drug development as well as in researches. Owing to the sensitivityof AMS analysis, BAMS application fields expand. The status being as useful as it is, additional modification and improvement may
contribute to the BAMS usefulness： the time taken for pre-treatment of samples needs to become as short as possible. A
conventional pre-treatment of a sample for BAMS analysis is performed through three steps； dehydration, combustion andreduction. In this study, we merged the combustion and the reduction to happen in series. We used an element analyzer（E/A）to
combust the dehydrated samples. The dump gas of the element analyzer was put to an additional column to totally discriminate
SO2, NO 2, and H 2O. Residual CO 2 gas was introduced to a reduction reactor that was selected using a selection switch valve.
Total 5 reaction chambers were constructed to perform graphite reduction in parallel. Overall treatment time taken for
combustion and reduction ranges 2 ~ 4 hours. In addition to the progress in time-saving feature, another advantage found in thecourse is； it became possible to deduce the density in the original sample by combining the E/A analysis to BAMS analysis； thecarbon concentration in the sample can be measured using E/A, while the drug concentration in the dehydrated sample ismeasured through BAMS analysis. In this contribution, details of the device design, the construction and the experiment result arepresented.

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 14: Ion-surface Collisions: Collision-induced Dissociation and Soft Landing

PTu-030

13:30 － 14:40

Soft-sputtering of protein molecules using various gas cluster ion beams

Kousuke Moritani, Kosuke Goto, Issei Ihara, Norio Inui, Kozo Mochiji
University of Hyogo

Keywords:
Soft-sputtering, SIMS, gas cluster ion beam, Ar cluster SIMS
Novel aspects:
The novel gas cluster ion projectiles besides Ar cluster were applied to SIMS for biomolecule measurements. The effect of cluster
species on soft-sputtering will be discussed.
Abstract:
A gas cluster ion beam（GCIB）provides an extremely-low energy projectile compared to conventional polyatomic projectiles for
SIMS. Because the kinetic energy of the cluster is shared by the constituent atoms, individual atoms have small kinetic energy in acluster composed of numerous atoms. Therefore, the damage of organic molecules can be substantially suppressed. Additionally,
numerous atoms simultaneously collide. Thus, the dense energy is deposited on the surface, causing the soft-sputtering of theprotein molecules.
We have investigated the damage formation on highly orientated pyrolytic graphite（HOPG）and the sputtering of DNA molecules
adsorbed on HOPG by Ar-GCIB.［ 1 ］This study has suggested that a kinetic energy per constituent atom of cluster（Eatom）was themost important variable to the damage formation on the sample. Moreover, it has been indicated that adjusting E atom of the GCIB
can suppress damage on the sample. Motivated by these experimental results, we have developed a size-selected gas-cluster
SIMS apparatus［2,3］, where Eatom can be controlled from several tens eV to below 1 eV by selecting the cluster size and theacceleration voltage of the gas-cluster ion. On applying this instrumentation to several protein and peptide molecules, wedemonstrated that the fragmentation is substantially suppressed when Eatom was decreased below ~ 5 eV and intact ions of some
protein molecules, for example, insulin（molecular weight： 5,808）and chymotripsin（molecular weight： ~25,000）, were detectedwithout using any matrix.［4,5］We have focused on the dependence of the emission of intact ions and fragment ions on theincident direction of the cluster ions as well as accumulated dosage of the cluster ions.［ 6 ］The bombardment at a lower angle
of incidence significantly enhanced the emission of intact ions. However, the yields of intact ion should be enhanced still more forpractical use.
Although there has been much progress in the use of Ar cluster ions as projectiles for the detection of intact ions of
biomolecules, details of the role of the cluster ions in soft-sputtering and ionization, remain unknown. Recently, it is reported that
insulin molecules were ionized and emitted to vacuum without matrix by neutral SO 2 cluster collision.［ 7 ］This result suggeststhat the chemical effect in cluster collision plays a role in soft-ionization of protein in addition to the physical sputtering processes.
We have investigated the soft-sputtering of protein molecules by Ar GCIB and some other gas cluster ion beam. In thispresentation, results of the biomolecule measurement in SIMS by various gas cluster ion projectiles will be presented.
［ 1 ］K. Moritani, S. Houzumi, K. Takeshima, N. Toyoda, K. Mochiji, J. Phys. Chem. C 2008； 112, 11357.
［ 2 ］K. Moritani, M. Hashinokuchi, J. Nakagawa, T. Kashiwagi, N. Toyoda, K. Mochiji, Appl. Surf. Sci. 2008； 255, 948.
［ 3 ］M. Hashinokuchi, K. Moritani, J. Nakagawa, T. Kashiwagi, N. Toyoda, K. Mochiji, J. Surf. Anal. 2008； 14 387.
［ 4 ］K. Mochiji, M. Hahinokuchi, K. Moritani, N. Toyoda, Rapid Commun. Mass Spectrom. 2009； 23, 648.
［ 5 ］K. Mochiji, J. Anal. Bioanal. Techniques 2011, DOI：10.4172/2155- 9872.S 2 -001.
［ 6 ］S. Oshima, I. Kashihara, K Moritani, N. Inui, K. Mochiji, Rapid Commun. Mass Spectrom. 2011； 25, 1070.
［ 7 ］C.R. Gebhardt, A. Tomsic, H. Schroder, M. Durr, K. L. Kompa, Angew. Chem. Int. Ed. 2009； 48, 4162.

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 14: Ion-surface Collisions: Collision-induced Dissociation and Soft Landing

PTu-031

11:10 － 12:20

Sensitive Ionization of Non-Volatile Analytes Using Multiply Charged
Primary Ions in Desorption Electrospray Ionization Mass Spectrometry

zhiqiang zhu, Qiliang Gong, Yafei Zhou, Ning Xu, Haiwei Gu, Huanwen Chen, Tenggao Zhu

Jiangxi Key Laboratory for Mass Spectrometry and Instrumentation, East China Institute of Technology, Nanchang,
Jiangxi Province, P. R.China

Keywords:
DESI, proteins, non-volatile analytes, ambient mass spectrometry, explosives
Novel aspects:
This is a novel and deformed DESI method with higher sensitivity for the non-volatile analytes.
Abstract:
Desorption electrospray ionization（DESI）is the first and the most popular ambient ionization technique and it can analysis
complex sample without sample pretreatment. DESI-MS has been successfully applied in numerous fields. However, for many
applications, especially for trace analysis, it is of interest to improve the sensitivity of DESI-MS.
In traditional mass spectrometry DESI, a mixture methanol/water/HAc is usually used to generate the primary ions. In this article,
dilute protein solutions were electrosprayed in DESI method to create multiply charged primary ions for the desorption ionizationof trace analytes on various surfaces（e.g., filter paper, glass, Al-foil）without any sample pretreatment. The ionic compounds weredetected by the LTQ XL mass spectrometer.
Compared with the methanol/water/acetic acid（49：49：2, v/v/v）solution, protein solutions significantly increased the signal
levels of non-volatile compounds such as benzoic acid, TNT, o-toluidine, peptide and insulin. For all the analytes tested, the limit
of detection（LOD）was reduced to about half of the original values which were obtained using traditional DESI. The results
showed that the signal enhancement is highly correlated with the molecular weight of the proteins and the selected solid
surfaces.
Our provided simple and novel DESI method is a universal and useful strategy for rapid and sensitive detection of trace amountsof strongly-bound and/or nonvolatile analytes, including explosives, peptides, and proteins. The results hit that it can further
improve the sensitivity by selecting larger protein and appropriate solid surfaces.

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 14: Ion-surface Collisions: Collision-induced Dissociation and Soft Landing

PTu-032

13:30 － 14:40

The Gas Phase Mechanism of 2-Furaldehyde Formation from Pentose
Sugars: A Mass Spectrometric and Theoretical Study

Federico Pepi1, Andreina Ricci2, Simona Piccolella2, Stefania Garzoli1, Rino Ragno1,
Alexandros Patsilinakos1, Pierluigi Giacomello1
1

University of Rome Sapienza, Rome, Italy, 2Second University of Naples, Caserta, Italy

Keywords:
Biomass, Pentoses, Dehydration Mechanism, Energy Resolved Mass Spectra, Triple Quadrupole
Novel aspects:
The gas phase mechanism of pentose sugars dehydration leading to 2 -furaldehyde was clarified by tandem mass spectrometry
and theoretical calculations.
Abstract:
Mass spectrometric techniques offer the possibility to investigate the gas phase mechanism of fundamental reactions in solution
allowing to clarify controversial mechanistic hypothesis and to propose simplified and generalised reaction models not influenced
by the precence of the condensed medium and of counter ions.
It is generally known in the carbohydrates chemistry that 2 -furaldheyde is the main product of the thermal acid catalyzed
dehydration of pentoses in solution. Taking into account the Kyoto protocol and the need of reducing society dependence on
imported crude oil, the scientific research has been recently focussed toward this process as an important chemicaltransformation of biomass for the production of sustainable energy and useful chemicals［ 1 - 2 ］.
The acid catalysed decomposition of D-xylose leading to 2 -furaldehyde has been investigated in a number of studies dating backto the first years of the XIX century, but its mechanism is still controversial.
In this work we report the first study of the gas-phase decomposition of protonated pentoses（xylose, ribose and arabinose）
performed by Electrospray Triple Quadrupole Mass Spectrometry.
Gaseous ionic species of interest, generated in the ESI source and structurally characterized by CAD mass spectrometry, wereallowed to undergo collisionally induced dehydration into the triple quadrupole collision cell. Energy threshold values of theconsecutive dehydration steps were derived by the energy resolved mass spectra of the ionic intermediates and were comparedwith dehydration energy barriers obtained by theoretical calculations.
The picture emerging from experimental and theoretical data allow the mechanism of protonated pentoses dehydration in thegas-phase to be identified and a reasonable new hypothesis for the reaction in solution to be proposed.
［ 1 ］Corma, A.；Iborra, S.； Velty A. Chem. Rev.2007, 107,2411-2502.
［ 2 ］Lange, J. P.； Van der Heide, E.； Van Buijtenen, J.； Price, R. Chem Sus Chem2012, 5, 150-166.

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 15: Mass Spectrometry for Nuclear Applications and Safety

PTu-033

11:10 － 12:20

SEPARATION AND MEASUREMENT OF URANIUM,PLUTONIUM AND
NEODYMIUM IN SIMULATE SPENT NUCLEAR FUELS BY
EXTRACTIONCHROMATOGRAPHY WITH ISOTOPE DILIUTION MASS
SPECTROMETRY

Jinying Li1,2,3, Guoping Xiao2,3, Lei Shi1,2,3

China Resources New Energy Group Co.,Ltd, 2China Institute of Atomic Energy, Beijing,China;, 3Chinese MS
Society,Beijing, China

1

Keywords:
MEASUREMENT OF URANIUM,PLUTONIUM AND NEODYMIUM；SIMULATE SPENT NUCLEAR FUELS；EXTRACTIONCHROMATOGRAPHY WITH ISOTOPE DILIUTION MASS SPECTROMETRY；
Novel aspects:
Using 144Nd as isotope spike, coupled Eichrom column methodologies have been employed to measure neodymium in simulate
nuclear fuel solution by ID-MC-ICP-MS. Uncertainty of concentration measurement for neodymium is 1.5%.
Abstract:
In this work, the concentration of uranium, plutonium and neodymium in simulate spent nuclear fuel resulting from nuclear fission
products was precisely determined by means of ID-TIMS and ID-MC-ICP-MS respectively.

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 15: Mass Spectrometry for Nuclear Applications and Safety

PTu-034

13:30 － 14:40

When was gold deposited ?

Nobuo Takaoka1,6, Nobutaka Shimada2, Masako Shima3, Tomoki Nakamura4,7, Keisuke Nagao5,
Hitoshi Sagawa6,8, Ryuji Okazaki4, Kazuhiko Shimada4

Kyushu Univertsity, Fukuoka, Japan, 2Kyushu University, Fukuoka, Japan, 3National Science Musium, Tokyo, Japan,
Faculy of Sciences, Kyushu University, Fukuoka, Japan, 5Faculy of Sciences, University of Tokyo, Tokyo, Japan, 6Facultyof Sciences, Yamagata Univercity, Yamagata, Japan, 7Faculty of Sciences, Tohoku University, Sendai, Japan, 8Human
Metabolome, Tsuruoka, Japan

1
4

Keywords:
double-beta-decay of Te-130, Te-Xe age determination, hydrothermal gold deposit
Novel aspects:
The Te-Xe dating is a new technique to measure ages of minerals formed through hydrothermal events.
Abstract:
130
Te decays to 130Xe with simultaneous emission of two electrons. The decay probability is extremely small： in half-life, as long as
50 billion times the age of universe（13.7 billion years）. Based on the newly determined double-beta-decay half-life of 130Te（7.0 ±
0.9stat± 1.1syst）× 1020 yr, we have measured the direct mineralization ages on tellurium-bearing minerals from some hydrothermal
gold deposits by the 130Te-130Xe method. The results are as follows： Calaverite（AuTe2）shows the age of 21.9±2.3 million years（Ma）from the epithermal Au-Te vein, Cripple Creek mining district, USA； Hessite-petzite（Ag2Te - Ag3AuTe2）34.9±5.4 Ma fromthe epithermal Au-Te vein, Cash mine, Boulder County, Colorado, USA； Altaite（PbTe）1,040±80 Ma from the Au-Te quartzcarbonate vein of the Mattagami Lake mine, Matagami, Quebec, Canada； Tellurobismuthite-tetradymite（Bi2Te3 - Bi2Te2S）9.4±1.2
Ma from Bi-Te quartz vein, Tsushima Island, Nagasaki Prefecture, Japan； Tellurobismuthite（Bi 2 Te 3 ）81.4±2.6 Ma from thehydrothermal Au-Te vein, Oya mine, Miyagi Prefecture, Japan； Tellurobismuthite（Bi2Te3）85±18 Ma from the Au-Te quartz vein,
Suwa mine, Ibaraki Prefecture, Japan； Native tellurium（Te） 2 ± 1 Ma from the epithermal Au-Te quartz vein, Rendaiji mine,
Shizuoka Prefecture, Japan. These ages are obviously younger than previously reported mineralization ages by other dating
methods such as K-Ar, Ar-Ar or Pb-Pb methods. For example, the age of altaite（PbTe）from the Mattagami Lake mine, Matagami,
Quebec, Canada is younger by ca. 1,500 Ma than the ages determined from Pb-Pb data for host rocks. This suggests that activities
of geothermal fluids that contained gold and other rare metals such as Te occurred long after the formation of the Archean crustof the region（ca. 2,600 to 2,700 Ma）, or that the altaite vein deposited at the Archean magmatic activities was heated to blocking
temperature and reset the isotopic system for 130Xe retention.

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 15: Mass Spectrometry for Nuclear Applications and Safety

PTu-035

11:10 － 12:20

Surface Desorption Atmospheric Pressure Chemical Ionization (DAPCI)
Source based on Graphite needle

Xinglei Zhang, Saijin Xiao, Junqing Yang, Xifang Huang
East China Institute of Technology,Nanchang,China

Keywords:
DAPCI Graphite needle
Novel aspects:
Garphite needle was used as a discharge needle and an adsorption material
Abstract:
A kind of green ionization source-Surface desorption atmospheric pressure chemical ionization source based on graphite needle
which can perform rapid and direct analysis of trace compounds in complex matrices was developed by East China Institute ofTechnology funded by the National Natural Science Foundation Science Foundation.
Graphite can be used as a discharge needle and an adsorption material. On the base of this, we debug and characterize thisintelligent ionzation using representative samples such as explosives, aromatic amines in different substrate surface, then we
establish a new intelligent direct chemical ionization source which can directly analyze the trace components in complex matices.
Combining with mechanical design, electronic information processing, process engineering, computer technology, automatic
control and many other technologies, we successfully developed an intelligent surface desorption atmospheric pressure chemical
ionization source [1-3] ,which coupled to the linear ion trap mass spectrometer, so as to achieve the function of Man-machinedialogue.
The novel ionization source can be coupled with conventional mass spectrometry with high sensitivity, high specificity and themethod can perform direct detection to powder, electrocuprol, suspending liquid, aerosols, especially vivo, real-time, on-line
detection and is applied on the rapid detection of various trace compounds in different surface, such as： food, drugs, explosive,
textile, leather, plastics. Therefore, it shows promising applications such as industry, agriculture, environmental monitoring, public
security and other areas of regarding people’s livelihood.

REFERENCES［ 1 ］Chen H., Jian Zheng, Zhang X., Luo M., Wang Z., Qiao X., Surface desorption atmospheric pressure chemical ionization mass
spectrometry for direct ambient sample analysis without toxic chemical contamination［J］, JOURNAL OF MASS
SPECTROMETRY,2007, 42： 1045-1056［ 2 ］Chen H., Zheng J., Zhang X., Luo M., Wang Z., Qiao X., Surface desorption atmospheric pressure chemical ionization mass
spectrometry for direct ambient sample analysis without toxic chemical contamination［J］, J. Mass Spectrom,2007, 42： 10451056［ 3 ］Zhang C., Chen H., Guymon A. J., Wu G., Cooks R. G., Ouyang Z., Instrumentation and methods for ion and reaction
monitoring using a non-scanning rectilinear ion trap［J］, International Journal of Mass Spectrometry,2006, 1 -10

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 15: Mass Spectrometry for Nuclear Applications and Safety

PTu-036

13:30 － 14:40

Rapid 90Sr detection by MALDI-TOF-MS with a new matrix, dipicolinicacid obtained from an eastern Japan-traditional food, Natto.

Masaki Koike, Kaori Chiba-Kamoshida

National Institute of Advanced Industrial Science and Technology, Tsukuba, Japan

Keywords:
strontium, isotope, MALDI, matrix, dipicolinic acid
Novel aspects:
MALDI-tof-ms analysis of radioactive strontium（90Sr）itself and its matrix, dipicolinic acid, are new. This method is much fasterthan the conventional radioanalytical analysis and practical.
Abstract:
Since the nuclear accident in Tokyo Electronic Power Company’s Fukushima No.1 nuclear power plant, measurement of theamount of radioactive materials have been quotidian concern not only for the people inside the disaster area but also for all thepeople in Japan. Initially, the acute effect of 131I（half-life is 8 days）was worried. Still now remains the fear of the effect on ahuman body caused by radioactive materials with longer lifetimes, 134 Cs（ 2 years）, 137 Cs（30 years）and 90 Sr（28.7 years）
contained especially in foods. In case of gamma-emitting nuclide, 134Cs and 137Cs, energy-resolved gamma-ray measurement allows
us a simple preparation method for variant analytes, e.g. foods, just cutting and putting into a regular package. On the other handfor a beta-emitting nuclide, 90Sr, conventional quantitative analysis takes long time（~ 3 weeks）. Broad line width of an energy
peak for the beta-emitting nuclide has prevented an energy-resolved direct measurement for 90 Sr, which forces us a depthfulpreparation process to prepare pure 90Sr from each analyte.
Based on the circumstance, we developed an entirely new and rapid method for analyzing the radioactive 90Sr by a commercial
MALDI-tof-mass spectrometer. As a new matrix, dipicolinic acid（2,6-pyridine dicarboxylate）, was introduced. Dipicolinic acid iscontained in a Japanese traditional food, Natto. Sumi et al.1 had presented that about 1 gram of dipicolinic acid can be obtained
from 1 litter culture medium of Bacillus subtilis Natto. And dipicolinic acid has high affinity to Sr ion. Furthermore, the molecular
structure of the dipicolinic acid was similar to that of 3 -hydroxypicolinic acid, a matrix for nucleic acid measurement in negativemode, which let us predict that the dipicolinic acid is ablated by the N 2 laser equipped in the conventional MALDI-tof-mass
instrument. Thus, we attempted to use it as a matrix for MALDI-tof-mass analysis of the Sr ion. In this presentation, we will
introduce a set of basic experiments to show the efficiency of the MALDI-tof-ms Sr detection using a combination of a stable
isotope, 88Sr and a new matrix, dipicolinic acid under the condition containing impurity ions, such as K+, Ca2+, Mg2+, Ba2+, 89Y3+ an
d 90Zr4+.
For MALDI-tof-mass experiments, Shimadzu AXIMA CFR+ was used. Before the experiment, a new stainless sample plate waswashed with 1 % formic acid, methanol, acetone, and distilled water, following the recommendation in the instrument manual.
Next, the sample plate was cleaned by UV cleaner（UV DRY STRIPPER / CLEANER MODEL UV- 1 ）to decrease the organic
impurities observed in the m/z region（85-95）for Sr analysis. The way for the sample preparation was simple. A sample solution
containing 88Sr and additional impurity ions, and a matrix solution including dipicolinic acid was mixed and spotted on the MALDItof-ms sample plate and dried. Measurement was performed in reflectron-mode.
The results were as follows.
1. Using the dipicolinic acid as a matrix, stable 86Sr, 87Sr and 88Sr peaks were observed depending on its abundance ratio, evenwith the other impurity ions in the sample solution.
2. All the alkali soil metals ions（Sr2+, Ca2+, Mg2+ and Ba2+）are bound to the dipicolinic acid and observed in the mass spectra as
singly-charged ions.
3. The other ions like 89Y3+ and 90Zr4+ were not observed at all in the reflectron spectra. It is presumably because those two ionswere not selected by the matrix, dipicolinic acid.
These results show that the combination of the ion-selection with the matrix molecule, dipicolinic acid, at the stage of theionization, and the mass-selection by the mass spectrometer realizes the rapid and practical analysis of radioacrive 90Sr.
［ 1 ］Japanese patent, application No. 2006-61039（P2006-61039A）

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 15: Mass Spectrometry for Nuclear Applications and Safety

PTu-038

13:30 － 14:40

Spatially Resolved Analysis of Uranium and Plutonium Isotope Ratios inEnvironmental Samples by Laser Ablation MC-ICP-MS

Siarhei Bulyha1, Stefanie Kappel2, Thomas Prohaska2
1

International Atomic Energy Agency, 2University of Natural Resources and Life Sciences BOKU, Vienna

Keywords:
Radioactive Contamination, Isotope Analysis, Mass Spectrometry
Novel aspects:
Assessment of variation of U and Pu isotopic composition in environmental micro-samples that have been contaminated withspent reactor fuels with different burn-up grades
Abstract:
Monitoring environmental contaminations containing actinides is of special interest for the assessment of hazards for humans,
including radiation hazards, and the impact on flora and fauna.
This presentation discusses the application of a multi collector inductively coupled plasma mass spectrometer（MC-ICP-MS）that
is equipped with three ion-counting multipliers and coupled to a laser ablation system（LA）for the rapid and sensitive
determination of 235 U/ 238 U, 236 U/ 238 U, 240 Pu/ 239 Pu and 242 Pu/ 239 Pu isotope ratios in micro-samples collected in an area that had
been contaminated with spent reactor fuel in the vicinity of the Chernobyl Nuclear Power Plant（NPP）. Micro-samples withdimensions ranging from ca. 0.1 mm to 1 mm and with surface alpha activities of 3 -38 mBq were first identified using nuclear
track radiography. U and Pu isotope ratios were then measured sequentially for the same micro-sample by LA-MC-ICP-MS. The
application of a zoom ion optic for aligning the ion beams into the ion counters allows fast switching between different isotope
systems, which enables all of the above mentioned isotope ratios to be measured for the same micro-sample within a total
analysis time of 15-20 min（excluding MC-IC-PMS optimization and calibration）.
A significant variation in uranium and plutonium isotope ratios in micro-samples was observed. 242Pu/240Pu and 240Pu/239Pu isotope
ratios showed a correlation, ranging from 0.007（ 2 ）-0.047（ 8 ）and 0.183（13）-0.577（40）for 242 Pu/ 240 Pu and 240 Pu/ 239 Pu,
whereas 235 U/ 238 U and 236 U/ 238 U ratios correlated inversely. The heterogeneity of the U and Pu isotopic composition of theindividual micro-samples is attributed to the different burn-up grades of uranium in the fuel rods from which the micro-samples
originated. In the Chernobyl RBMK-1000 reactor spent fuel was gradually replaced with freshly enriched U during operation, which
resulted in different irradiation histories and thus in varying burn-up grades of the fuel assemblies over the reactor core at the timeof the accident. Higher fuel burn-up results in generation of more 236U and Pu isotopes and therefore in higher 236U/238U, 242Pu/240P
u and 240Pu/239Pu isotope ratios. The measured Pu isotopic signatures were compared to the Pu isotopic composition distributionthat is typical for a RBMK-1000 reactor with initial 235U enrichment of about 2 %. A comparison of the measured Pu isotope ratios
in Chernobyl soils with Pu isotope ratios in RBMK spent-fuel yielded a good consistency of burn-up grades ranging from about 5
to 12 MWd/kg（U）.

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 16: New Approaches to Defining the Diversity of Glycans

PTu-039

11:10 － 12:20

MALDI MS analysis of N-glycan structures of a cell adhesion molecule,
CADM1, in various cancer cells

Tomoko Maruyama1, Mika Sakurai-Yageta1, Kaoru Kaneshiro2, Sadanori Skiya2, Shinichi Iwamoto2,
Koichi Tanaka2, Yoshinori Murakami1
1

The Institute of Medical Science, The University of Tokyo, Tokyo,Japan, 2Shimadzu Corporation, Kyoto, Japan

Keywords:
N-glycan, MALDI-MS, 3 -AQ-labeling, cancer, cell adhesion
Novel aspects:
Different N-glycan structures of a tumor related cell adhesion molecule, CADM1, was identified for the first time in various cancer
cells with high sensitivity by 3 -AQ-labeling and MALDI MS analysis.
Abstract:[Introduction] CADM 1 （Cell adhesion molecule 1 ）is a membrane glycoprotein whose expression is frequently down-regulated
in the progression of various cancers derived from epithelial cells, including the lung, kidney and breast（ 1 ）. On the other hand,
CADM 1 is overexpressed in adult T-cell leukemia（ATL）and small cell lung cancer（SCLC）, and promotes their invasion. The
molecular weight of CADM 1 was different in cancer cells possibly by tissue-specific alternative splicing and/or by different
glycosylation. In the extracellular domain（EC）of CADM1, 6 predicted N-glycosylation sites are present in 3 immunogloblin（Ig）-like loops（N 1 - N 6 ）. Here, we analyzed the structures of N-glycans of CADM 1 expressed in epithelial cells as well as those incancer cells such as renal cell cancers（RCC）, ATL and SCLC using glycopeptides and 3 -AQ-labeled N-glycans by MALDI mass
spectrometry.
[Method] CADM 1 -EC fragment was expressed in HEK293 cells as a fusion protein with Fc domain of mouse IgG（CADM 1 -EC-Fc）
and the secreted protein was purified using protein A beads. CADM 1 expressed in various cancer cells were purified byimmunoprecipitation using a specific antibody against the cytoplasmic domain of CADM 1 and separated by SDS-PAGE. To obtainglycopeptides, in-gel trypsin digestion of CADM 1 -EC-Fc was then performed and the digested polypeptides were dissolved in aliquid matrix, 3 -AQ/CHCA. For 3 -AQ-labeling of N-glycans, CADM 1 -EC-Fc or immunoprecipitated CADM 1 was in-gel digested
with PNGaseF and released N-glycans were labeled with 3 -AQ/CHCA（ 2 ）. The mass spectra were acquired using MALDI-DIT or
MALDI-QIT-TOF（SHIMADZU Corp.）.
[Results] We initially performed MADLI-DIT analysis of desialylated CADM 1 -EC-Fc glycopeptides, and identified tetra-antennarycomplex type of N-glycans at N2, N3, N 5 and N 6 by MS and MS2. By 3 -AQ-labeled N-glycan profiling, the same structure with asialic acid was mainly detected in N-glycans in CADM 1 -EC-Fc. The N-glycan profiling of CADM 1 in RCC, ATL and SCLC cells
suggests that cancer cells displayed heterogeneity in N-glycans of CADM1, and that the structures of N-glycans predominantly
seen in ATL/SCLC were distinct from that in epithelial cells. Different glycosylation patterns of CADM 1 in epithelial and ATL/SCLC
cells could be one of the molecular bases of distinct roles of CADM 1 in tumor suppression in epitheila and in cell invasion in ATL/
SCLC, and could provide possible molecular targets for the diagnosis and treatment of these malignant diseases.
Reference
1 ）Y. Murakami, Cancer Sci. 96, 543-52（2005）.
2 ）K. Kaneshiro, Anal Chem. 83, 3663- 7 （2011）.

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 16: New Approaches to Defining the Diversity of Glycans

PTu-040

13:30 － 14:40

Isolation and identification of glycopeptides with isomeric glycans bylectin-column chromatography and negative-ion MALDI-QIT-TOF MSn

Kazuko Hirose, Junko Amano

Laboratory of Glycobiology, The Noguchi Institute, Tokyo, Japan

Keywords:
Glycopeptide, MALDI, CID, Negative-ion, Pyrene derivatization
Novel aspects:
We proposed the useful method for analysis of glycan isomers on peptides, by combination of the lectin-column chromatography
and the on-plate PDAM derivatization followed by negative-ion MALDI-MSn.
Abstract:
Glycosylation is the most common post-translational modifications of proteins, which may have profound effects on protein
structure and function. Glycoproteins are heterogeneous with respect to their glycan structures. Various glycans can be attachedto the same position on the protein backbone. A distinct glycan structure depends on a particular site and it is referred to as sitespecific glycosylation. In fact, analysis of glycans is more complicated and harder than genomics or proteomics because glycans
have branched structures and the diversity of monosaccharides. In addition to the structural diversity, glycans on the proteinsshow dynamic changes in the course of development, differentiation, metabolic changes, malignancy, inflammation, or infection.
The structural changes should be associated with functional changes of the glycoproteins. Therefore, we have to know the
information of the alteration of glycan structure and the site of glycosylation on the protein as well as the protein. For this reason,
we have investigated glycopeptides without releasing glycans.
Mass spectrometry（MS）is a powerful tool for glycoproteomics. Generally, however, glycoprotein analysis using MS is not easycompared to proteins. There are some limitations that the hydrophilic nature of glycopeptides is disadvantageous for ionization,and the amount of each glycopeptide is smaller than nonglycosylated peptides because of heterogeneity. A step of enrichment ofglycopeptides is necessary. To discriminate glycan isomers, and also to concentrate the specific glycopeptides, lectin-column
chromatography is helpful for MS analysis of glycan structures on the proteins. Lectins are proteins, which specifically recognize
glycan structures. Therefore, the specific binding of lectins to glycan structures is used to characterize glycoconjugates.
Regarding sensitivity, conventional proteomics was difficult to detect altered glycosylation on proteins. Recently, our group has
established an on-plate pyrene derivatization method using 1 -pyrenyldiazomethane（PDAM）for highly sensitive matrix-assisted
laser desorption/ionization tandem mass spectrometry（MALDI-MSn）of glycopeptides in amounts of less than 100 fmol（J. Amanoet al., Anal. Chem., 82, 8738-8743（2010））. Collision-induced dissociation（CID）is effective for glycans structural determination. In
glycans analysis, negative-ion MSn spectra derived from such ions contain many fragment types（B and Y, C and Z, and A）and
therefore are more informative than the positive-ion MSn spectra derived from such ions usually consist mainly of B and Y fragment
types. In particular the internal ions（D- and E-type ions）provided useful information about the branching patterns of glycans.
In this study, we attempted to discriminate isomers of tri-antennary N-glycopeptides（NA 3 peptides）obtained from alpha 1 -acidglycoprotein or alpha 1 -antichymotrypsin. First, NA 3 peptides were separated from other glycopeptides and enriched by aPhaseolus vulgaris agglutinin（PHA）-L4-agarose column. PHA-L4 is a lectin that recognizes beta-6,2-GlcNAc branch of N-glycan andthe glycan can be separated from other isomers. Next, the enriched NA 3 peptides were treated with the on-plate PDAM
derivatization for highly sensitive MALDI-MS n analysis. The negative-ion CID of PDAM-derivatized NA 3 peptides showedcharacteristic spectral patterns. In addition, isomeric NA 3 on peptides could be discriminated based on their the negative-ion
MS3 spectra by selecting A ion as a precursor ion. The NA 3 containing beta-2,4,2-GlcNAc branch gave an abundant E-type ion（m/z 831）. The structure-depend fragment ion helped to identify NA 3 isomer. These data made it possible to discriminate beta-2,4,2and beta-6,2,2-GlcNAc branch of NA 3 on the specific glycosylation site.
Thus, we proposed the useful method for analysis of glycan isomers on peptides, by combination of the lectin-column
chromatography and the on-plate PDAM derivatization followed by negative-ion MALDI-MSn.

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 16: New Approaches to Defining the Diversity of Glycans

PTu-041

11:10 － 12:20

A workflow for identification of isobaric isoforms of glycans using off-line
MALDI-MSn system

Shuuichi Nakaya, Yuzo Yamazaki
Shimadzu Corporation, Kyoto, Japan

Keywords:
MALDI, MSn, Glycomics, Glycan
Novel aspects:
We provide a workflow for glycomics researches using offline LC-MALDI and improved software for identifying isobaric isoforms ofglycans.
Abstract:
Nowadays, analysis of glycosylation is one of the indispensable approaches for a development of antibody-drug and biomarker
discovery, because a large number of proteins in eukaryotes are glycosylated and they play various roles in physiological function
like a molecular recognizing. Therefore, a well-established workflow for characterization of glycosylation has been a one of thegrowing demands. The author and co-workers has reported a system for identification of glycan structures using an observational
MSn spectral library obtained by MALDI-QIT-TOF MS1),2). We demonstrates a practical workflow combined with the spectral libraryand off-line separation system for the glycan structure characterization.
In this study, glycans released from proteins with N-glycosidase were pyridylaminated and purified by cellulose and graphite
carbon tip. A small aliquot of a purified glycan mixture was subjected to MALDI MS analysis directly and a list of candidate
precursor ions was generated by the library search system, which is revised to correspond to various chemical labeling methods（Accurate Glycan Analyzer 2 ； AGA 2 / Shimadzu Corp.）. The remaining sample was provided to a nano HPLC for the
separation of isomers and eluted glycans were automatically fractionated onto a MALDI plate by a spotter（Accuspot； Shimadzu
Corp.）. Then, a Mass spectrum corresponding to each fraction time was obtained automatically, and the intensity maps of themass values as a set of candidates for next MSn analysis were created. Finally, MSn analysis was performed semi-automatically on
all the found precursors and isomer structures were identified by AGA2.
We applied the workflow to human immunoglobulin and erythropoietin, and obtained successful results.
The result of these glycoproteins indicates that our practical workflow could be applicable to multiple purposes including anbiomarker discovery, quality assurance study of antibody-drug, and other screening researches of glycosylation.

References：
1 ）A. Kameyama et.al. Anal. Chem., 77, 4719-4725（2005）
2 ）A. Kameyama et.al. J. Proteome. Res., 5, 808-814（2006）

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 16: New Approaches to Defining the Diversity of Glycans

PTu-042

13:30 － 14:40

Negative-ion fragmentation of neutral N-glycans derivatized with
3-aminoquinoline and other non-acidic reagents

Takashi Nishikaze, Kaoru Kaneshiro, Shin-Ichirou Kawabata, Koichi Tanaka

Koichi Tanaka Laboratory of Advanced Science and Technology, Shimadzu Corporation, Kyoto, Japan

Keywords:
MALDI, N-Glycan, On-target 3 AQ derivatization, Negative-ion mode, Fragmentation
Novel aspects:
The negative-ion fragmentation behavior of deprotonated or anion adducted N-glycans derivatized with non-acidic reagents
depends on the structure of the reducing-terminal GlcNAc residue（opened or closed）and the anions used.
Abstract:<Introduction>
Tandem mass spectrometry combined with collision-induced dissociation（CID）has been widely used to obtain structural
information of glycans. In general, positive-ion CID usually leads to cleavages of glycosidic bonds thereby providing information
simply on sequence and composition of N-glycans. Negative-ion fragmentation of underivatized N-glycans, on the other hand, hasbeen proven to be more informative as it defines structural features such as the specific composition of the two antennae, thelocation of fucose, and the presence or absence of the bisecting N-acetylglucosamine（GlcNAc）residue. For glycan analysis,
derivatization via reductive amination is often employed, but little is known about the influence of derivatization on negative-ion
fragmentation. In our previous study, we have developed an on-target derivatization method for highly sensitive detection ofglycans using 3 -aminoquinoline/alpha-cyano- 4 -hydroxycinnamic acid（ 3 AQ/CHCA）liquid matrix［Anal. Chem. 83（2011）36633667］. In this study, we evaluated the suitability of on-target 3 AQ-derivatized N-glycans for the structural characterization basedon negative-ion fragmentation. In addition, the fragmentation behavior of 3 AQ-derivatized N-glycans was compared with
N-glycans derivatized with other non-acidic reagents.
<Methods>
For on-target 3 AQ derivatization, underivatized N-glycan was mixed with 3 AQ/CHCA liquid matrix on a MALDI target, and thetarget was then left for 60min at 60 o C on a heating block. 2 -Aminobenzamide（ 2 AB）and 2 -pyridilamine（ 2 PA）derivatizedN-glycans were mixed with G3CA liquid matrix, which consists of p-coumaric acid and 1,1,3,3- tetramethylguanidine. Mass spectra
were acquired using a MALDI-QIT-TOF-MS（AXIMA-Resonance, Shimadzu/Kratos, UK）.
<Results and Discussion>
In this study, derivatization with acidic reagents such as 2 -aminobenzoic acid（ 2 AA）was omitted from consideration. It has been
indicated that negative-ion CID spectra of 2 AA derivatives lack many important“diagnostic“ product ions due to the localizationof the negative charge on the acidic group of the derivative. Firstly, we compared negative-ion low-energy CID spectra ofunderivatized, 2 AB-, 2 PA-, and on-target 3 AQ-derivatized NA 2 glycans using a MALDI ion trap instrument. Negative-ion MS2 of
on-target 3 AQ-derivatized N-glycans exhibited simple and informative spectra similar to those of underivatized N-glycans. It
showed product ions by cross ring cleavages of the chitobiose core and ions specific to two antennae, D and E ions. It was shownthat an interpretation of diagnostic product ions suggested for underivatized N-glycans could be directly applicable to on-target
3 AQ-derivatized N-glycans. On the other hand, conventional 2 AB- and 2 PA-derivatized N-glycans gave complicated spectra
which consist of extensive signals formed by dehydrations and multiple cleavages. These unfavorable product ions could be
eliminated by omitting the reduction process, which is involved in the conventional derivatization method via reductive amination,
resulting in spectra similar to that obtained from underivatized N-glycans. The difference in fragmentation behavior seemed tocome from the structural difference of the reducing-terminal GlcNAc ring（closed or opened）, rather than the structure of theaglycone moiety of derivatized N-glycan. On-target 2 AB or 2 PA derivatization approach was investigated as an alternative for the
on-target 3 AQ derivatization. However, complete derivatization was difficult, and ion yields were significantly low. There was noadvantage over the on-target 3 AQ derivatization. The fragmentation behavior of anionic adducts of 3 AQ-derivatized N-glycan
was also investigated and it was found that phosphate adduct shows important diagnostic fragments of N-glycans with highsensitivity. In conclusion, on-target 3 AQ derivatization using phosphate doped 3 AQ/CHCA liquid matrix was found to be suitablefor both sensitive detection and structural characterization of N-glycans based on negative-ion CID fragmentation.

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 17: Non-Covalent Ion–Molecule Interactions

PTu-043

11:10 － 12:20

Atomic metal anions: one-step dehydrogenation and defluoronation of
linear and cyclic hydrocarbons, alcohols and their fluoronated analogues

Paul M Mayer, Alex Mungham, Jeffery Butson, Jaleh Halvachizadeh, Dhiya Hassan, Sharon Curtis
University of Ottawa, Ottawa, Canada

Keywords:
metal, anions, dehydrogenation, defluorination, ligand
Novel aspects:
This work is the first extensive examination of the reactions of atomic metal anions, and demonstrates new reactions withalcohols, alkanes and fluorinated systems.
Abstract:
The generation and reactivity of gas-phase metal cations has been a focus of mass spectrometry related studies for quite sometime. This has mostly been related to adduct formation and their involvement as reagents in chemical ionization processes. Our
research group has recently discovered a way to generate a wide variety of metal anions（Na-, K-, Rb-, Cs-, Fe-, Ni-, Co-, Cu-, Ag-）
using a simple commercial electrospray mass spectrometer. The metal anions were all generated by the decomposition of metal
cation carboxylate complexes. Owing to the previous difficulties associated with generating these unique species, the idea ofreactive gas phase atomic metal anions has been relatively untouched. We will first present the results of theoretical calculations
on the precursor metal-oxalate anions with a view to understanding the formation of the metal anion upon CID of the complex.
The performance of a wide selection of methods was assessed and a robust computational approach to these systems wasestablished. Clear from the calculations is that the situation for the transition metal anions is complicated by several low-lying
electronic states, making the computational estimate of their properties a multi-determinant problem. None-the-less, evidence isseen for the insertion of metal atoms into the C-C bond in the oxalate dianion, a reaction that leaves the metal already primarily
negatively charged. In some cases, the calculated structure is essentially a MCO2 anion interacting with a neutral CO2 molecule atsome distance. Experimentally, this study investigates the previously unexplored reactivity of atomic metal anions produced in thesource of a triple quadrupole mass spectrometer with several volatile organic molecules. One class consisted of the reaction of
Fe- with the primary alcohols methanol through heptanol, secondary alcohols 2 -propanol and 2 butanol and the tertiary alcoholt-butanol. Dehydrogenation of the alcohols（except for methanol）was demonstrated through the observation of FeH 2 - in the
resulting mass spectrum. Labelling studies showed that this dehydrogenation takes place in the reaction of Fe - with a single
alcohol molecule, and not in the two-step process previously observed for metal carbonyl anions such as Fe（CO）2 -. In the caseof the smaller alcohols methanol, ethanol and propanol, FeOR - and FeOH - anions are observed. Fe - was also observed todehydrogenate alkanes in a similar manner. Theory has been applied to chart the mechanism of the dehydrogenation reaction
and the results will be presented in conjunction with rate constant measurements on an ion trap mass spectrometer. Metal anions
have also been allowed to react with fluorinated ring-systems such as per-fluorophenol and hexafluorobenzene. Here there isevidence for competitive deprotonation of the phenol OH group and F+ abstraction from the ring.

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 17: Non-Covalent Ion–Molecule Interactions

PTu-044

13:30 － 14:40

Energetics of dissociation of polycyclic aromatic hydrocarbons ininterstellar environments

Brandi J West1, Christine Joblin2,3, Valerie Blanchet3, Andras Bodi4, Balint Sztaray5, Paul M Mayer1
1
4

University of Ottawa, Ottawa, Canada, 2IRAP/CNRS, Toulouse, France, 3Universite de Toulouse UPS, Toulouse, France,
Paul Scherrer Institut, Villigen, Switzerland, 5University of the Pacific, Stockton, USA

Keywords:
RRKM interstellar ISM PAH energetics
Novel aspects:
Using Tandem MS and iPEPICO to accurately determine key bond strengths related to the interaction of H and H 2 with small
PAHs of interstellar interest
Abstract:
Recently, great interest has evolved in the chemistry of the interstellar medium（ISM）； especially the unidentified IR bands（UIRs）.
UIRs, observed in a variety of environments, are believed to derive from neutral and ionic large polycyclic aromatic hydrocarbons（PAH）and their fragments. If they are responsible for UIRs, then PAHs would account for up to 20% of the organic carbon foundin the ISM.
We are investigating small PAHs（naphthalene（NAP）, anthracene（ANT）, phenanthrene（PHE）and pyrene（PYR））as well assome dihydro- equivalents（1,2-dihydronapthalene（DHN）, 9,10-dihydroanthracene（DHA）and 9,10-dihydrophenanthrene（DHP））
using imaging photo-electron photo-ion coincidence spectroscopy（iPEPICO）and electron impact mass spectrometry（MS）； both
mass analyzed ion kinetic energy spectrometry（MIKES）and collision induced dissociation（CID）. Experiments were performed at
different ionization energies to produce breakdown diagrams for the various fragments. These diagrams are then fit using RRKM
theory to determine the zero Kelvin activation energy（E0）and the entropy of activation（ΔS）； these results are then comparedand discussed.
To date, all iPEPICO and MIKES results have been collected and the branching ratios have been measured in order to constructtheir breakdown curves. Looking at the fitted values of E0 for the molecules it can be seen that there are notable trends whenlooking at the PAHs, and when comparing them to the dihydro-PAHs.
H loss is the dominant fragment for all molecules studied. For PAHs E0 values are 4.20 +/- 0.04 eV and 3.92 eV and ΔS  are 2 +/- 2
JK-1mol-1, -0.03 JK-1mol-1 for NAP and ANT respectively. This indicates that as PAHs increase in size, there is a decrease in the E 0. Δ
S values close to zero shows that the fragments are formed through bond cleavage.
H loss from dihydro-PAHs is more complicated, with two fragmentation channels resulting in the same fragment. Both DHN and
DHA show a change in entropy； which is indicative of a different fragmentation pathway. For DHN, the two sets of values for H
loss are； E 0 values of 2.34 eV and 2.22 eV and ΔS  values of - 6 JK-1mol-1and -16 JK-1mol-1 for low and high energy respectively. Thischange has also been observed in DHA, which is still being investigated.
Another dominant fragment for PAHs is ethyne loss. The E0 and ΔS values for； NAP are 4.12 +/- 0.05 eV and 2 +/- 2 JK-1mol-1,
and for ANT are 3.82 eV and 4 JK-1mol-1. E0 again shows the inverse relationship to increasing size while there is little difference
between ΔS.
The organic fragment for the dihydro-PAHs is methyl loss. The E0 and ΔS values for DHN are 2.00 eV and -36 JK-1mol-1, and for DHA
are 1.72 eV and -30 JK-1mol-1. A large degree of rearrangement occurs during the dissociation； this can be partially explained by
the formation of the methyl fragment, but it is also possible that the resulting ion has a significantly different structure from theparent ion. This is still being investigated.
NAP also loses H2, both concertedly and consecutively as 2 H. E0 of the two pathways are； 4.77 +/- 0.062 eV for concerted loss
and 4.20 +/- 0.037 eV and 3.26 +/- 0.099 eV（giving E0total = 7.46 +/- 0.11 eV）for consecutive loss. This would indicate that it is morefavourable for both hydrogen atoms to be lost simultaneously, which would be a good indication of the loss of H 2. When ΔS iscompared, 12 +/- 4 JK-1mol-1 for concerted versus -18 +/- 7 JK-1mol-1 for the loss of the second hydrogen of the consecutive loss.
This also shows that the concerted loss is more favourable.

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 17: Non-Covalent Ion–Molecule Interactions

PTu-045

11:10 － 12:20

Gas Phase Acid-Base Chemistry of Cysteine and Serine Homologs

John C Poutsma, Corbin Muetterties, Vincent Yannello, Ashley Janiga
College of William and Mary, WIlliamsburg, VA, USA

Keywords:
kinetic method, amino acid
Novel aspects:
First experimental study of the gas phase acid base properties of homologs of cysteine and serine.
Abstract:
The gas-phase proton affinities and gas-phase acidities of extended homologs of cysteine（Cys）and serine（Ser）have beenobtained using the extended kinetic method in an ESI-triple quadrupole instrument. Previous photoelectron spectroscopy andhigh-level theoretical calculations have indicated that the preferred site of deprotonation for cysteine is the side chain thiol rather
than the carboxylic acid moiety. This arises from differential hydrogen bonding ability between the side chain S-H group and thecarboxylic acid O-H group. We have been interested in the relationship between the structure of amino acids and their acid-baseproperties. To this end, we have been determining gas-phase thermochemical properties of non-protein amino acid analogs of the
20 common amino acids. Previous studies from our lab on a series of lysine homologs showed that the proton affinity is highly
dependent on the length on the side chain due to difference in intramolecular hydrogen bonding capabilities. In contrast theacidities of these species do not show a clear trend with decreasing side chain length. They do however have very different
intramlolecular hydrogen bonding interactions. We anticipated that lengthening the side chain in cysteine homologs would affectthe relative stability of the different hydrogen bonding schemes.
We have measured the gas-phase acidity of two longer homologs, homocysteine（hCys）and 5 -mercapto-norvaline（hhCys）using
the extended kinetic method in an ESI triple-quadrupole instrument. In addition, we have carried out high-level hybrid density
functional theory calculations（B 3 LYP/ 6 -311++G**//B 3 LYP/ 6 -31+G*）on the gas phase acidity of the these two homologs andfind that a）the absolute acidity of hCys is the same（1397 kJ/mol）as that of cysteine（1396 kJ/mol）, b）that the COOH group inhcys is slightly more acidic than the thiol group（1.5 kJ/mol）, c）the acidity of hhCys is less than that of Cys and hCys（1408 kJ/mol）and d）the COOH moiety is the preferred site of deprotonation for hhCys. Proton affinities for these species have also beenmeasured and are in excellent agreement with those calculated at theB 3 LYP/ 6 -311++G**//B 3 LYP/ 6 -31+G* level of theory. We
find that the proton affinity increases from 903 kJ/mol for Cys to 934 kJ/mol for hCys to 943 kJ/mol for hhCys consistent withincreasing intramolecular hydrogen bonding ability for the longer chain homologs. Similar experiments and theoretical calculations
will be presented for homoserine（hSer）and 5 -hydroxynorvaline（hhSer）.

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 17: Non-Covalent Ion–Molecule Interactions

PTu-046

13:30 － 14:40

Mass spectrometric analysis of 1,3,5-Trinitroperhydro-1,3,5-triazine (RDX)

Sehwan Park1, Soo Gyeong Cho2, Eun Mee Goh2, Sungman Lee3, Sung-Suk Koh3, Jeongkwon Kim1
1
3

Chungnam National University, Daejeon, South Korea, 2Agency for Defense Development, Daejeon, South Korea,
Sensor Tech Inc., Kyunggi-Do, South Korea

Keywords:
RDX, Ion trap, ESI-MS, Explosive
Novel aspects:
Mass spectrometric analysis of RDX using ESI-MS to investigate adduct ions of RDX, such as RDX + formate, RDX + acetate, andRDX + benzoate
Abstract:
1,3,5-Trinitroperhydro-1,3,5-triazine（RDX）is one of the most widely used explosives. In this study, RDX was directly infused into anelectrospray ionization mass spectrometry（ESI-MS）system in negative ion mode. The RDX stock sample was prepared in aconcentration of 1 mg/mL in acetonitrile（ACN）. Then, the RDX stock sample was diluted in ACN（100-fold dilution）prior to theanalysis. Direct infusion of the diluted RDX sample provided an adduct ion of RDX + HCOO - where HCOOH is believed to beoriginated from the decomposition of RDX. In positive ion mode, no RDX signal was observed in the current direct infusion ESI-MS
analysis. The RDX samples spiked with acetic acid or benzoic acid were also analyzed, which provide an adduct ion of RDX +C2H4O2 -（m/z 280.9）, RDX + C7H6CO2 -（m/z 343.1）. Analysis of RDX spiked with ammonium acetate also generated an adduct ionof RDX + C2H4O2 -（m/z 281.1）. The detailed experimental procedures and results will be provided during the presentation.

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 17: Non-Covalent Ion–Molecule Interactions

PTu-047

11:10 － 12:20

Formation of hydrated amino acid ions and estimation of their firsthydrated shell

Mami Sakai, Kanako Sekimoto, Mitsuo Takayama
Yokohama City University, Yokohama, Japan

Keywords:
amino acid, first hydrated shell, collision-induced dissociation, ambient ionization
Novel aspects:
First hydrated shells for pos/neg phenylalanine ions were estimated by using the breakdown graphs obtained from CID spectra ofhydrated phenylalanine ions.
Abstract:
Introduction
Many of the hydrogen bonds between water and individual amino acids lead to the native state of protein along the folding
pathway. In recent years, investigating the hydrated structure of amino acids has become an interesting subject. High-pressure
mass spectrometry is one of the ideal techniques for evaluating the enthalpy H for sequential hydration reaction and investigating
the stability of hydrated amino acid ions. However, especially deprotonated amino acids have never been studied and only small
water cluster can be measured by high-pressure mass spectrometry. Here we attempted to make an ambient ionization system
for the formation of both positive and negative ions of phenylalanine and those hydrated clusters. Furthermore, the ambient
ionization system coupled with a triple-quadrupole mass spectrometer was combined to collision-induced dissociation（CID）
experiments to estimate the water molecule detachment energy of protonated and deprotonated phenylalanine, and for
estimating its first hydrated shell.
Methods
Mass spectra were acquired on a triple-quadrupole mass spectrometer TSQ-7000（Thermo-Fisher-Scientific, San Jose, CA）.
Ambient ion source was constructed from a needle（ca. 1 μm in the tip curvature radius）for micro-plasma ionization and a microceramic heater for rapid vaporization. The gap between the needle and the orifice plate of mass spectrometer was 3 mm, andthe needle angle to the axis of orifice hole was varied in the range of 0 -π/2. DC voltages applied to the needle were -2.2kV and+3.4kV. The discharge was performed in the laboratory air with relative humidity of 28 % -65 % at 298 K and 760 torr. The orificetemperature was varied in the range of 333-373K. The pressure of argon used for CID was 1.5mTorr.
Results
The micro ceramic heater placed in the laboratory air became feasible to vaporize a powder of amino acids putted on theceramic plate, i.e., Jv > 0 in the expression for MS ionization Ji = I Jv. The resulting amino acid vapor diffused into a micro-plasma
region was positively and negatively ionized by ion/molecule reactions in ambient air, i.e., I >0. The resulting atmospheric ions
H3O+ and O2 -, originated from ambient air, were used for ion/molecule reaction as reactant ions. Positive and negative ion mass
spectra of phenylalanine（Phe）at the orifice temperature of 353K and a humidity of 60 % showed peaks corresponding to+（H（Hprotonated and deprotonated molecules and their water cluster series［Phe+H］
2 O）
n（n= 0 -18, m/z 166-490）and［Phe-H］
2 O）
n（n= 0 -21, m/z 164-542）, respectively. The CID spectra of the individual precursor ions［Phe±H］±（H2O）m were obtained, and thebreakdown graphs for precursor ions were drawn on the basis of the CID spectra. The detachment energy En,n-1 of one watermolecule from hydrated cluster ions［Phe±H］±（H2O）n under the CID conditions were estimated from the breakdown graphs. In+（H
the case of the precursor ion［Phe+H］
2 O）
15 , the detachment energy E n,n-1（n = 1 - 8 ）could be estimated and the discontinuous+（H
gap in detachment energy between E4,3 and E5,4 was observed. The result obtained suggested that［Phe+H］
2 O）
4 was morestable than larger cluster size above n = 5. Furthermore, the average values of E n,n-1 obtained by five precursor ions+（H
［Phe+H］
2 O）
13-17 was linearly correlated with that in reliable H n-1,n values obtained with a high-pressure mass spectrometry. In（H
the case of the precursor ion［Phe-H］
2 O）
14 , the detachment energy E n,n-1（n = 1 - 7 ）could be estimated and the discontinuous+（H
gap in detachment energy between E3,2 and E4,3 was observed. This result suggested that［Phe+H］
2 O）
3 was more stable thanlarge cluster size above n = 4.

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 17: Non-Covalent Ion–Molecule Interactions

PTu-048

13:30 － 14:40

Mass Spectrometry Studies of Metal Triflates and Triflimides and Their
Interactions with Lewis Bases

Jean-Francois Gal1, Ilaria Monfardini1, Claudio Iacobucci1, Lionel Massi1, Sandra Olivero1,
Elisabet Dunach1, Kalev Kasemets2, Peeter Burk2
1

University of Nice-Sophia Antipolis, Nice, France, 2University of Tartu, Tartu, Estonia

Keywords:
Triflates； Triflimides； Catalysts； Lewis acids； ESI-MS.
Novel aspects:
Mass spectrometric characterization by ESI-MS of metal triflates and triflimides； Ligand competition method； Quantitative
parameter for the interaction between ligand and Lewis acids.
Abstract:
Metal trifluoromethanesulfonates（triflates M（CF3SO3）n）and bis（trifluoromethanesulfonyl）imide salts（triflimides
M［（CF 3 SO 2 ）2 N］n ）are regarded as Lewis“superacids“. This feature is attributed to the specific electronic structure of theanions： a highly delocalized negative charge, and a resulting low coordination power, leaving a highly reactive cation. Such salts
have remarkable catalytic properties.[1]Exploration of new preparation routes of these salts is an active field, and methods for their thorough characterization aredesirable. Under specific conditions（nitromethane as solvent, addition of strong neutral ligands）, electrospray ionization mass
spectrometry（ESI-MS）and collision induced dissociation（CID）can afford useful analytical data on these salts.[2] In this method,
the formation of positive ions is based on the displacement of one anion in the salt by neutral Lewis bases, such as phosphoryl
compounds or amides. For example, the bonding of two molecules of triphenylphosphine oxide to indium triflate produces asingly charged positive ion by expulsion of one anion from the salt：+（Ph
In（CF3SO3）3 + 2 Ph3PO -->［In（CF3SO3）23 PO）
2 ］ + CF 3 SO 3
This kind of adduct ion is often the base peak in the ESI（+）mass spectrum of mixtures of the phosphine oxide and metal triflates
in nitromethane. Similar ions were observed in the Fast Atom Bombardment mass spectra of rare earth triflates adducts withdifferent strong Lewis bases.[3] From these observations, with the aim of ranking the ligand affinities for various metal centers, acompetition method using mixtures of two different ligands was developed. The relative intensities of the ionic species containing
the different combinations of ligands generated an order of ligand relative affinities for several metal salts.[4]This semi-quantitative method was recently improved. Modeling the effect of the ligand concentrations on the signal intensities
led us to propose a quantitative parameter describing the relative affinity for the metal center.[5]Quantum chemical calculations using DFT are currently carried out, in an attempt to better understand the meaning of theseaffinities.
In this communication, these ESI-MS results and the current quantitative developments of the ligand competition method will bepresented.
References［1］（a）Kobayashi, S.； Sugiura, M.； Kitagawa, H.； Lam, W. W. L.； Rare-Earth Metal Triflates in Organic Synthesis； Chem. Rev.,
2002, 102, 2227-2302；（b）Antoniotti, S.； Dalla, V.； Dunach, E.； Metal Triflimidates： Better than Metal Triflates as Catalysts in
Organic Synthesis - The Effect of a Highly Delocalized Counteranion； Angew. Chem. Int. Ed.2010, 49： 7860-7888；（c）Gal, J.-F.；
Iacobucci, C.； Monfardini, I.； Massi, L.； Dunach, E.； Olivero, S.； Metal triflates and triflimides as Lewis“superacids“：
preparation, synthetic application and affinity tests by mass spectrometry； J. Phys. Org. Chem,submitted.
［ 2 ］Monfardini, I.； Massi, L.； Tremel, P.； Hauville, A.； Olivero, S.； Dunach, E.； Gal, J.-F.； Mass spectrometric characterizationof metal triflates and triflimides（Lewis superacids catalysts）by electrospray ionization and tandem mass spectrometry； Rapid
Commun. Mass Spectrom. 2010, 24, 26112619.
［ 3 ］Tsuruta, H.； Yamaguchi, K.； Imamoto, T.； Tandem mass spectrometric analysis of rare earth（III）complexes： evaluation of
the relative strength of their Lewis acidity； Tetrahedron, 2003, 59, 10419-10438.
［ 4 ］Monfardini, I.； Massi, L.； Dunach, E.； Olivero, S.； Gal, J.-F.； Relative basicities toward metal triflates Lewis acids byelectrospray mass spectrometry； Chem. Commun., 2010, 46, 8472-8474.
［ 5 ］Gal, J.-F.； Iacobucci, C.； Monfardini, I.； Massi, L.； Dunach, E.； Olivero, S.； A Quantitative Approach of the Interaction
between Metal Triflates and Organic Ligands Using Electrospray Mass Spectrometry； ChemPhysChem, submitted.

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 17: Non-Covalent Ion–Molecule Interactions

PTu-049

11:10 － 12:20

Mass spectrometric approaches to enantioselective complexation system
in solution

Motohiro Shizuma1, Hirofumi Sato1, Takashi Nakakoji2, Hideya Kawasaki2, Kaori Asano3,
Tsuyoshi Matsuzaki3, Takeyuki Suzuki3, Keiji Hirose4, Yoshito Tobe4, Ryuichi Arakawa2, Daisuke Ono1
1
4

Osaka Municipal Tech. Res. Inst., Osaka, Japan, 2Kansai University, Osaka, Japan, 3ISIR, Osaka University, Osaka, Japan,
Graduate School of Engineering Science, Osaka University, Osaka, Japan

Keywords:
Chirality, Complexation, Isotope-Labeling, Enantiomer
Novel aspects:
Effects of ionization method such as FAB, ESI and MALDI on enantioselective complexation equilibrium system were clarified using
the deuterium-labeled enantiomer method, comparing with that in solution evaluated by NMR.
Abstract:
Enantioselective complexation equilibrium system in solution, in which a chiral crown ether（enantiopure）binds to an enantiomersof chiral ammonium ions, was examined using FAB, ESI and liquid-MALDI mass spectrometry coupling with the enantiomer labeled
guest method, referring to the results of NMR analysis of the solution state. The chiral crown ether（enantiopure, H）binds toammonium ion of isopropyl（R）-D-phenylalanate（G R + ）at K R = 2 M -1 and（S）-L-phenylalanate（G S + ）at K S = 1 M -1 indichloromethane-d2/chroloform-d（v/v = 1 / 2 ）at 298 K, respectively. The concentration ratio of the resulting complexes changes
theoretically depending on the association constants（KR and KS）, the initial concentration of H, GR + and GS +.
In order to observe the corresponding complex ions in mass spectrometry, three component solution including an equimolar
mixture of deuterium-labeled（d7）GS + and unlabeled GR +, and H was used, and the resulting diastereomeric complex ions,（H +GR）+ and（H + GS-d7）+, were monitored in mass spectrometry and the relative peak intensity of the complex ions was comparedwith the concentration ratio in solution.
The chiral crown ether was synthesized as reported before. Deuterium-labeled（S）-phenylalanine isopropyl-d7 ester hydrochloride
was prepared by esterification of amino acid with deuterated isopropyl alcohol（d7 ）. Unlabeled（R）-phenylalanine isopropyl
ester hydrochloride was synthesized using isopropyl alcohol.
Sample solutions were prepared by mixing H solution and equimolar solution of GR + Cl - /G S-d7 + Cl - .［G R + ］0 =［G S-d7 + ］0 were theconstant concentration conditions and only［H］0 was changed in each ionization mass spectrometric experiment.
In FAB mass spectrometry（matrix： 3 -nitrobenzyl alcohol）, the relative peak intensity of the complex ions,（H + G R）+ and（H +GS-d7）+, changed in the same manner of the concentration ratio of the complexes in solution. On the other hand, in ESI（methanol/
chloroform）and liquid-MALDI（tetra（n-butyl）ammonium salts）mass spectrometry, the changes of the relative peak intensity of
the complex ions against the initial concentrations of H, G R + and GS-d7 + were not in agreement with those of the concentration
ratio of the complexes in solution.

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 17: Non-Covalent Ion–Molecule Interactions

PTu-050

13:30 － 14:40

Characteristic hydration of saccharides isolated from liquid beams

Namiki Toyama

Genesis Research Institute, Inc., Chiba, Japan

Keywords:
saccharide, hydration, hydrogen bond, liquid beam, IR-laser ionization
Novel aspects:
Hydration structure of conformational isomers of saccharides was distinguished by distribution of the number of water molecules
in a cluster ion produced from an aqueous solution of the saccharide.
Abstract:
Functions and properties of saccharides have been widely studied because of their importance in biology, chemistry andpharmacology. The nature of the saccharides is influenced by interaction with surrounding water molecules, which form ahydrogen-bond network not only in a bulk solution but also in a binding site of a sugar binding protein in vivo. Inter- and intramolecular hydrogen-bond interactions of the saccharides in aqueous solutions have been investigated by means of spectroscopic
and thermodynamic measurements. The complexity of the hydrogen-bond structure is enhanced by structural variation andfluctuation of the saccharide molecules in the solution.
Microscopic information on the hydrogen-bond interaction in hydrated molecules has been obtained by an isolation of clusters
and molecules from solutions into vacuum. A soft ionization method has been developed by utilizing a lamellar liquid flow in thevacuum（liquid beam）in combination with an IR laser ablation technique［ 1 - 3 ］. We have studied isolation of various neutral
molecules from a liquid beam of aqueous solutions and proposed a mechanism for ejection of molecular species from thesolutions underirradiation of an IR laser resonant to an OH stretching vibration of liquid water［4,5］. We have reported
aggregation of arginine zwitterions and their hydrophobic hydration structure［ 4 ］and proton transfer between weakly hydrogenbonding water molecules in liquid water induced by an IR-multiphoton excitation［ 5 ］.
Here, I report about formation and characterization of hydrogen bonded cluster ions produced from a liquid beam of an aqueous
solution of saccharides（M）under irradiation of an IR laser on the liquid beam. Hydrated D-glucose cluster ions, H 3O+Mm（H2O）n+and OH-Mm（H2O）n, were observed with the hydrated oxonium and hydroxide ions, H 3O（H2 O）
n and OH（H 2 O）
n , in time-of-flightmass spectra for the sample solution of 1 -100 mM D-glucose. Ion intensity of the cluster ions was analyzed as a function ofconcentration of D-glucose in the source solution. Increase in the ratio of the D-glucose cluster ions was found with the+concentration of D-glucose, whereas the ratio of H 3O（H2 O）
n and OH（H 2 O）
n decreased and the total intensity of the ions wasconstant. On the basis of the result, I propose a reaction between glucose molecules and a ion-pair, H3O+---OH-, produced by an
OH-vibrational excitation of a water molecule in an aqueous solution of D-glucose. The product ions, H 3 O + M and OH - M, areejected from the liquid beam with accompanied neutral molecules surrounding the ion core.
This ionization method was applied to investigate effects of the OH group conformation on hydration of monosaccharides. The
number of water molecules（n）in the cluster ion has been compared for the conformational isomers of D-glucose, D-mannoseand D-galactose, which are different at conformation of OH（ 2 ）and OH（ 4 ）groups from that of D-glucose, respectively. The
distribution of n is distinguished in the galactose cluster ions from other monosaccharides for both of oxonium and hydroxide ion
adducts. The difference of the n distribution in the cluster ions is interpreted as a difference in anisotropy of hydration for themonosaccharides.
1. A. Charvat, E. Lugovoj, M. Faubel, B. Abel, Eur. Phys. J. D 20（2002）573
2. W. Kleinekofort, J. Avdiev, B. Brutschy, Int. J. Mass Spectr. 152（1996）135
3. J. Kohno, N. Toyama, M. A. Buntine, F. Mafun, T. Kondow, Chem. Phys. Letters, 420（2005）18
4. N. Toyama, J. Kohno, F. Mafun, T. Kondow, Chem. Phys. Letters, 419（2006）369
5. N. Toyama, J. Kohno, T. Kondow, Chem. Phys. Letters, 420（2006）77

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 17: Non-Covalent Ion–Molecule Interactions

PTu-051

11:10 － 12:20

Evaluation of intermolecular association of glycosaminoglycan
oligosaccharides using nanoelectrospray ionization mass spectrometry

Yuya Otsuka, Toshikazu Minamisawa
Seikagaku corporation, Tokyo, Japan

Keywords:
Glycosaminoglycan, Oligosaccharide, Non-covalent complex
Novel aspects:
Non-covalent intermolecular association between glycosaminoglycan oligosaccharides could be evaluated quantitatively using
nanoelectrospray ionization MS. Structural requirement for efficient association could be discussed.
Abstract:
Considerable interests have recently been paid for mass spectrometric evaluation of non-covalent interactions between
biomolecules. Several studies have successfully demonstrated the potential of MS to analyze non-covalent complexes generating
in solution. An outstanding example is the use of cold-spray ionization（CSI）-MS, which is powerful to detect weakly-associatedcomplex or cluster consisting of nucleic acids, amino acids, steroids, and so on.1)
The challenge of the present study is to observe glycan-glycan interactions quantitatively, especially focusing on
glycosaminoglycans（GAGs）. GAG is widely distributed in vertebrate tissues and body fluids, and it is well known to play crucial
roles in many biological phenomena such as regulation of growth factors or cytokines, cell-cell recognition, and infection, throughintermolecular association.
According to the past literature, some specific interactions were suggested to occur between hyaluronan（HA）and chondroitin
sulfate（CS）, both of which are representative GAGs2). However, no evidence has been shown so far to confirm direct binding of
HA with CS.
A nanoelectrospray ionization MS was employed to analyze associations between HA tetrasaccharide and other structuralvariations of mono- and oligosaccharides. Each combination of saccharide samples was dissolved in water and diluted withmethanol at 50 % before MS analysis. MS experiments were performed on LCMS-IT-TOF system（Shimadzu）with negative ion
mode. Interaction strength of each saccharide combination was estimated by the required concentration of formamide added into
the sample solution to dissociate the non-covalent complex. A successful result of this study was observation of the complex of
HA and CS tetrasaccharides, suggesting HA can interact with CS directly in solution. It was also revealed that the affinity of eachsaccharide to HA tetrasaccharide was different depending on the saccharide length, or substitution of hydroxyl group. Based onthese data, the present method was considered applicable to evaluate non-covalent interaction between GAG oligosaccharides.
Our study will also provide useful information to propose a novel saccharide structure to achieve much potent association.

References
1 ）K. Yamaguchi. BUNSEKI KAGAKU. 59, 985-1001（2010）2 ）Turley A. E. et al., Nature. 283, 268-271（1980）.

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 17: Non-Covalent Ion–Molecule Interactions

PTu-052

13:30 － 14:40

Enantioselective binding characteristics of amino acid to chiral copper(II)complex using electrospray ionization mass spectrometry coupling withthe deuterium-labeled method

Takashi Nakakoji1, Hirofumi Sato2, Hiroyuki Miyake3, Hideya Kawasaki1, Ryuichi Arakawa1,
Daisuke Ono2, Motohiro Shizuma2

Kansai University, Osaka, Japan, 2Osaka Municipal Technical Research Institute, Osaka, Japan, 3Osaka City University,
Osaka, Japan

1

Keywords:
Enantioselective coordination, Amino acid, Chiral ligand, Chiral-discriminating ability, Metal complex
Novel aspects:
We prepared chiral ligand/metal complexes to discriminate enantiomers of amino acids, and the chiral discrimination ability of themetal/chiral ligand complex toward amino acid evaluated by using ESIMS.
Abstract:
Mass spectrometry is one of the powerful and useful tools for clarifying the coordination of metal complexes in organometallic
chemistry field. We have studied an enantioselective coordination of chiral compound to chiral organometallic complex usingmass spectrometry. Recently, we found that amino acids enantiodifferentiatingly formed the three component complex with chiralligand/metal complex and the chiral-discriminating ability toward amino acid was evaluated using electrospray ionization mass
spectrometry（ESIMS）coupling with the deuterium-labeled enantiomer method.
In this research, we designed novel chiral tetradentate ligands, of which backbone were ethylenediamine N,N’-diacetic acid. Twotypes of the ligands derived from S-alanine in four steps. These differed in terms of different carbonyl terminal structure of
S-alanine,（S,S）L1 and（S,S）L2,（CH3XCOC*HCH3N（CH3）CH2-）2［X = O, L1； X = NH, L2］.
Copper（II）chloride was chosen as center metal on the basis of pre-examination using mass spectrometry. R-amino acids（R-AA）（alanine, valine, leucine, methionine, phenylalanine）were purchased. n represents
and deuterium-labeled S-amino acids（S-AA-dn）
the number of deuterium atoms.
Sample solutions were prepared by mixing copper（II）chloride, ligand（L）, and equimolar mixture of R-AA/S-AA-dn in methanol,and the concentration was adjusted at 100 mM. Mass spectra of the sample solutions were measured with a Finnigan
LCQDECA（Thermo Quest）equipped with ESI source. The operating conditions were set as follows： spray voltage, 5 kV； capillarytemperature, 200 ºC； sheath gas flow rate, 30 arbitrary unit. Sample solutions were injected at a flow rate of 3 mL/min.
In all measured mass spectra, the complex ions,［Cu/（L）/（R-AA）］+ and［Cu/（L）/（S-AA-dn）］+, were detected successfully.
The relative peak intensity of the complex ions,（I［Cu/（L）/（R-AA）］+ /I［Cu/（L）/（S-AA-d n ）］+ ： I R /I S value）, enhanced asincreasing the content of the amino acid componentin the prepared sample solution, and eventually reached a constant value
under the condition of more than 4 equivalent amino acid mixture for［Cu/（L）］. As the IR/IS value may reflect the concentration
ratio of the complex ion in the solution, the IR/IS value would be reasonably regarded as an index of chiral discrimination ability ofthe［Cu/（L）］toward the amino acid. When the mass spectra were measured for the given amino acids under the ratio（［R-AA/
S-AA-dn］/［Cu/（L）］= 1 / 4 ）, the IR/IS value were larger than 1.00. Thus,［Cu/（L）］preferred R-amino acids in forming the threecomponent complex.
The stoichiometry and structure of complexes were clarified using UV-vis, CD, and NMR spectroscopy in detail. As a result, thechiral-discriminating of the complexes toward amino acids would be primarily occurred due to steric hindrance between thecoordinating ligand and amino acid.

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 17: Non-Covalent Ion–Molecule Interactions

PTu-053

11:10 － 12:20

MECHANISTIC INVESTIGATION OF ECO-FRIENDLY IODINATION OF
AROMATIC DERIVATIVES BY MASS SPECTROMETRY

Diogo Oliveira-Silva, Andre F Rodrigues-Oliveira, Marcelo Serigatti, Cristiano Raminelli
Universidade Federal de Sao Paulo - Campus Diadema, Diadema, Brazil

Keywords:
iodination, mechanism, phenol
Novel aspects:
Mechanistic elucidation of a well established aromatic iodination reaction.
Abstract:
Aromatic iodinated compounds have been used as valuable chemicals in pharmaceutical and agricultural chemistry and as
building blocks in organic synthesis, especially for C-C and C-N bond forming reactions.1  ,  2 Some iodoarenes are also biologically
active molecules used in medicine as drugs and diagnostic aids as radioactively labeled markers or contrasts.3
Among the approaches to synthesize halogenated aromatic compounds, the iodination promoted by iodide or iodine in
combination with an oxidizer has stood out. 4  ,  5 Following the growing concern over environmental pollution and sustainable
development,“greener“ iodination synthetic methods were recently developed, in particular aerobic oxidative procedures.3
Although several protocols have been published regarding aqueous-medium for aromatic iodination, there is a lack of mechanistic
evidences about this functionalization.
In this contribution we present the preliminary results of a mass spectrometry systematic mechanistic investigation of eco-friendly
phenol iodination.
The protocol explored as study model was recently communicated as an efficient iodination method of phenols and otheraromatic derivatives, employing iodine（I 2）and hydrogen peroxide（H2O2 30 %）in water.6 Briefly, we treated phenol（ 1 mmol）
with I2（ 2 mmol）and H2O2 30%（ 4 mmol）in water（ 5 mL）at 50 oC for 24 h（obtained 2,4,6-triiodophenol, yield about 80%）. To
evidence the product ion of proposed iodine reactive species, blank reactions（without phenol）were also carried out in the same
conditions（I2/H2O2/Water and temperature）. Samples were collected at the beginning of the reaction and after 1, 3 and 18 h.
Each sample was immediately analyzed by ESI-MS on a LC-MS8030 Triple-Quad mass spectrometer（Shimadzu）.The MS scan（m/z= 100 - 700）and product ion scan（m/z = 50 - 700）analyses were performed in positive and negative modes. The source
parameters were screened to avoid any influence in detected species.
For the best of our knowledge all studies involving oxidative iodination protocols in aqueous medium have considered theiodonium ion（I +）as the key intermediate for the electrophilic aromatic substitution. However, in our preliminary studies no I +related species was found by MS experiments. Additionally, we could detect several iodide containing structures（I-, I3 -,［I-H2O］and［I-H 2 O 2 ］- ）. These findings are partially in agreement with some proposed reaction pathways described in the literature.
Further investigations are been conducted in order to find other involved intermediates.
Acknowledgments： FUNDECT（MS）and SHIMADZU DO BRASIL.
1 - Hanson, J. R. J. Chem. Res.-S2006, 277.
2 - Kandepi, V.； Narender, N. Synthesis2012, 44, 15.
3 - Carniti, P.； Colonna, S.； Gervasini, A. Catal. Lett.2010, 137, 55.
4 - Stavber, S.； Jereb, M.； Zupan, M. Synthesis2008, 1487.
5 - Gallo, R. D. C.； Gebara, K. S.； Muzzi, R. M.； Raminelli, C. J. Braz. Chem. Soc. 2010, 21, 770.
6 - Gallo, R.D. C.； Ferreira, I. M.； Casagrande, G. A.； Pizzuti, L.； Raminelli, C. In 14th BMOS, Brazil, 2011.

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 17: Non-Covalent Ion–Molecule Interactions

PTu-054

13:30 － 14:40

Comparing the dissociation energies of non-covalent complexes measured
with BIRD and RRKM modelling of CID

Justin B Renaud1, Lan Liu2, John Klassen2, Paul M Mayer1
1

University of Ottawa, 2University of Alberta

Keywords:
non-covalent complex, travelling wave ion mobility, CID, RRKM, BIRD
Novel aspects:
The effects of ion mobility upon dissociation energy measurements is explored in depth for a number of different and biologically
relevant complexes
Abstract:
Several flavours of tandem MS dissociative methods are commonly used to investigate non-covalent complex stability in the gas
phase, however the mechanisms can be quite different and therefore can give rise to different results. CID spectra at various
collision energies were obtained for a number of non-covalent complexes on a Waters G 2 Synapt mass spectrometer. Complexes
included a short chain-variable fragment antibody with several related tri-saccharides, β-Lactoglobulin with fatty acids and ahelical polysaccharides with fatty acids. A theoretical breakdown energy model based on RRKM theory was matched toexperimental observations, yielding the 0 K activation energy（E 0) and the entropy of activation. All systems studied havepreviously been analyzed using blackbody infrared radiative dissociation（BIRD）techniques, allowing for the relative stability
rankings obtained using these 2 dissociative mechanisms to be compared.
The effects of ion mobility upon the internal energy distributions of the reacting non-covalent complexes is also explored,
specifically in regards to the pressure of Helium, which is shown to have a drastic cooling effect upon the complexes.

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 17: Non-Covalent Ion–Molecule Interactions

PTu-055

11:10 － 12:20

Molecular recognition in gas phase: a method for quantitative analysis ofprotein-ligand interactions energy by CID-MS.

Andrey Dyachenko1, Sergio Madurga1,2, Ernest Giralt1,21Institute for Research in Biomedicine, Barcelona, Spain, 2Department of Organic Chemistry, Universidad de Barcelona,
Barcelona, Spain

Keywords:
Native mass spectrometry, energy transfer model, non-covalent interactions, protein-ligand interactions, molecular recognition
Novel aspects:
New model for calculation of gas-phase binding energy using CID mass spectrometry.
Abstract:
Molecular recognition is a process crucial for the majority of cellular functions. Better understanding of factors that play key rolesin the interactions of biomolecules can provide means for broadening our knowledge about living organisms and developing new
potent drugs. Two such major key-factors are the dynamics of the interacting molecules［ 1 ］and their interplay with aqueous
environment［ 2 ］. The latter will be addressed here, both experimentally and theoretically. Vascular endothelial growth factor（VEGF）, being an important therapeutical target related to the suppression of the tumor growth, was used as a model protein
for this study.
Mass spectrometry provides a perfect opportunity to address the question about the role of the solvent in protein-ligand
interactions because it allows studying molecules in the absence of their aqueous environment. For the present study, the libraryof 5 cyclicpeptidic ligands was prepared. Their affinities to VEGF in solution were determined with NMR chemical shift
perturbation（CSP）technique. Then VEGF-ligand complexes were analyzed using the Electrospray ionizationquadrupole time-of
flight（ESI-qTOF）mass spectrometer using collision induced dissociation（CID）in order to establish the ranking of the complexes
according to their stability in gas-phase. It turned out that rankings of ligands in solution and gas phase are mutually opposite, i.e.
the peptide with lowest solution affinity to VEGF forms the strongest complex in the gas phase, and vice versa. After calorimetricstudy of the system it was concluded that the enthalpic component of Gibbs energy of binding, that plays dominant role instability of the noncovalent complex in gas phase, grows with decrease of affinity in solution, thus allowing the weakest ligand toform the strongest complex in gas phase［ 2 ］.
To be able to compare directly the enthalpy binding and gas-phase binding energy a method is needed to calculate the latterfrom experimental data. Unfortunately, to-date there is no method to quantify gas-phase stabilities of noncovalent protein-ligand
complexes from CID data. We propose a model that uses readily available properties of the interacting molecules, such as
collision cross section or density of energy states, as input data, and allows modeling the“slow heating“ of molecular complex
during the CID［ 3 ］. The model takes into account that both excitation and relaxation of the system can take place upon collision
with buffer gas molecule. The model can predict the amount of kinetic energy of the complex that is transferred to the internalenergy under the certain conditions（buffer gas type, pressure, path length etc.）If the calculation was done under conditions,
under which 50 percent of the complex are decomposed in the gas phase, the result of calculation can be taken as a gas phasestability of the complex. The model was verified with molecular dynamics simulations.

References
1. A. V. Smrcka, N. Kichik, T. Tarragó,a M. Burroughs, M. S. Park, N. K. Itoga, H. A. Stern, B. M. Willardson and E. Giralt. NMR analysisof G-protein βγ subunit complexes reveals a dynamic Gα-Gβγ subunit interface and multiple protein recognition modes. Proc.
Natl.Acad.Sci.USA, 107, 639-44（2010）
2. A. Dyachenko, M. Goldflam, M. Vilaseca, E. Giralt, Molecular Recognition at Protein Surface in Solution and Gas Phase： Five
VEGF Peptidic Ligands Show Inverse Affinity When Studied by NMR and CID-MS, Biopolymers（Pept Sci）94： 689700, 2010
3. A. Dyachenko, S. Madurga, E.Giral, Unpublished results

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 17: Non-Covalent Ion–Molecule Interactions

PTu-056

13:30 － 14:40

Gas-phase basicity of 2-furaldehyde

Simona Piccolella1, Andreina Ricci1, Federico Pepi2, Alexandros Patsilinakos2, Rino Ragno2,
Stefania Garzoli2, Pierluigi Giacomello2
1

Second University of Naples, Caserta, Italy, 2’Sapienza’ University of Rome, Rome, Italy

Keywords:
2 -furaldehyde, extended kinetic method, proton affinity, gas-phase basicity, mass spectrometry
Novel aspects:
This study represents the first estimate of the gas-phase basicity of 2 -furaldehyde, determined by the extended Cooks’s kinetic
method from ESI-TQ tandem mass spectrometric experiments along with theoretical calculations.
Abstract:
2 -Furaldehyde（ 2 -FA）, also known as furfural or 2 -furancarboxaldehyde, is an heterocyclic aldehyde that can be obtained fromthe thermal dehydration of pentose monosaccharides. This molecule can be considered as an important sustainable intermediate
for the preparation of a great variety of chemicals, pharmaceuticals and furan-based polymers. Although the great importance of
this molecule, its gas-phase basicity（GB）has never been measured. In this work the GB of 2 -furaldehyde was determined by theextended Cooks’s kinetic method from ESI-TQ tandem mass spectrometric experiments along with theoretical calculations. As
expected, computational results identify the aldehydic oxygen atom of 2 -FA as the preferred protonation site. The geometries of
O-O-cis and O-O-trans 2 -furaldehyde and of their six different protomers were calculated at the B 3 LYP/aug-TZV（d,p）level oftheory； PA values were also calculated at the G 3 （MP2, CCSD（T））level of theory. From the experimental proton affinity（PA）（115.09 ± 10 J mol-1）of 2 -FA a GB value of 813.6 ± 2 kJ mol-1 at 298 K and a（847.9 ± 5 kJ mol-1）and protonation entropy（ΔpS）
ΔH°f value of  533.0 ± 6 kJ mol-1 for protonated 2 -furaldehyde were derived.

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 17: Non-Covalent Ion–Molecule Interactions

PTu-057

11:10 － 12:20

Non-covalent interaction between peptides and its effect on the
dissociation process of Bradykinin Molecule

Chuan-Fan Ding, Chen Chen, Xiaodan He
Fudan University, Shanghai, China

Keywords:
Non-covalent Interaction； Peptides； Conformation； Collision-induced dissociation mechanism； Mass spectrometry.
Novel aspects:
The non-covalent interaction between peptides and the conformation mechanism of Bradykinin, and also the effect of noncovalent interaction on the collision-induced dissociation process of protein molecules.
Abstract:
The study of the gas-phase property of non-covalent complexes by the mass spectrometer is very important since it allows the
exploration of non-covalent interactions in the absence of solvation effects. It permits a better understanding of the protein
conformation and recognition mechanism1-5.
The structure and conformation of proteins determine their biological functions. The interaction between peptides within a protein
molecule is one of the major factors that influence its structure and conformation. In this work, Bradykinin（RPPGFSPFR）was
chosen as a model to investigate the non-covalent interaction between peptides within the amino acid chain.
For investigating the possible non-covalent interaction between peptides of a Bradykinin molecule, several peptides which havethe same amino acid sequence were synthesized. In the first fracture mode, we synthesized two peptides RS- 6 （RPPGFS）and
PR- 3 （PFR）according to our requirement； In the second fracture mode, two peptides RF- 5 （RPPGF）and SR- 4 （SPFR）were
obtained. By removing the arginine at N-terminal or C-terminal, we designed and synthesized another four kinds of polypeptide,
including PS- 5 （PPGFS）, PF- 2 （PF）, PF- 4 （PPGF）and SF- 3 （SPF）. In our experiments, any two kinds of these eight
polypeptides  were mixed in a molar ratio of 1 ：1, respectively, in 90/10 methanol/water solution and then reacted at room
temperature for 12 hours.
The evaluation of the non-covalent interaction by mass spectrometric experiments was performed using Thermo Finnigan LTQ ion
trap mass spectrometer equipped with an electrospray ionization source. In the experiments, the electrospray capillary voltage
was set to 3.5kV and the capillary temperature was maintained at 350&#8451；. The sample was introduced via a syringe pump at
a flow rate of 3 µL/min.
Electrospray ionization mass spectrometry（ESI-MS）results revealed that the two peptides obtained in each mode can form noncovalent complex. In the first fracture mode, RS- 6 and PR-3, PS- 5 and PR- 3 can form 1 ： 1 complexes, while there is not any noncovalent binding between peptides RS- 6 and PF-2,PS- 5 and FP-2. And in the second fracture mode, there is always 1 ： 1 noncovalent binding formed between two peptides, RF- 5 and SR-4, PF- 4 and SR-4,RF- 5 and SF-3,PF- 4 and SF-3, no matter whether
the N-terminal or C-terminal arginine was removed.
The non-covalent binding between two peptides was further confirmed by collision-induced dissociation（CID）in a tandem mass
spectrometer. It is demonstrated that the binding strength of RS- 6 + PR- 3 and RF- 5 + SR- 4 complexes is much stronger thanother peptide complexes. These results are in accordance with the conformation of Bradykinin.
This study provides important and reliable information for the non-covalent interaction between peptides and the conformation
mechanism of Bradykinin, and also the effect of non-covalent interaction on the collision-induced dissociation process of protein
molecules.

Reference
1. Chu,Y.Q.； Dai, X.H.； Jiang, D.； Jiang, G.Y.； Fang, X.； Ding, C.Y.； Rapid Commun. Mass Spectrom., 2010, 24, 2255
2. Loo, J. A. Mass Spectrom. Rev.1997, 16, 1
3. Syka, J. E. P.； Coon, J. J.； Schroeder, M. J.； Shabanowitz, J.； Hunt, D. F.； PNAS 2004, 101, 26, 9528
4. Lee J.； Jayathilaka L. P.； Gupta S.； Huang J. S. ； Lee B. S. J. Am. Soc. Mass Spectrom, 2012,23, 942
5. Wang Q.； Chu Y.Q.； Zhang K.； Dai X.H.； Fang X.； Ding C.F.； Acta Phys. Chim. Sin.2012, 28（ 4 ）, 971

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 18: Advances in Resolution and Accuracy of Isotope Ratio Analyses

PTu-058

13:30 － 14:40

A continuous flow system for quantitatively preparation and isotopic
measurement of radiogenic argon.

Tatiana Velivetckaia, Aleksandr Ignatev, Sergey Budnitskiy

Far East Geological Institute FEB Russian Academy of Sciences, Vladivostok, Russia

Keywords:
isotope ratio mass spectrometer
Novel aspects:
new method for measurement of 36Ar and 40Ar isotopes for potassium argon（K/Ar）geochronology
Abstract:
Isotope composition of noble gas is widely used for geochemical and geochronological studies. Argon isotope ratios aremeasured with specialized mass spectrometers. Some new models of this technique have been produced in recent years. Having
high-technology achievements for measurement isotope ratios of ultra-small quantity noble gas, these technique keeps significant
requirement for vacuum system to achieve extreme high vacuum and measurements of isotope ratios in static mode. Maintenanceof running conditions is a challenging task. Preparation systems for liberate argon from samples with the extremely low blank arenot simple.
Here we present a new method for measurement of 36Ar and 40Ar isotopes for potassium argon（K/Ar）geochronology that canbe realize with conventional isotope ratio mass spectrometer operating in a dynamic mode. We also present a laser preparation
system where the extraction and cleaning argon is performed in a continuous flow（CF）of helium. The preparation system is
attached to a mass spectrometer（MS）via open-split mode. The CF-MS method is an alternative to measurement of argon
isotopes in a static mode and can be used for the analysis of radiogenic argon of nx10-12 g with the same sensitivity and accuracy
as a static mode. The method of argon isotope measurement in a continuous flow is simpler and more reliable compared to ameasurement method operating in a static mode.
CF-MS system includes a mass spectrometer MAT-253（Thermo Fisher Scientific）equipped with Faraday detectors at m/z 36, 37,
38, 39 and 40 connected to high-gain electrometers of 1 x10 11 Ohms for 40 Ar and 1 x10 12 Ohms for all other. The sample andreference argon enters into the ion source of the MS with a helium flow through fused silica capillary. The reference is a mixture of
air argon and helium in the ratio of about 1 ：100 stored in gas-cylinder under pressure ~ 80 atm. It is connected to a dosing system
to produce equal doses of the reference. A single reference dose contains 0.652 ± 0.001 ng of 40Ar. The reference is used to
calculate a quantity of radiogenic argon liberated from samples. This is provided by comparing the peak areas of the reference
and sample, which are following each other in order of reference sample reference with a minimum time interval within a single
chromatogram. This method, using of air argon as the reference gas has an advantage over conventional method of 38-Ar isotopic
dilution： 1 ）there is no contamination of the sample by 40Ar/36Ar incoming from a tracer 38-argon； 2 ）aquantity of argon in adose is invariable for several years 3 ）availability of 40Ar/36Ar air ratio monitoring by reference gas.
Pretreatments techniques. Rapid and quantitative liberating of argon from samples is provided by the CO 2 laser heating within amultiple-charge chamber. The sample chamber is equipped with a built-in heater to remove a sorbed gas and flushed with astream of clean He ~50 mL/min. Laser liberating sample argon are transferred by He flow to a cryogenic purification system andthen to a cryo-focusing trap where sample argon is collected by a molecular sieve at liquid nitrogen temperature. After defrostingof cryo-focusing trap the short-pulsed argon are transferred to a capillary column. The column separates the argon fromcontaminations and produces a shape of peak to integrate ion currents of masses 36 and 40 by multiple Faraday detectors. Due tothe short-pulsed output of argon, the value of signal / instrumental noise relationship is increased if to compare its with ameasurement in a static mode for same sample sizes. All-around analysis take up 10-15 min. The background signals were 5 x10 -4ng
for 40A rand 6 x10-6ng for 36Ar.

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 18: Advances in Resolution and Accuracy of Isotope Ratio Analyses

PTu-059

11:10 － 12:20

High precision analysis stable isotopes of sulfur (32S, 33S, 34S) using UV
laser fluorination continuous He flow mass spectrometry

Aleksandr V Ignatev, Tatiana Velivetckaia

Far East Geological Institute, FEB RAS, Vladivostok, Russia

Keywords:
mass spectrometry, stable isotope
Novel aspects:
New method high precision analysis stable isotopes of sulfur（32S, 33S, 34S）using UV laser fluorination irm-GC-MS
Abstract:
During the last decades, significant advances in the field of stable isotope geochemistry have resulted from the development andapplication of systems, which are based on in situ laser extraction procedures. Current laser techniques make it possible toanalyze 34S/32S isotope ratio with good spatial resolution（Crowe et al., 1990； Kelley and Fallick, 1990）. Nevertheless, systematic
experimental work has shown that the most commonly applied laser extraction technique results in a systematic fractionation of
the d34S values. Another recent advance is the development of continuous He flow techniques that use a UV laser ablation andgas chromatograph for online purification of gases, which allows to achieve precise analysis stable sulfur isotopes at nanomole
level samples（Ono et al., 2006）. The technique involves fluorination of sulfide（silver sulfide or pyrite）, and separation of productgas by gas chromatography. The basic sources of error with continuous helium flow is found to be tailing of the major ion beam
（32SF5+）into beams（33SF5+）, which results in distortion of the measured δ33S.
We have developed a method for determination of 34S/32S, 33S/32S isotope ratios in sulfur-bearing minerals using UV laser ablation.
Our equipment consists of small volume stainless steel chamber, a nickel reactor and six ports Valco valve with a trap for
cryogenic separation SF 6 . Helium（flow 40 cm 3 /min）passes through the chamber and nickel reactor. A photoablation of thesample surface formed an aerosol which in a continuous He flow passes through the reactor with ~ 2 % admixture of BrF5. Aerosol
are fluoridated at 350 0C. Total SF6 are condensed to a trap with liquid nitrogen. The trap is heated stepwise and cryogenicallypurified SF 6 are entered through chromatographic capillary column in the ion source. Purification of microquantity of SF 6 fromother gases was carry out by cryogenic separation and chromatographic capillary column. Between the ion source is placed a
special interface. The interface allows you to enter SF 6 after pumping helium and avoid distortion of the measurement results
δ33S. Measuring the ratios of sulfur isotopes are carried out on a MAT-253 mass spectrometer.
Samples for in situ analysis were prepared as polished, 1 - to 2 -mm thick small slabs, up to 1.5x1.5 cm. in largest dimension. The
slabs were placed in the sample. Reference SF6 gas, with known isotopic composition, was used for standardization. Additionally,
the chamber is loaded with standards IAEA-S1, IAEA-S2, and IAEA-S3. For making spot analysis of individual standards IAEA,
samples of IAEA were placed into stainless steel（alloy SS316）cups with 2 mm hole in diameter. NewWave UP213 Nd：YAG laser（ 4 ns pulse length, wave-length 213 nm）was used as the ablation source for analysis of solid samples. The repetition rate of thelaser we generally used 10 Hz. Ablation spot diameter ranges was between 50 and 200 mm. In our system to obtain the amplitudeof 1 Volt signal（for pyrite）150 laser pulses is sufficient at a resolution of 100 mm and range of fluences 46 J/cm2. For all othercases 300 pulses are required at the same power. Sulfur isotope data are presented using standard notation δ34S, δ33S and Δ33S
where Δ33S =［ln（ 1 +δ33S/1000）-λ*ln（ 1 +δ34S/1000）］*1000. Δ33S describe the deviation of a δ33S from a reference massdependent fractionation line. Data are presented relative to V-CDT. We report results to perform high precision（±0.1 - 0.2‰, SDfor δ34S, δ33S and ±0.05 ‰ for Δ33S）in situ sulfur isotope measurements by LA in a suite of well-characterized, isotopicallyhomogeneous natural sulfide minerals, pyrite, sphalerite, galena and standards IAEA-S1, IAEA-S2, IAEA-S3.

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 18: Advances in Resolution and Accuracy of Isotope Ratio Analyses

PTu-060

13:30 － 14:40

MC-ICPMS: enhanced ion yields for high precision measurement of sub-fg
Pu and high mass resolution for S isotope measurements.

Jerome A Johemko1, Charles B Douthitt2, Johannes Schwieters3, Claudia Bouman3, Nicholas Lloyd3

Thermo Fisher Scientific, Honolulu, USA, 2Thermo Fisher Scientific, San Jose, CA, USA, 3Thermo Fisher Scientific GmbH,
Bremen, Germany

1

Keywords:
MC ICP-MS, sub fg Pu, Nd in seawater, S isotopes
Novel aspects:
New geometry cones and improved pumping for greater ionization efficiency. 10e12 ohm feedback resistors.
Abstract:
Significant improvements in analytical performance of the Thermo Fisher model Neptune-Plus high-resolution multiple-collector
inductively coupled plasma mass spectrometer（HR-MC-ICPMS）include improvements in sensitivity and enhancements to the
dynamic range.
Improvements to the pumping system and cone design lead to increased ion yields, in some cases up to 4 %（relative to nominal
values around 0.1 %）. Significant increases in sensitivity have been observed for both wet plasmas（from solutions）and dry
plasmas（from laser ablation of solids）. The enhancements to sensitivity, when combined with the many favorable characteristicsof MC-ICPMS, open up a challenging class of analytical problems which require analysis of low concentrations in small samples（“measuring nothing in not very much“）,as demonstrated by the accurate and precise quantitation and measurement ofisotope ratios of sub-fg levels of Pu in 100-micro liter samples. Other recent examples will be shown, including high precision
measurement of Nd in seawater at low（ 5 ppb）concentrations. The increase in sensitivity also leads to improvement in counting
statistics, leading to higher precision and, in some cases, allows measurements to be moved from SEMs into Faraday cups, also
resulting in higher precision. This will be illustrated by measurements of the variation of 235 U/ 238 U caused by mass dependent
fractionation, a measurement which also exploits the large dynamic range of the Neptune-plus.
The high mass resolution capability of the Neptune-Plus HR-MC-ICPMS（to remove polyatomic interferences）combined with theenhanced sensitivity（to compensate for the reduction in sensitivity that accompanies increase in resolution）allow exploration of
the measurement of S isotopes for elemental and isotopic analysis. Using high mass resolution and amplifiers with 10e12 ohm
resistors for isotope dilution measurements, researchers report 50 times higher detection efficiency and 100 times better precision
for sulfur and δ 34S than the single-collector high-resolution sector field type ICP MS（HR-ICP-SFMS）. Reduction of blanks by> 1 order of magnitude relative to ID-TIMS suggests that ID-HR-MC-ICPMS is the best method for measurement of very low
concentrations of S. Coupling GC with MC-ICPMS has allowed, for the first time, compound specific δ34S isotope analysis（CSIA）
of volatile organic molecules, and the technique is currently being extended for more general use in the measurement of S isotope
systematics.

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 18: Advances in Resolution and Accuracy of Isotope Ratio Analyses

PTu-061

11:10 － 12:20

A COMPARATIVE STUDY OF CALCIUM AND STRONTIUM METABOLISM
IN THE SHEEP MODEL USING CA-41 AND SR-84 ISOTOPIC TRACERS

Fransiska Dewi1,2, Tim Schulze-Koenig3, Annette Liesegang4, Johanna Irrgeher5, Claudia Weigt6,
Gisela Kuhn6, Hans-Arno Synal3, Thomas Prohaska5, Thomas Walczyk1,2,7

NUS Graduate School for Integrative Sciences and Engineering, National University of Singapore, Singapore,
Department of Chemistry (Science), National University of Singapore, Singapore, 3Laboratory of Ion Beam Physics,
Swiss Federal Institute of Technology (ETH), Switzerland, 4Institute of Animal Nutrition, Vetsuisse Faculty, University of
Zurich, Germany, 5Division of Analytical Chemistry, University of Natural Resources and Life Sciences (BOKU), Austria,
6
Institute of Biomechanics, Swiss Federal Institute of Technology (ETH), Switzerland, 7Department of Biochemistry(Medicine), National University of Singapore, Singapore
12

Keywords:
bone, calcium, strontium, stable isotope, isotope tracing
Novel aspects:
The use of strontium stable isotopes as surrogate markers of calcium, which is safer and less expensive compared to calcium
radio- and stable-isotopes.
Abstract:
41
Ca, a quasi stable calcium（Ca）isotope, is a promising tracer for labeling human skeleton that can be detected at high sensitivity
using accelerator mass spectrometry（AMS）. Once the skeleton is labeled, changes in urinary tracer excretion can be used toassess Ca bone balance and turnover. Stable strontium isotopes（84Sr and 86Sr）may serve as surrogate markers due to similarities
in Ca and Sr metabolism. They are non-radioactive and more easily detected than 41Ca. This study was conducted in an adultsheep to compare Ca and Sr in terms of urinary excretion and bone deposition.
A 2 year old sheep received a single intravenous dose of 41Ca（50 nCi）and 86Sr（40 mg）. Over a period of 180 days, spot urine
samples were collected to follow clearance of both tracers. Samples were analyzed for 41Ca by AMS at ETH Zurich and for 86Srand element content at NUS by thermal ionization mass spectrometry（TIMS）and atomic absorption spectrometry（AAS）,
respectively. After sacrifice, relevant bones were obtained（tibia, radius, femur, metatarsus, metacarpus, vertebrae）. Bones were
cut into slices and samples of about 2 x 2 x 2 mm taken to assess spatial differences in tracer deposition using the same
techniques. To achieve even higher spatial resolution（with spot size as small as 10 µm）, similar pieces of bone that came fromadjacent areas to those analyzed earlier were subjected to Ca and Sr elemental analysis and Sr isotopic analysis using laser
ablation inductively coupled plasma mass spectrometry（LA-ICP-MS）at BOKU.
Analysis by using TIMS and AAS showed that deposition and excretion of both elements were highly correlated but they did notequal each other. In the skeleton, we found that Ca was preferentially deposited in bone relative to Sr and that more of bothtracers were incorporated in parts of the skeleton with higher remodeling rates（more active sites）. The observed linear
correlation between Ca and Sr deposition indicated that no site-specific discrimination occurred. Higher incorporation of tracers in
the more actively remodeled sites emerged from LA-ICP-MS analysis. As the sheep reached early adulthood when the tracers
were given, we found a higher tracer deposition along both sides of, but not directly on, the growth plate in the metaphyseal area
as well as between the inner and outer circles of the shaft.
With regards to tracer excretion, Sr is known to be less well absorbed in the gut than Ca and excreted preferentially over Ca in
urine. These differences were also reflected in our findings when the Ca and Sr tracer excretion was plotted against time postdose. Three-compartmental model（SAAM II）was used to describe the long-term kinetics of uptake, distribution and excretion of
Ca based on the excretion data collected.
In this study we have demonstrated the ability to detect minute amounts of both tracers in the skeleton and urine over extended
periods of time. Deposition of both tracers in the skeleton was also highly correlated with each other. Their excretion in the sheep
model was found to follow closely the compartmental model that was established earlier for human Ca metabolism. Thesefindings would speak for a possible use of Sr stable isotopes as surrogate markers of Ca in human studies.

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 18: Advances in Resolution and Accuracy of Isotope Ratio Analyses

PTu-062

13:30 － 14:40

Recent advance in MC-ICP-MS as a tool for the absolute determination of
isotopic composition of heavy elements

Masaharu Tanimizu

Japan Agency for Marine-Earth Science and Technology

Keywords:
atomic weight, absolute isotopic composition, MC-ICP-MS, mass-dependent discrimination
Novel aspects:
summarize recent advance in MC-ICP-MS for atomic weight determination
Abstract:
The precision of isotope ratios is now easily accessible to the level of better than 0.01 % using TIMS and MC-ICP-MS. Especially,
isotope ratios of various elements can be now determined by MC-ICP-MS through its high ionization efficiency of the ion source.
The ion transmission of the MC-ICP-MS from sample uptake to the detection of ion current is also improved by the several ordersof magnitude against requirements for natural geological and environmental samples. However, there are several data regarding
mass-independent behavior of MC-ICP-MS（Nd and W）. This feature is fundamentally different from the mass-dependent ion
transmission point of view which has been accepted for a long time. This is not so serious to the relative isotopic analyses against
an isotopic reference, but important to determine absolute isotope ratios, which have been determined through the evaluation ofmass-dependent discrimination factor（K factor）from gravimetrically prepared calibration solutions. This is also potentiallyaffected to the element doping technique to correct the K factor using isotope ratios of a neighbor element to analyte.
In this presentation, I will introduce several recent advances in MC-ICP-MS to the determination of standard atomic weight and its
uncertainty originated from natural isotopic variation, and its limitation and future direction will be suggested from a personal
point of view.

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 18: Advances in Resolution and Accuracy of Isotope Ratio Analyses

PTu-063

11:10 － 12:20

MEASUREMENT OF IRON TRANSFER FROM DIET TO BRAIN IN THE RAT
MODEL

Jiehua Chen, Shahreena Shahnavas, Nadia Singh, Wei Yi Ong, Thomas R Walczyk
National University of Singapore, Singapore

Keywords:
Brain iron uptake； Negative thermal ionization mass spectrometry； Stable iron isotope techniques
Novel aspects:
This is to the best of our knowledge the first study investigating the link between dietary iron and brain iron uptake in an adult rat
model using stable isotope techniques.
Abstract:
Neurodegenerative disorders are on the rise with Alzheimer’s disease（AD）and Parkinson’s disease（PD）being expected toreach epidemic proportions in the coming decades. Iron deposits in brain in AD and PD patients led to the suggestion that iron ispossibly involved in the pathogenesis of neurodegenerative disorders. At the same time, economic welfare and improved
production methods have led to a sharp increase in consumption of dietary or supplementary iron over the past 50 years inindustrialized countries and more recently in the developing world. Many lines of evidence point to iron induced oxidative stressand free radical damage as mechanisms that may lead to neurodegeneration. It remains to be seen, however, if an excessive ironintake is a risk factor to healthy individuals and if high body iron is paralleled by high iron deposits in brain.
In this study we describe a novel methodological approach using stable iron isotopes to determine directly the fraction of iron in
the diet that has been transferred to the brain and other organs over time in the adult rat model. We used for the first time ever acontinuous feeding approach in which a constant amount of a stable iron isotope（57Fe）was administered into the drinking water
daily with the rat chow diet to seven 6 - 8 month old adult male Wistar rats over 4 months. Iron isotopic analysis of organ tissues
was conducted using Negative Thermal Ionization Mass Spectrometry（NTI-MS）.
Isotopic enrichment for the brain is 7 %. Inter-individual variations between rats were relatively small as indicated by the relatively
small standard deviations obtained for the brain across animals（ 8 % RSD）. The most surprising finding in our study is theobservation that iron uptake by brain is considerable in normal adult rats. Although only 0.000537 ± 0.000076% of dietary iron intake
could be traced in brain on average, which may appear negligible, this amount constitutes ca. 9 % of total brain iron with figures
being of similar order of magnitude for the other tissues under investigation（11.5-36.3%）. The abovementioned ca. 9 % of brainiron could be traced back to the diet over a period of four months, a time span that compares to 7 -10 human years.
Our knowledge of brain iron homeostasis and uptake is still very limited. Attention has been paid in the past primarily to thegrowing brain and impact of iron deficiency on brain development with radioisotope techniques. To our knowledge, no studies
have been published to date focusing on iron accumulation in the brain of the adult rat. This knowledge gap can now be closedwith the methodological approach we are presenting here. Contrary to radioisotopes, stable isotopes do not decay. This does not
only eliminate health hazards and practical limitations in conducting feeding trials. Tracers can be fed over years, if deemednecessary, and samples stored without any time limit. Furthermore, long term continuous feeding studies allow us to determine
brain iron balance reliably as opposed to the conventional single dose iron tracer administration with short-term observational
period.

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 18: Advances in Resolution and Accuracy of Isotope Ratio Analyses

PTu-064

13:30 － 14:40

Elemental mapping of Eu, Yb, Hf, Pb, Th and U for zircon using highresolution laser ablation-ICP-Mass spectrometry

Kentaro Hattori1, Shu-hei Sakata1, Hideki Iwano2, Takafumi Hirata1
1

Kyoto University, Kyoto, Japan, 2Kyoto Fission Track Co.Ltd., Kyoto, Japan

Keywords:
mass-spectrometry, laser ablation, imaging, zircon
Novel aspects:
Two-dimensional mapping for isotopes with high resolution LA-ICP-MS techniques would reveal the trace elemental distribution
mechanism during zircon formation in granites.
Abstract:
The laser ablation system attached to an ICP-mass spectrometry（LA-ICP-MS）has been widely used for elemental and isotopic
analyses for solid and liquid samples. The laser ablation system utilizing a shorter wavelength and pulse width can minimize anelemental fractionation during both laser ablation and ionization processes, and improve transport efficiency of sample from theablation area to the ICP ion source. Moreover, magnetic sector type ICP-MS instruments can provide both higher sensitivity andlower background counts, resulting in high spatial resolution of the LA-ICP-MS analyses. The high resolution ICP-MS would enable
to conduct elemental and isotopic analyses and imaging with a laser pit size less than 10 μm, which may be comparable to an
ion-beam size of secondary ion mass spectrometry（SIMS）. In this study, we demonstrate that two-dimensional images forisotopes of 153 Eu, 172 Yb, 178 Hf, 204 Pb, 206 Pb, 207 Pb, 208 Pb, 232 Th, and 238 U, and 206 Pb/ 238 U ratio were yielded from the OD- 3 zircon
crystal with the LA-ICP-MS using a 2
μm laser spot size, and discuss the elemental distribution within zircon comparing thecathodo luminescence（CL）images.
The OD- 3 zircon was collected from Mihara body of Kawamoto granodiorite distributed in Shimane prefecture, Japan. The
zircons are euhedral to subhedral crystals with reddish color. The age of the Kawamoto granodiorite was dated to be 33 Ma
based on the systematic comparison of zircon fission track and（SHRIMP, LA-ICP-MS or TIMS）U-Pb dating. The imaging was carriedout using a Nu AttoM magnetic sector field-single collector-ICP-MS（Nu instruments, Wrexham, UK）equipped with a NWR193 laser
ablation system（New Wave Research, Fremont, CA, USA）, which utilizes 193 nm ArF excimer laser. The result shows that 153Eu, 17
2
Yb, 178 Hf, 206 Pb and 238 U were heterogeneously distributed within the zircon. The signal intensity of 232 Th shows non-linear
correlation with that of 238U, which was negatively correlated with the CL luminosity. Moreover, distribution of 178Hf was clearly
different from both the distribution of 238U and CL images. The non-linear correlation could be explained either by changes in theratio of the distribution coefficients（DU/DTh）, or by diffusion properties of these elements during the crystal growth of zircon.

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 18: Advances in Resolution and Accuracy of Isotope Ratio Analyses

PTu-065

11:10 － 12:20

The estimation of the effect of initial 230Th disequilibrium on U-Pb datingof young zircon crystal by LA-ICPMS

Shu-hei Sakata1, Hideki Iwano2, Kenshi Maki1, Takaomi D Yokoyama1, Takahumi Hirata1
1

Kyoto University, Kyoto, Japan, 2Kyoto Fission Track Co. Ltd., Kyoto, Japan

Keywords:
zircon chronology, LA-ICPMS U-Pb age, 230Th disequilibrium, high-sensitivity measurement
Novel aspects:
Developments of high-sensitivity LA-ICPMS technique revealed the contribution of disequilibrium on 230Th onto the resulting U-Pb
ages for the young zircons.
Abstract:
Recent developments in the laser ablation-ICP-MS technique make it possible to apply U-Pb dating method to young zircon
crystals which are younger than 1 Ma. For the dating of young zircons, however, we have to consider a possible systematic error
which can be originated from the effect of initial loss or enhancement of 230Th in both the zircon crystal and source magma. In thiscase, the true 238U-206Pb age（T）can be obtained through the following equiation：
206
Pb/238U =［（exp（λ238T）- 1 ）+（λ238/λ230）（f - 1 ）（ 1 - exp（-λ230T））exp（λ238T）］,
where λ230 and λ238 are decay constant of 230Th and 238U, respectively,“ f“ represents the level of loss or enhancement of 230Th,
which is defined by the degree of Th-U fractionation between zircon and source magma［（Th/U）zircon/（Th/U）magma］. In this study,
the 206 Pb/ 238 U, 207 Pb/ 235 U and 208 Pb/ 232 Th ratios for several young zircon crystals whose ages varies from 0.5 to 1 Ma, were
measured by means of a high-sensitivity ICP-sector field-mass spectrometer coupled with laser ablation sample introduction
technique. Based on the measured ratios, we tried to estimate the contribution of disequilibrium on 230Th onto the resulting U-Pb
ages for the young zircons through two different combination of the U-Pb and Th-Pb ages. First one is the conventional concordiaplot, and the second is the pseudo-concordia diagram； plot of 207 Pb/ 235 U ratio against 208 Pb/ 232 Th ratio. If there is the effectof 230 Th disequilibrium, resulting data would leave from the concordia-curve and be plotted on the concordia-curve on
the 207 Pb/ 235 U vs 208 Pb/ 232 Th diagram because the contribution of disequilibrium nuclide should be smaller for the 235 U- 207 Pb
and 232Th-208Pb decay series. Further details about the result will be discussed in our presentation.

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 19: Mass Spectrometric Diagnosis

PTu-066

13:30 － 14:40

Developing a human cancer diagnostics system based on the probe
electrospray ionization-mass spectrometry and Bayesian statistics

Kentaro Yoshimura1, Lee Chuin Chen2, Mridul Kanti Mandal3, Kunio Tanabe1,5, Michio Hara4,
Hideki Fujii4, Masayuki Takeda6, Kenzo Hiraoka3, Sen Takeda1

Department of Anatomy and Cell Biology, Interdisciplinary Graduate School of Medicine and Engineering, University of
Yamanashi, 2Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi, 3Clean Energy
Research Center, University of Yamanashi, 4Department of Surgery, Interdisciplinary Graduate School of Medicine andEngineering, University of Yamanashi, 5Faculty of Science and Engineering, Waseda University, 6Department of Urology,
Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi
1

Keywords:
PESI, Diagnostics, Cancer, Electrospray, Ambient
Novel aspects:
We established a rapid human cancer diagnostics system based on ambient ionization/mass spectrometry and statistics. Our
system is applicable to raw specimens resected from patients without any pretreatments.
Abstract:
Probe electrospray ionization（PESI）is an ambient ionization technique that uses a fine stainless needle for pickup and ionizationof samples［Hiraoka et al., 2007］. The needle has a diameter of 140 μm and its tip radius is sub-micrometer, and moves up anddown along a vertical axis by a linear actuator to capture the sample from surface of specimen and generate the electrospray［Yoshimura et al., 2009］. The strength of PESI-mass spectrometry（PESI-MS）lies in low invasiveness, rapidity and no need forparticular pretreatments, especially when applied to biological samples. In the present study, we constructed a prototype ofcancer-diagnostic system using quadrupole mass spectrometer（LCMS-2020, Shimadzu）. The liquid chromatograph unit isdisassembled and replaced with the PESI unit.
Previously, we reported real-time analyses of living mice that delineate the differences in lipid composition of hepatocytes
between normal and steatotic mice［Yoshimura et al., 2011］. As a next step, we analyzed various human tissues to test its
potential application to medical diagnosis. When we analyze the surgically extirpated human kidney, liver, pancreas, gall bladder,
lymph node, and mucosal epithelium, unique mass spectral patterns were obtained from each specimens. These spectra weremainly derived from lipid components. Next, to ascertain whether or not cancer specific mass spectra exist, human clear cell renal
cell carcinoma（ccRCC）was used as a positive control because of its well-known feature of lipid storage in the cytoplasm. As
expected, we could successfully obtain distinct profiles of lipids in cancerous region, in which TAG dominated. Subsequently, mass
spectra of non-cancerous and cancerous regions from 8 cases were subjected to PCA. Principal component scores from both
specimens were clearly separated, the results suggesting the usefulness of lipid profiles for cancer diagnosis［submitted］.
To further sophisticate our diagnosis system, we employed the dual penalized logistic regression machine（dPLRM）, which is a
Bayesian inference-based learning machine and automatically give a probabilistic prediction by referring the training data set［Tanabe et al., 2001］. Acquired raw data of ion intensities from ccRCC are exported and stored on a database format. This
database is then used as training data set for dPLRM-based diagnostic algorithm. We performed a single-blind evaluation for
ccRCC, and predictions by dPLRM were completely consistent with pathohistological diagnosis.
This algorithm does not require the annotation of cancer specific molecules（cancer markers）for prediction of cancer probability,
because it recognizes the relative variations in the whole mass spectral pattern. To demonstrate the potential of our system oncancer diagnosis, we measure the mass spectra of human hepatocellular carcinoma（HCC）, which does not show any prominent
peaks as seen in the RCC. Surprisingly, our system showed coincidence of diagnosis with that by pathohistology. These results
collectively demonstrate the usefulness of PESI-MS to the human cancer diagnosis［in preparation］.

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 19: Mass Spectrometric Diagnosis

PTu-067

11:10 － 12:20

Ultrasensitive Multiplexed Detection of miRNAs Using Gold Nanoparticles
and LDI-TOF MS

Hyunook Kang, Seol-Hye Hong, Woon-Seok Yeo
Konkuk University, Seoul, Korea

Keywords:
miRNA, LDI-TOF MS, nanoparticles, quantification
Novel aspects:
This poster presents a new method for miRNA detection with both multiplexing and quantification capabilities. In addition, thelimit of detection of the method is well below picomolar（pM）.
Abstract:
In this poster, we describe a simple, fast, and sensitive method for the multiplexed detection of miRNAs without the need of PCRor a labeling by combining self-assembled monolayers（SAMs）on gold and laser desorption/ionization time of flight mass
spectrometry（LDI-TOF MS）. In our strategy, the target miRNA is captured specifically by the complementary DNAs immobilized
on gold nanoparticles（AuNPs）and gold chips. AuNPs carry a large number of small molecules, called Am-tag, as a reporter forthe detection of the target miRNA. The presence of miRNA in a solution is verified by mass peaks corresponding to Am-tag using
LDI-TOF MS. For the multiplexed detection, we designed and synthesized four Am-tag molecules containing 0, 4, 8, 12 isotopes sothat they have same molecular properties but different molecular weights. By observing and analyzing mass signals of these Amtags on AuNPs decorated with different probe DNAs, miRNAs in samples can be easily discriminated and the relative amounts ofmiRNAs can be quantified. As a model case, we tested four miRNA, mir-365, mir-142- 3 p, mir-1207- 3 p, and let- 7 c. We believe that
this strategy will provide a biological important tool for accurate, sensitive, rapid, and low-cost multiplexed detection of miRNA.

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 19: Mass Spectrometric Diagnosis

PTu-068

13:30 － 14:40

Mapping longitudinal proteomic expression levels in brain tissue for an
amyloid Alzheimer’s disease mouse model using shotgun based mass
spectrometry

Ganna Shevchenko1, Kim Kultima2, Jonas Bergquist3, Kina Höglund4,5, Lars Andersson5,
Magnus Wetterhall6
1
4

Uppsala University, Uppsala, Sweden, 2Uppsala University, Uppsala, Sweden, 3Uppsala University, Uppsala, Sweden,
AstraZeneca, Södertälje, Sweden, 5AstraZeneca, Södertälje, Sweden, 6Uppsala University, Uppsala, Sweden

Keywords:
Alzheimers； Transgenic mouse； proteomics； CPE
Novel aspects:
The proteomic expression levels in brain tissue of an Alzheimer’s disease mouse model were longitudinally studied using
optimized extraction conditions and label free quantification
Abstract:
Alzheimer’s disease（AD）is the most common form of dementia amongst the elderly, accounting for 60% of all diagnosed cases.
Typically, diagnosis of AD cannot be made until the disease has progressed to the point that dementia is present. The
neuropathology of AD is characterized by the aggregation and deposition of misfolded peptides and proteins, in particular,
aggregated β-amyloid peptide in the form of extracellular plaques and hyperphosphorlylation of the tau protein in the form of
intracellular neurofibrillary tangles in specific brain regions. Another key feature in the bimolecular processes underlying AD
pathobiology is alteration of the membrane interactions in the brain tissue. This is confirmed by the fact that protein interactions
are altered as a result of abnormalities in the membrane protein（MP）interactions. In the same way, degradation products fromMPs may serve as diagnostic markers of disease. The advances in proteomics technologies, especially in liquid chromatographytandem mass spectrometry（LC-MS/MS）, provide opportunities for the identification of disease-specific proteins and biomarker
discovery. Despite a great progress has been made in brain research, comprehensive analysis of brain MPs remains a challenge
due to their hydrophobicity, heterogeneity and low abundance.
We have successfully optimized cloud point extraction（CPE）methodology for the simultaneous extraction and enrichment ofboth the MPs and hydrophilic proteins from brain tissue followed by“shotgun“ based MS analysis. High preconcentration
coefficients, good selectivity and possibility to combine with various methods of downstream analysis are attractive features
promoting the use of non-ionic surfactants to extract hydrophobic proteins which can be potential molecular markers of thedisease from biological samples.
The present study was designed to longitudinally characterize the proteomic profile in the AD disease amyloid mouse model（Tg2576 APPSWE）to find proteins linked to AD. Transgenic mouse models of AD have served as valuable tools for investigating
pathogenic mechanisms related to Aβ accumulation. The Tg2576 model, which have been used in this study develops manyneuropathological features of Alzheimer’s, including amyloid plaques, dystrophic neuritis and inflammatory changes, and it is oneof the most well characterized strains of transgenic animals over-expressing human APP.
Brain tissue from the Tg2576 APPSWE mouse model and litter mate controls at the age of 12, 15, 18 and 22 months were cloud
point extracted and phase separated using the non-ionic surfactant Triton X-114. The extracted protein fractions were delipidated
and precipitated with tri-n-butylphosphate/acetone /methanol（ 1 ：12： 1 ）. The proteins were tryptically digested on 3 kDa spin
filters. The detection of peptides and analysis of their expression levels were done with nanoLC-MS/MS using a 7 T hybrid LTQ
FTMS instrument together with a label-free approach. This longitudinal study of the Tg2576 mouse model gave 480-720 uniqueproteins identified/animal. The utilized CPE method yields a phase separation of the proteins into either a hydrophilic or ahydrophobic phase, which can be processed and analyzed individually. Eight out of 13 suggested synaptic degradation markers
were identified in all samples. Label free quantification and multivariate data analysis of the extracted samples showed a goodage correlation within each studied age group. However, clear age related trends could be found for the AD model. These trends
were most distinct in the hydrophobic（membrane）fractions showing a clear decrease of the number of proteins and expression
levels with age. The obtained results show the potential of longitudinal monitoring of neurodegenerative processes in diseaseprogression.

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 19: Mass Spectrometric Diagnosis

PTu-069

11:10 － 12:20

Serum metabolome analysis for early detection of colorectal cancer

Masaru Yoshida1,2,3, Shin Nishiumi1, Yoshihiro Izumi1, Atsuki Matsubara1, Takeshi Azuma1

Division of Gastroenterology, Department of Internal Medicine, Kobe University Graduate School of Medicine, Kobe,
Japan, 2The Integrated Center for Mass Spectrometry, Kobe University Graduate School of Medicine, Kobe, Japan,
3
Division of Metabolomics Research, Kobe University Graduate School of Medicine, Kobe, Japan
1

Keywords:
serum metabolomics, GC/MS, colorectal cancer
Novel aspects:
GC/MS-based serum metabolomics could be used as a novel method for colorectal cancer screening tests.
Abstract:
To improve the quality of life of colorectal cancer patients, it is important to establish new screening methods for early diagnosisof colorectal cancer. We performed serum metabolome analysis using gas-chromatography/mass-spectrometry（GC/MS）. First,
the accuracy of our GC/MS-based serum metabolomic analytical method was evaluated by calculating the RSD% values of serum
levels of various metabolites. Second, the intra-day（morning, daytime, and night）and inter-day（among 3 days）variances ofserum metabolite levels were examined. Then, serum metabolite levels were compared between colorectal cancer patients（N=60； N=12 for each stage from 0 to 4 ）and age- and sex-matched healthy volunteers（N=60）as a training set. The
metabolites whose levels displayed significant changes were subjected to multiple logistic regression analysis with the stepwise
variable selection method, and a colorectal cancer prediction model was established. The validity of the prediction model was
confirmed using colorectal cancer patients（N=59）and healthy volunteers（N=63）as a validation set.
The prediction model was composed of the 4 metabolites selected, and its AUC, sensitivity, specificity, and accuracy were 0.9097,
85.0%, 85.0%, and 85.0%, respectively, according to the training set data. In contrast, the sensitivity, specificity, and accuracy of CEA
were 35.0%, 96.7%, and 65.8%, respectively, and those of CA19- 9 were 16.7%, 100%, and 58.3%, respectively. At the validation set, thesensitivity, specificity, and accuracy of the prediction model were 83.1 % , 81.0 % , and 82.0 % , respectively, and these values were
almost the same as those obtained with the training set. In addition, the model displayed high sensitivity for detecting stage 0 - 2
colorectal cancer（82.8%）.
Our prediction model established via GC/MS-based serum metabolome analysis is valuable for early detection of colorectal
cancer and has the potential to become a novel screening test for colorectal cancer.

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 19: Mass Spectrometric Diagnosis

PTu-070

13:30 － 14:40

Analysis of Drugs and Steroid Hormones in Human Saliva and Serum using
Column-switching Ultra-fast LC/MS/MS with On-line Analyte Purification

Yukiko Hirabayashi1, Takashi Shinagawa2, Joji Suzuki2, Toshio Kaji2
1

Central Research Laboratory, Hitachi, Ltd., Tokyo, Japan, 2Hitachi Yokohama Hospital, Hitachi, Ltd., Kanagawa, Japan

Keywords:
Saliva, Serum, Drug, Steroid-hormone, Column-swiching
Novel aspects:
We developed an convenient analytical method for drugs and steroid hormones in saliva, substituting for blood, using columnswitching LC/MS/MS and showed the correlation in those concentrations in saliva and serum.
Abstract:
Introduction：
Saliva which has a similar composition to blood can be used as a substitute for blood in medical screening, such as therapeutic
drug monitoring and biomedical examinations. It is generally believed that concentrations of components in saliva depend on
concentrations of protein-unbound components in plasma, and examining the correlation in concentrations is important. We havetherefore analyzed several oral drugs and steroid hormones in saliva and serum of test subjects dosing the drugs by columnswitching LC/MS/MS using an ultra-fast LC system and compared concentrations of the drugs and hormones in saliva and serum.
Methods：
Saliva and serum were collected from healthy adult volunteer-subjects. Informed consent was obtained from every subject prior
to the dosage and collections at Hitachi Yokohama Hospital. After dosing an oral-drug with 100-mL water, saliva and serum werecollected from subjects at 15 and 3 times, respectively, within about 9 hours. The unstimulated saliva were accumulated in theoral cavity and spat into plastic sample tubes in several batches for 15 minutes.
The saliva and serum were kept frozen at -30 &#8451； and thawed at room temperature. To analyze the drugs, the saliva and thetenfold-dilution serum with water were added to an equal amount of an LC-mobile phase solution, and vortex mixed. Samples
were centrifuged and supernatant fluids were used for analysis. A series of saliva-based calibrator solutions and a series of diluted
serum-based calibrator solutions were treated in the same way. To analyze the steroid hormones, the saliva and the tenfolddilution serum with water were added to LC-mobile phase solution containing an internal standard and other preparations were
almost the same as those used in drug analysis.
A LachromUltra ultra-fast liquid chromatograph system（Hitachi Hi-Technologies, Tokyo, Japan）, a 3200 Q TRAP tandem mass
spectrometer system equipped with an ESI ion source（AB SCIEX, Massachusetts, USA）, and a Model 3011 six-port switching valveunit（Shiseido, Tokyo, Japan）were used in this analysis. The pre-columns for protein removal were Capcell Pak MF-C 8 or MF-SCX
cartridges（4.0 mm i. d., 10 mm long, Shiseido, Tokyo, Japan）, the intermediate trapping column was an OASIS HLB column（2.1mm 1.d., 20 mm long, Waters, Massachusetts, USA）. The main analytical column was a LachromUltra C18 column（2.0 mm i.d., 20
mm long, Hitachi Hi-Technologies, Tokyo, Japan）. The sample was introduced into the pre-column with the mobile phase forpretreatment（aqueous solution containing 0.1 % formic acid, 10 mM ammonium formate, or 5 mM ammonium acetate）, after
removing proteins the flow channel was switched by the valve then the sample injected into the trapping column. After trapping
objective substances, the substances were flashed with the mobile phase for analysis（water/methanol（30：70, v/v）containing 0.1% formic acid, 10 mM ammonium formate, or 5 mM ammonium acetate）by flow channel-switching, using backflash method, thenintroduced into the analytical column and separated. The separated substances were ionized and measured by MRM.
Results and discussion：
The LLOQ of Sodium valproate, Etizolam, and Ibuprofen were 10, 0.05, and 1 ng/mL, respectively, with a signal-to-noise ratio of
10：1. The coefficients of variation（CVs）of Sodium valproate, Etizolam, and Ibuprofen（saliva-based calibrator solutions）were 1.6,
3.9, and 4.1 %, respectively. The LLOQ of progesterone was 0.025 ng/mL. The CV of progesterone was 2.3 %.
Correlation coefficients of concentrations in saliva and serum for Sodium valproate, Etizolam, and Ibuprofen were 0.77, 0.72, and0.74. The correlation coefficient of concentrations in saliva and serum for progesterone was 0.83.
Conclusions：Concentrations of components in saliva and serum showed good correlation. This method for saliva-screening has merits foryoung patients, such as babies and children.

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 19: Mass Spectrometric Diagnosis

PTu-071

11:10 － 12:20

Acylglycine: A Potential Biomarker for the Clinical Diagnosis of Inborn
Metabolic Disorders

Bonnie Fong1,2, Sidney Tam2, Kelvin Leung1

1
Department of Chemistry, Hong Kong Baptist University, Hong Kong, 2Department of pathology and Clinical
Biochemistry, Queen Mary Hospital, Hong Kong

Keywords:
Acylglycines, Inborn errors of metabolism
Novel aspects:
We developed a LC-MS/MS method for acylglycines determination with isomers and set up the reference intervals in pediatric
Chinese.
Abstract:
Urinary organic acids, plasma amino acids and acylcarnitine profile analyses are the main tools used to diagnose inborn errors of
metabolisms（IEM）. However, without metabolic decompensation, these parameters are often not helpful. On the other hand, incases of IEM, acylglycines are consistently raised even when patients appear to be in remission. This study aims to set-up a liquid
chromatography-electrospray ionization-tandem mass spectrometry（LC-ESI-MS/MS）method for the determination of urine
acylglycines, complementary to organic acid and acylcarnitine profiles, for the diagnosis of IEM. In addition, local reference
intervals for various acylglycines are established by using this method. Acylglycines were isolated by solid-phase extraction,
derivatized with n-butanol, separated by HPLC, and detected by ESI-MS/MS. Acylglycines were quantified with deuterated internal
standards. Mean recoveries of acylglycines ranged from 90.2 to 109.3%. Within- and between-run imprecisions for all acylglycines
have CVs less than 10%. Linear regression coefficients were greater than 0.99. Reference intervals were established according to
CLSI guidelines by analyzing 204 samples from apparently healthy individuals less than 18 years of age. Partitioning into age groupreference intervals was not indicated, according to the Harris and Boyd approach. In this context, a single reference interval foreach acylglycine could be used. This method of urine acylglycines analysis is a powerful diagnostic tool, complementary to urineorganic acids and plasma acylcarnitine profiling, for detecting certain inborn errors of metabolism.
Reference：
Fong, B.M.W.； Tam, S.； Leung, K.S.Y. Talanta, 2012, 88, 193-200

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 19: Mass Spectrometric Diagnosis

PTu-072

13:30 － 14:40

Clinical application of rapid bacterial identification using mass
spectrometry

Kazuyuki Sogawa1, Masaharu Watanabe1, Fumio Nomura1,2
1

Chiba University Hospital, Chiba, Japan, 2Chiba University, Chiba, Japan

Keywords:
rapid bacterial identification, MALDI-TOF MS
Novel aspects:
Clinical application of rapid bacterial identification using mass spectrometry
Abstract:
Background： In a clinical diagnostic microbiology laboratory, the current method of identifying bacterial isolates is based mainlyon  phenotypic characteristics, such as growth patterns on different media,  colony morphology, Gram stain, and biochemical
reactions.  Collectively, these techniques can identify most  bacterial isolates accurately, but they are costly  and time-consuming. We
prospectively   assessed the performance of MALDI-TOF MS in identifying bacterial strains that are routinely isolated fromclinical  samples（Anal Bioanal Chem 2011； Anal Bioanal Chem 2012）. We also examined positive blood culture bottles for which a
rapid decision was required.
Methods： Bacterial colonies obtained from a total of 468 strains of 92 bacterial species isolated at the Department of ClinicalLaboratory at Chiba University were directly placed on target MALDI plates, followed by addition of CHCA matrix solution. For
identification of bacteria from positive blood culture bottles, samples of approximately 3 mL were recovered from positive blood
culture bottles and injected with a syringe into venous blood collection tubes that were then centrifuged. Bacteria were separated
at the surface of the gel and pellets were directly deposited on target MALDI plates. All plates were analyzed by MALDI-TOF MS
and the microorganisms were identified by pattern matching with the libraries in BioTyperTM 2.0 software.
Results： Escherichia coli and Pseudomonas aeruginosa were used to evaluate colony to colony differences in the MALDI-TOF MS
data. The scores were 2.371±0.044 and 2.377±0.039, respectively. Prevotella buccae and Clostridium perfringens were usedto
investigate the influence of the culture time. The scores on day 1 and day 3 were 2.341 and 2.352, respectively, for Prevotella
buccae and 2.435 and 2.411, respectively, for Clostridium perfringens. The identification rates at the species and genus levels were
91.7%（429/468）and 97.0%（454/468）, respectively. By adding data for the clinical isolates to BioTyperTM 2.0, the identification rateincreased. The identification rate was 86.5 %（32/37）at the species in cases with monomicrobial isolate in the positive blood
culture bottle. S. aureus and Staphylococcus haemolyticus were identified in a case with polymicrobial isolate in the bottle, inwhich the polymicrobial isolate could not be identified simultaneously using MALDI-TOF MS methods.
Conclusions： MALDI-TOF MS is a rapid, simple and high-throughput proteomic technique for identification of bacterial species.
Since colony to colony differences and effects of culture duration on the results are minimal, this method can be implemented in aconventional laboratory setting. For some pathogens, preanalytic processes should be refined and the current database requires
improvement to obtain more accurate results. However, in general, MALDI-TOF MS performs equally well to conventional methods
and is a promising technology for use in clinical laboratories.

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 19: Mass Spectrometric Diagnosis

PTu-073

11:10 － 12:20

Lectin-capturing and peptide affinity enrichment-based identification ofcancer biomarker TIMP1 by high-resolution FT-ICR MS

Yeong Hee AHN, Kwang Hoe KIM, Park Min SHIN, Eun Sun JI, Jong Shin YOO
Korea Basic Science Institute, Cheongwon, Korea

Keywords:
lectin-capturing, cancer biomarker, FT-ICR MS
Novel aspects:
TIMP 1 in human serum was identified to be a potent CRC biomarker by lectin-capturing and peptide affinity enrichment-based
approach with MALDI FT-ICR MS.
Abstract:
Various aberrant glycosylation patterns have been known to be connected to cancer. Thus, an aberrant glycosylation state isimportant in studying pathological mechanisms involved in cancer or in developing cancer biomarkers. Cancer blood sample
containing glycoproteins aberrantly glycosylated can be used as a target for development of a serological cancer marker. The
lectin-capturing method has been widely used to fractionate protein glycoforms that have a specific glycan structure active to thelectin used. Therefore, this method is very attractive as a tool to capture a specific protein glycoforms of interest. Among a varietyof lectins being employed for capturing an unique glycan form involving with cancer progression, L-PHA was used for capturing theaberrant glycoforms of glycoproteins up-regulated as a result of aberrant addition of β-1,6-N-acetylglucosamine（β-1,6-GlcNAc）by
N-acetylglucosaminyltransferase-V（GnT-V）, which is known to be up-regulated in invasive/metastatic colorectal cancer cells. By
using lectin-coupled/FT-ICR MS with peptide affinity enrichment（SISCAPA）technique, it is investigated that a target protein, tissue
inhibitor of metalloproteinase 1 （TIMP 1 ）can be a potent serological colorectal cancer marker. Since exact mass measurementof multiplex isotopic peaks of the target peptide surrogating glycoprotein TIMP 1 can be accomplished by virtue of high mass
resolution, robust identification of the target peptide is achievable with 15 T FT-ICR MS. Whether TIMP 1 in human serum can be apotent CRC biomarker will be investigated by using FT-ICR MS. Additionally it is explored that high-resolution FT-ICR MS can be auseful tool toward identifying potential protein biomarker.

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 19: Mass Spectrometric Diagnosis

PTu-074

13:30 － 14:40

Relating ESI-MS response with polar-, structural- and primarycharacteristics of neuropeptides: Developing a deeper understanding

Birthe V Nielsen, Daniel A Abaye, Frank Pullen, Minh T Nguyen

University of Greenwich, School of Science, Chatham Maritime, Kent, ME4 4TB, UK

Keywords:
Neuropeptides, ESI-MS response, HDX MS
Novel aspects:
Reports exist on ESI response of small（m/z 300 to 1,600Da）or large proteins however, none on medium-sized molecules（1500 to
3500Da）. Our research is aimed at filling this gap.
Abstract:
The search for key pain biomarkers is a hot topic within the pharmaceutical industry and it is clear from scientific literature thatneuropeptides are implicated as potentially key markers. The detection and quantification of this class of peptides is best
achieved using mass spectrometry（MS）but a deep understanding of how they ionise and behave in the gas phase is not yet fully
understood. Currently, little is known about the factors affecting responsiveness of this size of peptides in positive electrosprayionization（ESI）MS though the intensities（selectivity and signal suppression）of the ions depends heavily on analyte basicity insolution,（pKb）, sampling and instrumental conditions and on secondary structure. Other factors may be equally important andour research seeks to develop this understanding.
Neuropeptides are an important class of chemical messengers that modulate nervous system response. They act as neuronalmessengers in the brain and central nervous system, influencing many neurobehavioral functions. Our main interest is in theneuropeptides that are relevant to the perception of pain, including substance P（SP）, calcitonin gene-related peptide（CGRP）,
vasoactive intestinal peptide（VIP）and also corticotropim releasing factor（CRF）and prolactin releasing peptide（PrRP）.
In positive ESI of peptides, multiply charged ions are typically produced. Multiple protonation is an important attribute in structure
identification and accurate molecular weight determination in MS. To achieve protonation, the analytes must be basic both insolution（low pH）and in the gas phase. It is often expected that the primary sequence of the analytes and the pKb of theconstituent ionisable amino groups of the peptide will be prominent factors in determining the number and intensity of
protonation during the positive ESI response. However, this is not always the case［1,2］. In addition to the mass spectrometersettings, sample processing prior to the ESI stage is crucial to obtaining an adequate spectrum of ions and to achieving
protonation. Our research indicates that the multiple protonation of specific neuropeptides cannot be controlled purely by varying
pre-ESI parameters（pH of the mobile phase, flow rate（nano- and micro LC）, concentration of organic modifier, peptide
concentration）, but that some charge states might be a result of the conformational state, peptide polarity and the position ofvery basic amino acids（arginine）.
Here we present our investigation on the link between peptide characteristics and ESI response to allow robust quantitation ofthese important biomarkers especially during label-free quantitation. Factors investigated include peptide polarity, peptide
conformation in solution（mobile phase）and the influence of the sequence position of specific amino acids（arginine）, effects ofelectrolyte, and effect of conformation in solution（circular dichroism analysis and hydrogen-deuterium exchange HDX MS）, andtheir detection in a saliva matrix.
（ 1 ）Daniel A. Abaye, Frank S. Pullen and Birthe V. Nielsen. Peptide polarity and the position of arginine as sources of selectivity
during positive electrospray ionisation mass spectrometry（2011）. Rapid Commun. Mass Spectrom. 25, p. 3597
（ 2 ）Daniel A. Abaye, Frank S. Pullen and Birthe V. Nielsen. Practical considerations in analysing neuropeptides, calcitonin
generelated peptide and vasoactive intestinal peptide, by nanoelectrospray ionisation and quadrupole timeofflight mass
spectrometry： monitoring multiple protonations（2011）. Rapid Commun. Mass Spectrom. 25, p.1107

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 19: Mass Spectrometric Diagnosis

PTu-075

11:10 － 12:20

Fabrication of a titanium based MALDI bacterial chip for rapid, sensitiveand direct analysis of pathogenic bacteria

Hui-Fen Wu1,2,3, Judy Gopal1,2, Nazim Hasan1

Department of Chemistry, National Sun Yat-Sen University, 2Center for Nanoscience and Nanotechnology, National Sun
Yat-Sen University, 3Doctoral Degree Program in Marine Biotechnology, National Sun Yat - Sen University,

1

Keywords:
MALDI-MS, Biochip, mass sensor, pathogenic bacteria
Novel aspects:
We report a titanium chip for MALDI-MS produced from a simple, cost effective and rapid heat treatment process. It can bereused many times for direct analysis of pathogenic bacteria.
Abstract:
For the first time, we report the fabrication of a titanium bacterial chip for MALDI-MS produced from a simple, cost effective andrapid heat treatment process. This bacterial chip can be reused many times and is highly versatile. Besides, this bacterial chip canbe applied for direct analysis of pathogenic bacteria. When immersed into a solution containing pathogenic bacteria, it is able tolead to rapid and sensitive capture of the pathogenic bacteria from the solution and hence act as a bacterial sensor. We have
optimized the ideal chip which exhibits accelerated capture efficacy as the 800°C heat treated chip. These bacterial chips serve
dual roles；（ 1 ）They can be applied as MALDI-MS target plates for direct and highly sensitive bacterial analysis.（ 2 ）They can beused as bacterial sensors for direct analysis of the captured bacteria using MALDI-MS. The sensitivity of these chips when used asbacterial sensors is < 103cfu/mL. The lowest detectable concentration by direct MALDI-MS analysis was found to be 104 cfu/mL.
The results were further justified by using standard plate counting method combined with Tukey Kramer statistical analysis andfluorescence imaging followed by image processing for fluorescence quantification using ImageJ software to substantiate the
MALDI-MS results.

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 19: Mass Spectrometric Diagnosis

PTu-076

13:30 － 14:40

An optimized membrane proteins enrichment strategy for the bottom-up
mass spectrometry approach

Sravani Musunuri, Ganna Shevchenko, Magnus Wetterhall, Jonas Bergquist
Uppsala University, Uppsala, Sweden

Keywords:
Brain, Membrane proteins, Cloud point extraction, Two dimensional gel electrophoresis, Mass spectrometry
Novel aspects:
Application of the cloud point extraction and 2 -DE separation to increase the coverage of membrane proteins by reducing thecomplexity of brain proteome for the mass spectrometry analysis.
Abstract:
Neuroproteomics is a continuously emerging field with the intention to identify biomarkers in various neurodegenerative disorders.
The usage of mass spectrometry in neuroproteomics has become a powerful tool for the analysis of these very complex samples.
However many challenges arise due to the diverse nature of the tissue and the high dynamic nature of the proteome. The
complexity of biological samples can be reduced by introducing various pre-fractionation techniques during sample preparation,
which determine the end result for the downstream proteomic analysis. With the aim to identify proteins in a highly complex brain
tissue we tested various detergents, organic solvents and organic acids for the selective extraction of membrane bound proteins（MPs）. The protocols where initially tested in mouse brain and the optimized extraction conditions were further applied tohuman brain tissue.
In this study, brain proteins extracted by various protocols were delipidated, on-filter digested and analyzed by reversed phasenanoliquid chromatography in combination with electrospray ionization tandem mass spectrometry using a 7 Thybrid LTQ-FT
mass spectrometer. Detergent based lysis buffers produced high yield of proteins and high percentage of MPs（25-30%）which is inaccordance with the genome expression levels. Among the tested detergents, Triton X-114 and n-Octyl-β-D-glucoside detergents
provided the highest total number of identified proteins as well as the highest numbers and percentages of identified membrane（29 %）and transmembrane proteins（52 %）. An additional advantage of Triton X-114 is temperature induced pre-fractionation
separation called as cloud point extraction（CPE）. In CPE technique using Triton X-114 when the temperature is raised above 36ºC, separation of hydrophilic and hydrophobic fractions was achieved. By exploiting this unique trait of polyoxyethylene non-ionicdetergents, CPE was performed on human brain tissue to characterize the low abundant MPs as well as transmembrane proteins.
A total of 638 proteins were identified in the human brain extract, using a gel free on-filter tryptic digestion prior to mass
spectrometry. All proteins were classified according to their sub-cellular localizations. Among them 30 % were identified as MPsfrom which 20% are found exclusively in the hydrophobic fraction, 5 % in the hydrophilic fraction and the remaining 5 % in both.
Hydrophilic and hydrophobic phases of the human brain CPE extracts were subjected to further separation by 2 -DE and theprotein spot patterns were analyzed. The hydrophobic membrane fraction shows unique spots in 2 -DE that are typically masked
by high abundant proteins in the hydrophilic fraction. The preliminary data suggests the potential application of pre-fractionationand separation steps that are compatible with“shotgun“ based mass spectrometry approach to increase the coverage of low
abundant MPs. The optimized extraction conditions mentioned here can be used for studying the potential biomarkers of interestin neurodegenerative disorders.

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 19: Mass Spectrometric Diagnosis

PTu-077

11:10 － 12:20

alpha-Tocopherol suppresses lipid peroxidation and behavioral
impairment in mouse model of Down syndrome

Mototada Shichiri1,2, Noiriko Ishida1, Yoshihisa Hagihara1, Yasukazu Yoshida1, Hiroshi Tamai2,
Etsuo Niki1

National Institute of Advanced Industrial Science and Technology (AIST), Osaka, Japan, 2Department of Pediatrics,
Osaka Medical College, Osaka, Japan

1

Keywords:
Ts65Dn mouse, Down syndrome, oxidative stress, lipid peroxidation, vitamin E
Novel aspects:
alpha-Tocopherol supplementation from an embryonic stage may be an effective treatment for the cognitive deficits associatedwith Down syndrome
Abstract:
It is widely accepted that oxidative stress is involved in the pathogenesis of Down syndrome, but the effectiveness of antioxidant
treatment remains inconclusive. In the present study, we tested whether chronic administration of α-tocopherol, the biologicallymost active form of vitamin E, ameliorates the cognitive deficits exhibited by Ts65Dn mice, a mouse model of Down syndrome.
Ts65Dn mice carry a segmental trisomy of mouse chromosome 16. Many of the genes in human chromosome 21 are conserved inmouse chromosome 16. Although Ts65Dn mice do not have all the features associated with Down syndrome（such as congenital
heart defects）, they display many Down syndrome-like features, including significant performance deficits in specific behavioral
tasks. α-Tocopherol was administered to pregnant Ts65Dn females from the day of conception throughout the pregnancy, and topups during their entire life, from birth to the end of the behavioral testing period. Lipid peroxidation products, includinghydroxyoctadecadienoic acid, 7 -hydroxycholesterol and 8 -isoprostane were measured by using mass spectrometry. First,
cognitive deficits were confirmed for Ts65Dn mice fed on the control diet, revealing reduced anxiety or regardlessness in theelevated plus maze task test and spatial learning deficit in the Morris water maze task test. However, supplementation with α-tocopherol attenuated both cognitive impairments. In addition, we found that the levels of 8 -isoprostane in the brain tissue andhydroxyoctadecadienoic acid and 7 -hydroxycholesterol in the plasma of Ts65Dn mice were higher than those in control mice.
Supplementation with α-tocopherol decreased these lipid peroxidation products in Ts65Dn mice. Furthermore, we demonstrated
that supplementation with α-tocopherol improved hypocellularity in the hippocampal dentate gyrus region of Ts65Dn mice. In
addition, we found that the lipid peroxidation product acrolein was stained in the dentate gyrus region of Ts65Dn mice. These
results indicate that lipid peroxidation closely relates to hypocellularity in the dentate gyrus region of Ts65Dn mice. These results
imply that chronic α-tocopherol supplementation from the embryonic stage may be an effective treatment for the cognitive
deficits associated with Down syndrome.

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 19: Mass Spectrometric Diagnosis

PTu-078

13:30 － 14:40

PGRN: a novel therapeutic target and biomarker for insulin resistance andobesity identified by differential proteome analysis

Matsubara Toshiya1,2, Minami Kohtaro2, Yokoi Norihide2, Nishimura Osamu2, Seino Susumu2
1

Shimadzu Co., Kyoto, Japan, 2Kobe University Graduate School of Medicine, Hyogo, Japan

Keywords:
insulin resistance, obesity, differential proteome analysis, diabetes, NBS method
Novel aspects:
Identification of a novel therapeutic target and biomarker for insulin resistance and obesity identified by differential proteome
analysis.
Abstract:<Background>
Insulin resistance is a characteristic feature of obesity and type 2 diabetes. Adipose tissue is now recognized as not only anenergy-storage tissue, but also an endocrine tissue that secretes a variety of bioactive substances（adipokines）. Defects inadipokine secretion accompanying adipose tissue dysfunction contribute to the pathophysiology of insulin resistance and obesity.
The relationship between inflammatory process and insulin resistance has recently drawn considerable attention. For example,
TNF-alpha, a proinflammatory cytokine, has been shown to contribute to the development of insulin resistance. On the other
hand, glucocorticoids, which are known to have an anti-inflammatory action, also induce insulin resistance in human and animals.
Dexamethasone, a glucocorticoid, has been reported to impair insulin signaling and insulin-stimulated glucose uptake in adipose
tissue, liver, and skeletal muscle. Since TNF-alpha and dexamethasone both induce insulin resistance despite their opposite
inflammatory properties,we reasoned that there might be a common key mediator responsible for the cellular basis of insulin
resistance induced by TNF-alpha and dexamethasone.
In the present study, we searched for a novel adipokine（s）that play a key role in developing insulin resistance using 3 T 3 -L 1
adipocytes treated with TNF-alpha or dexamethasone. For this purpose, we utilized a method of differential proteome analysis
based on stable isotope labeling of proteins with chemical reagent 2 -nitrobenzenesulfenyl chloride（NBSCl）incorporating six 13C（13C6）or six 12C（13C0）in the tryptophan residues.
<Aim>
The aim of this study is to search for a novel adipokine（s）that play a key role in developing insulin resistance.
<Methods>
To identify proteins associated with insulin resistance in adipocytes in vitro, differential proteome analysis using the NBS method
was performed in 3 T 3 -L 1 adipocytes in which insulin resistance was induced by TNF-alpha or dexamethasone. The relative
quantification and the identification of differentially expressed proteinswere performed using LC-MALDI-TOF MS.
<Results>
We found that 37 and 43 proteins were upregulated by TNF-alpha treatment and dexamethasone treatment, respectively, among
which 21 proteins are common in the two treatments. We also found that 11 and 7 proteins were downregulated by TNF-alphatreatment and dexamethasone treatment, respectively, among which 2 proteins are common in these treatments. Identification of
haptoglobin, serum amyloid A- 3 （SAA 3 ）protein precursor and nicotinamide phosphoribosyltransferase, all of which are known
as the adipokines to be upregulated by such treatment, confirmed the validity of the method. After excluding known adipokines
among the 23 proteins identified, we confirmed the results of differential proteome analysis on 8 proteins by immunoblot analysis
using antibodies currently available. We finally selected progranulin（PGRN）because it is the only protein with both secretory andproinflammatory properties and further analyzed the protein functions.
PGRN was upregulated in both TNF-alpha-treated（1.66-fold vs. control）and dexamethasone-treated（3.01-fold vs. control）
adipocytes. PGRN in blood and adipose tissues was markedly increased in obese mouse models and was normalized withtreatment of pioglitazone, an insulin-sensitizing agent. Ablation of PGRN prevented mice from high fat diet（HFD）-induced insulin
resistance, adipocyte hypertrophy, and obesity. PGRN deficiency blocked elevation of IL-6, an inflammatory cytokine, induced by
HFD in blood and adipose tissues. Insulin resistance induced by chronic administration of PGRN was suppressed by neutralizing
IL- 6 in vivo.
<Conclusion>
PGRN is a key adipokine that mediates HFD-induced insulin resistance and obesity through production of IL- 6 in adipose tissue,
and may be a promising therapeutic target for obesity. Differential proteome analysis based on the NBS method described here
should be useful for identifying proteins involved in insulin resistance in various tissues.
<Reference>
Matsubara T. et al., PGRN is a key adipokine mediating high fat diet-induced insulin resistance and obesity through IL- 6 in adipose
tissue. Cell Metab.（2012）15, 38-50.

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 19: Mass Spectrometric Diagnosis

PTu-079

11:10 － 12:20

Direct molecular analysis by pico-drop sweat trapping from a single glandon a finger

Harue Hiramoto1, Kanako Honda1, Naohiro Tsuyama1, Hajime Mizuno1, Iwao Sakane2, Sachiko Date3,
Tsutomu Masujima1,3
13Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima, Japan, 2ITO EN Ltd., Shizuoka, Japan,
Quantitative Biology Center, RIKEN, Japan

Keywords:
sweat, analgesic diagnosis, single sweat gland, live single-cell MS
Novel aspects:
Many compounds from a single sweat gland of a finger were successfully identified and drug kinetics in sweat was found to be
similar to that in plasma.
Abstract:
Introduction
Development of diagnosis methods which is analgesic, non-invasive, highly sensitive and rapid is expected for future medicine.
Since sweat includes biological and chemical compounds originated from blood, it reflects the concentration of these materials inblood. To prove sweat can be used for the analgesic diagnosis to monitor blood content such as drug, alcohol level, and also for
doping, we picked a sweat droplet from a single sweat gland with a nanospray tip followed by direct submitting to nanoESI MS.
We succeeded to achieve pico-drop sweat-nano mass spectrometry which enables direct sampling of sweat droplets of picolititer volume in analgesic way.
MethodWe selected a finger’s sweat gland as the region of picking sweat. The subjected volunteer humans were those who took
medicine（troxipide, a stomach-medicine, KYORIN pharmaceutical Co., Ltd.）or not. First, we flushed over the surface of the finger
and wiped water off with a tissue and then waited about 2 minutes until the temperature of finger surface recovered to theoriginal temperature. Then we observed sweat from finger’s sweat glands by a stereomicroscope, and sucked sweat quickly after
secretion by a nanospray tip. We added 2 µL of ionization solvent of 70% MeCN aq from the back of the tip, then we analyzed
that tiny amount of sweat by the mass spectrometer, LTQ-Orbitrap（Thermo Fisher Scientific）. The ion spray voltage was 1000V
for positive ion detection mode. Aligned peaks were applied with PCA and t-test for sweat specific peak detection. We also tried
to detect time-dependent change of the peak intensity of the medicine in sweat and compared to that of plasma concentration.
ResultWe detected many peaks from a sweat droplet and identified. For example, urea, lactic acid, ornithine, urocanic acid（which is
the metabolic intermediate of L-histidine）, sodium chloride, and 15 amino acids such as alanine, valine, proline, threonine,phenylalanine from the sweat of both subjects. The peak at m/z 295.1652 was also detected only in the drug-administered person.
It was identified as troxipide by the exact mass and MS/MS. Sequential analysis of troxipide showed the highest intensity around2 - 3 hours after taking medicine and the intensity lowered gradually. It was very similar to the previous report of plasma
dynamics. We tried to calculate the concentration of this medicine in the sweat by making standard curve. First, we optimized thecomposition of ionizing solvent for sensitive detection of the drug. We prepared solvents with different blend ratio and analyzed
the standard troxipide, and we found that 70% MeCN aq was the best. Next, we dissolved the standard medicine with the solvent
and then analyzed. We found that the peak intensity was correlated highly with the concentration of medicine. The standard
curve was linear between the concentration of 10nM to 1 µM with the correlation coefficient of 0.99, and the slope of the standard
curve didn’t change when we added sweat to troxipide solution. When we calculated troxipide concentration in sweat droplet
with the standard curve, it was about 30nM. It was significantly lower than plasma concentration. These data suggest thatmolecular content can be quantitatively analyzed from directly isolated extremely tiny sweat using this method. If the analgesic
diagnosis comes to be real by this method, it will greatly contribute the establishment of new clinical laboratory system.

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 19: Mass Spectrometric Diagnosis

PTu-080

13:30 － 14:40

Identifications of amyloidogenic proteins in FFPE tissue sections by
MALDI-imaging coupled with on-tissue digestion andimmunohistochemical/microscopic examinations

Toyofumi Nakanishi1, Minako Ito1, Takashi Nirasawa2, Teruo Ueno1, Motomu Tsuji1, Takayuki Takubo1
1

Osaka Medical College, Takatsuki, Japan, 2Bruker Daltonics K.K., Yokohama, Japan

Keywords:
MALDI-MS imaging, AL-type amyloidosis, On-tissue trypsin digestion, Immunoglobulin lambda
Novel aspects:
MALDI-imaging coupled with on-tissue digestion technique enables us to identify amyloidogenic proteins in FFPE sliced tissue
sections without any procedure for retrieval of epitopes before on-tissue digestion.
Abstract:
Matrix-assisted laser desorption time-of-flight ionization（MALDI）-imaging MS（IMS）with MSMS analysis using on-tissue tryptic
digests is a powerful tool for identification of disease-related proteins in formalin-fixed paraffin-embedded（FFPE）tissue sections.
We applied these techniques to identify tryptic peptides of deposited amyloidogenic proteins in amyloidosis tissue sections.
Sequence determination of tryptic peptides was performed using MALDI-MSMS analysis directly from Congo-red positive regionsin sliced tissue sections without any procedure for retrieval of epitopes before on-tissue digestion.
A tryptic peptide, m/z=1073.5, was identified with the sequence, YASWYQQK, from 48 th to 56 th position of the Ig lambda chain.
Heat-map image at m/z=1073.5 was overlapped with Congo red positive regions. Immunohistochemistry of FFPE tissue sections
were confirmed to react with anti-amyloid P component and anti-lambda chain antibodies, not an anti-serum amyloid A antibody.
From results of IMS analysis and immunohistochemistry, we diagnosed AL-type of lung-amyloidosis in this case. We also identified
amyloidogenic proteins at the other 2 cases of amyloidoses. In conclusion, the protocol enables us to identify insoluble deposited
proteins in amyloid plaques using FFPE in the same way as fresh frozen tissues.

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 19: Mass Spectrometric Diagnosis

PTu-081

11:10 － 12:20

LC-MS/MS based basal and ACTH-stimulated serum concentrations ofadrenal and gonadal steroid homones and its application on heterozygote
CYP21A2 mutations

Alexandra E Kulle1, Juergen Hedderich2, Dorothee Roessner1, Jessica Schmitz1, Lena Niermeyer1,
Paul-Martin Holterhus1, Felix G Riepe1

University Hospital Schleswig-Holstein / Children’s Hospital, Kiel, Germany, 2University Hospital schleswig-Hosltein/
Institute of Mediacal Informatics and Statistics, Kiel, Germany

1

Keywords:
adrenal and gondal steroid concetrations, heterozygote carriers of 21OHD, ACTH stimulation test
Novel aspects:
Multisteroid assay for basal and ACTH stimulated steroid hormones, identifiying heterozogote carriers for 21-hydroxylase
deficiency
Abstract:
Background:Congenital Adrenal Hyperplasia（CAH）is composed of several inherited defects of steroid biosynthesis that impair cortisol
biosynthesis. 21-hydroxylase deficiency（21OHD）is the most common form of CAH and has a high prevalence of asymptomaticheterozygote carriers in the general population. A screening method and case detection of heterozygote carriers of 21OHD istherefore greatly useful. The key steroid of 21OHD is 17-hydroxyprogesterone, which is highly elevated in homozygote carriers
while heterozygote carriers have normal to slightly elevated values. Both derivatives 11-deoxycortisol and cortisol are humiliated in
21OHD.
Objective:
To determine basal and ACTH-stimulated values of progesterone, deoxycorticosterone （DOC）, corticosterone,17hydroxyprogesterone（17OHP）, 11-deoxycortisol（11S）, 21-deoxycortisol（21S）, cortisol（F）, cortisone, androstenedione,
testosterone and dihydrotestosterone genotypic normal subjects and heterozygote carriers of CYP21A 2 mutations, to generatereference ranges for these hormones, and to determine a distinctive basal or ACTH-stimulated parameter for separation ofheterozygote carriers of CYP21A 2 mutations from normal controls.
Methods and Materials:
We established an assay for measuring all hormones in small sample volume. 0.1 mL plasma was extracted by solid phaseextraction（SPE）and analyzed using an UPLC-MS/MS in MRM mode. We compared our assay to different commercial RIA assays
and a RIA with prior Extraction.
The study included 58 heterozygote carriers of CAH（35 males, 23 females, age range 6 -78 years）and 44 control subjects（25males, 19 females, age range 8 -58 years）. For all probands basal steroid hormone values, ACTH-stimulated values after 30 minutes
and after 60 minutes were determined. Quotients of 17OHP+21S/F and 17OHP/11S were calculated.
Results:
The coefficients of determination for the comparison were between 0.82 for DOC RIA vs LCMSMS and 0.99 for 17OHP RIA vsLCMSMS. With this validated assay basal and ACTH stimulated steroid concentrations for controls and heterozygote carriers were
determined. Basal steroid concentrations in controls were 0.06-2.84 ng/mL（17OHP）, 0.09-1.2 ng/mL（11S）and 40-370 ng/mL（F）.
ACTH-stimulated values after 30 min in controls were 0.06-2.94 ng/mL（17OHP）, 0.09-1.8 ng/mL（11S）, and 40-474 ng/mL（F）. The
ACTH-stimulated values after 60 min were 0.09-2.81 ng/mL,（17OHP）, 0.09-2.3 ng/mL（11S）and 100-534 ng/mL（F）. We foundsignificantly lower basal values for F in heterozygote carriers（p<0.003）. The ACTH-stimulated values of 17OPH after 30 min and 60
min were significantly higher in heterozygote carriers（P<0.001）. The quotient 17OHP/11S was significantly higher in heterozygote
carries both, at baseline as well as after ACTH-stimulation（p<0.001）. The quotient 17OHP/F was significantly higher inheterozygote carriers after ACTH-stimulation（p<0.1）. However all values showed an overlap between heterozygote carriers andnormal control subjects.
Conclusion:
We determined basal and ACTH reference ranges. We could distinguish between heterozygote carriers of CYP21A 2 mutations
and normal controls on the basis of the stimulated 17OHP and the quotient of 17OHP+21/11S with a specificity and sensitivity of90%.

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 19: Mass Spectrometric Diagnosis

PTu-082

13:30 － 14:40

Discovery of novel urinary biomarker candidates for diagnosis of prostatecancerKenji NAKAYAMA1, Takahiro INOUE2, Sadanori SEKIYA3, Minoru SUZUKI3, Hiroki TSUMOTO1,
Shin-Ichiro KAWABATA3, Shinichi IWAMOTO3, Kazuharu SHIMIZU1, Osamu OGAWA2,
Gozo TSUJIMOTO1, Koichi TANAKA3

Department of Cancer Omics Research, World-Leading Drug Discovery Research Center, Graduate School of
Pharmaceutical Sciences, Kyoto University, Kyoto, JAPAN, 2Department of Urology, Graduate School of Medicine, Kyoto
University, Kyoto, JAPAN, 3Koichi Tanaka Laboratory of Advanced Science and Technology, Shimadzu Corporation,
Kyoto, JAPAN1

Keywords:
MALDI-TOF/MS, prostate cancer, urine, peptidomics, biomarker candidates.
Novel aspects:
We discovered novel urinary biomarker candidates for diagnosis of prostate cancer. The amino acid sequences of the candidates
were identified using a MALDI-DIT-TOF/MS.
Abstract:
Background: For men in the United States, prostate cancer（PCa）is the most common type of cancer and the second highest
cause of cancer death［ 1 ］. In Japan, recently, mortality rates for PCa have been increasing dramatically as well［ 2 ］. Large-scaleclinical detection of prostate specific antigen（PSA）levels in serum as a biomarker of PCa has been carried out since the 1990s［ 3 ］. While the over all benefits and risks of population PSA screening for PCa continues to be assessed, the problem with PSA
not being cancer specific marker will continue to be a clinical problem［3,4］. For detecting PCa, the serum PSA cutoff value of 4.1
ng/mL is used globally, yielding only a sensitivity of 20.5% though a specificity of 93.8%［ 5 ］. Therefore, it is supposed that there isno cutoff value of PSA with both high sensitivity and high specificity for monitoring healthy men for PCa, but rather a continuum of
PCa risk at all values of serum PSA. Therefore, great emphasis has been placed on the need to discover novel biomarkers fordiagnosis of PCa. We focused on urine samples voided after prostate massage, because the urine samples after prostate massage
could be expected to contain many peptides and protein-fragments secreted from prostatic microenvironments, enabling
detection of secreted prostate products as potential sources of PCa specific biomarkers［ 6 ］. Therefore, we conductedpeptidomic and proteomic analyses of the samples using a matrix assisted laser desorption/ionization time-of-flight mass
spectrometer（MALDI-TOF/MS）in order to discover novel biomarkers for diagnosis of PCa.
Methods: This study was conducted with the permission of the Ethics Committee of Graduate School of Medicine, Kyoto
University. All examinations were performed with the informed consent of all subjects. Urine samples were collected as follows：
the clinician performed a digital rectal exam and gently massaged each side of the prostate gland three times； and the prostate
massages stimulated release of prostate fluids into the urethra. The prostate fluids were collected when the subjects voided urine
following the massage. The collected samples were filtrated with 100 μm cell strainers and were centrifuged at 2,000 x g for 10 min
at 20&#8451； to exclude urinary debris. After centrifugation, the supernatants were filtrated with the strainers again. The filtrated
supernatants were stored at -80&#8451； until the analyses. In this study, we developed a new analytical system： urinary peptides
and protein-fragments captured by ion-exchange resin were applied to the analyses using a MALDI-TOF/MS. This system wascomposed of ion-exchange capturing and concentrating processes of them in the urine samples, and of a desalting of theconcentrated biomolecules. CM-Sepharose as an ion-exchange resin and home-built MALDI digital ion trap（DIT）TOF/MS nmanufactured by Shimadzu Corporation were utilized. Differential analyses of mass profile data between the two groups, PCa
samples and non-cancer samples, were performed with a reflectron pass-through mode of MALDI-DIT-TOF/MS. Identification ofpeaks was analyzed with an ion-trap mode of the TOF/MS2. The peak lists obtained from the MS2 spectra were used to identify
amino acid sequences using the Mascot search engine. We targeted intact peptides and protein-fragments between 1,000 and
5,000 Da in the urine samples, because the sequences of many of them can be confirmed using MALDI-DIT-TOF/MSn in thismolecular weight range.
Results and Discussion: Novel urinary biomarker candidates were discovered for diagnosis of PCa. In this conference presentation,
we would like to discuss the relationships between these candidates and the diagnostic possibilities of PCa.
References:［ 1 ］Urol Clin North Am 2002；29：173.［ 2 ］Jpn J Clin Oncol 2005；35：690.［ 3 ］J Proteomics 2009；72：907.［ 4 ］NEngl J Med 2001；344：1373.［ 5 ］JAMA 2005；294：70.［ 6 ］World J Urol 2007；25：557.

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 19: Mass Spectrometric Diagnosis

PTu-083

11:10 － 12:20

Mass spectrometric determination of carcinoid syndrome tumor marker
5-HIAA in human serum

Niina Tohmola1, Outi Itkonen2, Sakari Joenvaara1, Risto Renkonen1,2, Esa Hamalainen2
1

University of Helsinki, Helsinki, Finland, 2Helsinki University Central Hospital, Helsinki, Finland

Keywords:
5 -hydroxyindole acetic acid, Carcinoid syndrome, Diagnostics
Novel aspects:
A HPLC-electrochemical detection assay is usually used to monitor the disease. We developed a simple and sensitive LC-MS/MS
method for the quantification of serum 5 -HIAA.
Abstract:
Aim:
In patients with carcinoid disease, urinary 5 -hydroxyindole acetic acid（ 5 -HIAA）is generally used to diagnose and monitor the
disease. To avoid the cumbersome 24h-urine collection, we developed and validated a liquid chromatography tandem mass
spectrometry（LC-MS/MS）method for the quantification of serum 5 -HIAA.
Materials and Methods:
We used serum samples from healthy volunteers. Samples were spiked with 5 -HIAA-D 2 internal standard and extracted using
WAX µElution plates（Waters）.The assay was performed using a Kinetex C18 column（Phenomenex）coupled to an Agilent
Technologies 1200 HPLC and an AB Sciex 4000 QTRAP MS with an ESI ion source in positive ionization mode. The duration of one
run was 3 min.
Results and Discussion:
The assay was sensitive with a limit of detection of 7.8 nmol/l and limit of quantification of 9.5 nmol/l and had a wide assay rangeof 10-5000 nmol/l. The within-assay variation was 3.8-5.3% and the day-to-day variation 5.8-10.5%. When 5 -HIAA was added to threeserum samples containing 99-252 nmol/l endogenous 5 -HIAA, the mean recovery was 90 %（spiked with 200 nmol/l）and 97 %（spiked with 500 nmol/l）showing good accuracy.
Conclusions:
We have developed a sensitive and accurate LC-MS/MS method for determination of serum 5 -HIAA. The assay has a short runtime and a wide assay range. Compared to 24h-urine collection, blood sampling is well controlled and fast. Therefore, this assay
may provide faster and more accurate diagnosis and follow-up of carsinoid syndrome as compared to determination of urinary
5 -HIAA.

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 19: Mass Spectrometric Diagnosis

PTu-084

13:30 － 14:40

Chip-based electrospray ionization tandem mass spectrometry a novel
tool for rapid diagnostic of Fabry disease

MIRELA GALUSCA1, CORINA FLANGEA1, CRISTINA MOSOARCA1,2, CLAUDIA COZMA3,
ALINA D ZAMFIR1

1
Aurel Vlaicu University, Arad, Romania, 2Physics Department, West University Timisoara, Vasile Parvan Blvd. 4, 300223,
Timisoara, Romania, 3Laboratory of Analytical Chemistry and Biopolymer Structure Analysis, Department of Chemistry,
University of Konstanz, 78457 Konstanz, Germany

Keywords:
Fabry disease； chip-based nanoESI； CID； ETD； rapid diagnosis
Novel aspects:
We developed here a novel platform for rapid and reliable diagnosis of Fabry disease based on chip-nanoESI CID and ETD
multistage MS in combination with enzyme assay.
Abstract:
Fabry disease is an inherited condition belonging to the group of lysosomal storage diseases（LSDs）characterized by the absenceor reduction of alpha-galactosidase A activity in lysosomes, that results in accumulation of globotriaosylceramide（Gb 3 ）andrelated neutral glycosphingolipids in blood vessel walls. The clinical onset occurs in childhood and over time, microvascularaffliction of the kidneys, heart, and brain progresses, leading to early death. One of the major problems in the management ofFabry disease is the lack of a standardized protocol for early diagnosis, before a major deterioration of the tissues to arise.
Therefore, in the last years important efforts were invested in development of methods for reliable detection of the disease. In
terms of rapid diagnosis, Fabry disease remains to date one of the LSDs, which benefited the most from the advantages of mass
spectrometry（MS）. The current MS-based strategies［1,2］target either the detection of non-degraded Gb 3 and lyso-Gb 3 inplasma, urine and tissue extracts or monitoring the alpha-galactosidase activity from dried blood spots（DBS）.
Although capable to provide early detection of the disease, occasionally, DBS-MS approaches generated false negative results. A
cause of these errors is the lack of MS systems able to ensure the level of reproducibility necessary for comparative screening
patient vs. healthy control. A reduced sensitivity and throughput of the MS experiments as well as the carry-over from sample tosample which might occur during successive MS analyses could also influence the obtained data. To eliminate these drawbacks,
we propose here a novel platform for reliable diagnostic of Fabry disease, based on enzyme assay and fully automated chipnanoelectrospray（nanoESI）MS, collision-induced dissociation（CID）and electron transfer dissociation（ETD）multistage MS（MSn）performed on a high capacity ion trap（HCT）MS, in-laboratory coupled to a NanoMate 400 robot. In our approach the
enzyme assay products derived from DBS of 11 healthy donors and two Fabry disease patients were automatically infused by
NanoMate robot into HCT MS, in a high throughput regime, under identical conditions. The cleavage of substrate GLA-S generated
a product, GLA-P, which could be quantified related to an internal standard GLA-IS. Due to the high sensitivity and reproducibilityof the experiments provided by chip-nanoESI MS, the comparative analysis patient vs. control indicated a 13 fold reduction in
GLA-P：GLA-IS ratio in the case of the subjects suffering from Fabry disease. GLA-P：GLA-IS ratios calculated according to theabsolute intensity of the MS signals showed 13 times lower relative values in Fabry patients as compared to controls, which clearly
indicates the dissimilarity in enzyme occurrence or activity. To substantiate this diagnostic, GLA-P and GLA-IS corresponding ionswere confirmed by CID and ETD used complementarily. This first employment of ETD in rapid diagnosis of a LSD was possible dueto the ability of chip-nanoESI to form multiply charged cations. The high performance of NanoMate-HCT MSn in terms of sensitivity
and reproducibility was also crucial in discriminating the Fabry cases where alpha-galactosidase is completely absent from thosein which the enzyme is present at the trace level. Additionally, the method demonstrated the capability to distinguish the Fabry
patients unable to synthesize the alpha-galactosidase from those producing an inactive enzyme. Both aspects have a major clinicalimportance for adequate therapy orientation. These results recommend chip-nanoESI MS as a valuable tool for detection andmonitoring of rare diseases.
［ 1 ］Z. Spacil, S. Elliott, S.L. Reeber, M.H. Gelb, C.R. Scott, F. Turecek, Anal Chem. 15（2011）4822-4828.
［ 2 ］X.K. Zhang, C.S. Elbin, W. L. Chuang, S.K. Cooper, C.A. Marashio, C. Beauregard, J.M. Keutzer, Clin. Chem. 54（2008）1725-1728.

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 19: Mass Spectrometric Diagnosis

PTu-085

11:10 － 12:20

Performance Evaluation of an Analytical LC-MS/MS Method for Accurate
Measurement of 25-OH Vitamin-D in Serum

Heather Gamble, Changtong Hao, Sha Joshua Ye, George Scott
IONICS Mass Spectrometry, Toronto, Canada

Keywords:
LC-MS/MS, Vitamin D, New Methods
Novel aspects:
Development of a rapid, highly sensitive method for quantitative analysis of 25（OH）vitamin D in human serum with limited sample
preparation requirements.
Abstract:
Background
Vitamin D is a group of fat-soluble hormones. The metabolites of vitamin D have a critical physiological function in maintaining
calcium and phosphate homeostasis. Vitamin D deficiency can best be diagnosed using 25（OH）vitamin D as opposed to theother vitamin D metabolites because 25（OH）vitamin D levels in serum reflect the body’s storage levels of vitamin D and correlatewith the clinical symptoms of vitamin D deficiency. We have developed a fast and accurate LC-MS/MS method to simultaneously
measure 25（OH）vitamin D 3 and 25（OH）vitamin D 2 covering an analytical concentration range of 0.1-100 and 0.4-125 ng/mL,
respectively.
Methods
25（OH）vitamin D was purchased from Sigma（Milwaukee, WI）and vitamin D free human serum was purchased from Golden
Western Biologicals（Temecula, CA）. The 25（OH）vitamin D spiked serum was deproteinized by mixing one volume of serum,
one volume of 0.1% formic acid solution and two volumes of acetonitrile. The mixture was vortexed for one minute followed bycentrifugation for 15 min. The supernatant was transferred to a clean vial for quantitation by LC-MS/MS. Two µL of supernatantwere loaded on a Fortis C18 column（50x2.1mm, 3 µ）coupled to an IONICS 3 Q 200 Series triple quadrupole detector with a
Shimadzu Prominence UPLC system. The binary gradient program used to elute 25（OH）vitamin D at a flow rate of 0.4 mL/min
was as follows： starts at 95% A（water：methanol = 95：5, v/v, with 0.1% formic acid and 5 mM ammonium acetate）； at 0.01 min,changes to 85% B（water：methanol = 5 ：95, v/v, with 0.1% formic acid and 5 mM ammonium acetate）； at 0.3 min, increases to
100 % B at 3 min, holds for 1 min, then decreases to 5 % B and equilibrates the column for two minutes. The quantifier masstransitions used for 25（OH）vitamin D 3 and 25（OH）vitamin D 2 are 401.3/257.2 and 413.2/355.2, respectively. All the solvents usedin this method are HPLC grade.
Results
This method covers a concentration range of 3 orders of magnitude from 0.1and 0.4 ng/mL for 25（OH）vitamin D 3 and 25（OH）
vitamin D2, respectively, while maintaining excellent linearity（R2 = 0.998）. The intra-day and inter-day variabilities for both 25（OH）
vitamin D range from 1 to 10 % . The intra-day variability is determined by processing 4 replicates of each QC sample and theinter-day variability is determined with 4 replicates in 3 batches.
Conclusion
A 6 -min, sensitive, and reliable LC-MS/MS method was developed for quantitative determination of 25（OH）vitamin D in humanserum. The sample preparation for this LC-MS/MS method is simple and well suited for routine clinical analysis of 25（OH）vitaminD.

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 19: Mass Spectrometric Diagnosis

PTu-086

13:30 － 14:40

Phosphoproteomic analysis of an EGFR/HER2 targeted drug-resistantgastric cancer cell line

Yong Yook Lee1, Minjueng Kang1, Byoung-Kyu Cho1, Hwang-Phill Kim2, Tae-You Kim2, Eugene C Yi1
Molecular Medicine and Biopharmaceutical Sciences, Seoul National University, Seoul, Korea, 2Cancer ResearchInstitute, Seoul National University Hospital, Seoul, Korea

1

Keywords:
Lapatinib, gastric cancer cell line, SUN216, phosphoprotein, LC-MS
Novel aspects:
Studies were performed with Q-Exactive LC-MS tandem mass spectrometry in lapatinib resistant gastric cancer cell line for aquantitative phosphoproteome evaluation.
Abstract:
Lapatinib, a dual kinase inhibitor of EGFR and HER 2 tyrosine kinases, has shown promising in vitro results in inhibiting the growthof HER 2 -overexpressing cancer cells. However, like other EGFR-targeted drugs, lapatinib drug resistance remains a major clinicalchallenge in targeted cancer therapy due to poor understanding of underlying mechanisms. To study the cellular mechanisms ofthe targeted cancer drug resistance, we generated lapatinib-resistant HER 2 -overexpressing human gastric cancer cell line（SNU216
LR）incubating lapatinib-sensitive SNU216 cells for a prolonged period with lapatinib. To identify signaling networks that are crucial
for maintaining the acquired drug resistance in SNU216 LR, we performed a quantitative phosphoproteome evaluation for the
SNU216 LR cells using a phosphopeptide enrichment method followed by Q-Exactive LC-MS tandem mass spectrometry analysis.
Several phosphoproteins involved in the downstream signal transduction of Ras/Raf/mitogen-activated protein kinase（MAPK）
pathway were found to be upregulated in SNU216 LR compared to its the parental cell line. These data suggest that activation of
the Ras/Raf/MAPK signaling pathway could be a potential mediator of resistance to lapatinib.

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 19: Mass Spectrometric Diagnosis

PTu-087

11:10 － 12:20

A Fast Method for Quantitative Determination of Methotrexate in Human
Serum by a LC-MS/MS

Sha Joshua Ye, Heather Gamble, George Scott
IONICS Mass Spectrometry, Toronto, Canada

Keywords:
Drug Monitoring, New Methods, LC-MS/MS
Novel aspects:
Development of a rapid, sensitive, accurate LC-MS/MS method for quantitation of methorexate in serum requiring little sample
preparation.
Abstract:
BackgroundIn the present study, an LC-MS/MS method was developed and verified for quantitation of methotrexate in human serum.
Methotrexate, a classical antifolate drug which has been in use for many years, is one of the oldest chemotherapy drugs. It iseffective in treating various neoplastic diseases such as acute leukemia, non-Hodgkin lymphoma, breast carcinoma, and more
recently autoimmune disease at much lower concentrations. Fast LC-MS/MS with high selectivity and sensitivity is a preferredanalysis technique.
Methods
Methotrexate and human serum were purchased from Sigma（Milwaukee, WI）. The methotrexate spiked serum was cleaned byprotein precipitation by mixing one volume of serum, one volume of 0.1% formic acid solution and two volumes of acetonitrile. The
mixture was vortexed for one minute followed by centrifugation for 15 min. The supernatant was transferred to a clean vial for
quantitation by LC-MS/MS. Five µL of supernatant were loaded on an Imtakt C 8 column（75x2.0mm, 3 µm）coupled to an IONICS
3 Q 200 Series triple quadrupole detector with a Shimadzu Prominence UPLC system. The mass transitions of 455.1/134.0 and
455.1/308.0 are used for methotrexate quantitation. The total LC cycle time is 3 min at a liquid flow rate of 0.4 mL/min. All thesolvents used in this method are HPLC grade.
Results
This method covers a concentration range of 0.06 to 275 µmol/L while maintaining excellent linearity（R2 = 0.999）. The intra andinter-day variability of three level QCs for this experiment were < 7 and 8 %, respectively. The intra-day variability is determinedby processing 4 replicates of each QC sample and the inter-day variability is determined with 4 replicates in 3 batches.
Conclusion
A fast, sensitive, and accurate LC-MS/MS method was developed for methotrexate in serum. The LLOQ achieved is 0.06 µmol/Lwith 5 µL sample injection. This LC-MS/MS method requires little sample preparation and is well-suited for routine therapeutic
drug monitoring of methotrexate.

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 19: Mass Spectrometric Diagnosis

PTu-088

13:30 － 14:40

Chip Based MicroLC-MS for Small Molecules Analysis

Remco van Soest1, Xiang Zhu1, Xiaohong Chen2, Nicole Hebert1, Hua-Fen Liu2
1

Eksigent/AB SCIEX, 2AB SCIEX

Keywords:
nanoLC chips chromatography steroids lc-ms
Novel aspects:
Use of chips in nanoLC-MS for analysis of small molecules with increased sensitivity.
Abstract:
Sensitive nanoLC-MS is widely used in a variety of research areas. Although providing excellent sensitivity, the low flow rate（200300 nl/min）reduces sample throughput which is important for small molecule analysis in clinical research. 200 um ID chip based
columns at proportionally higher flow rates（1.5 uL /min）were used to perform small molecule analysis. The delay time can begreatly reduced compared to nanoLC while still providing higher sensitivity with smaller sample volumes than conventional HPLC
methods. In this study the applicability of chip based microLC-MS for the analysis of small molecules（panel of steroids）incomplex biological matrices）is investigated.
The system used consisted of a NanoLC-Ultra（Eksigent, part of AB SCIEX）nanoLC system, cHiPLC Nanoflex and a QTRAP 5500（ABSCIEX）mass spectrometer. Separation was performed on a 200 um x 5 cm C18 chip column. Mobile phase A is H2O（0.1% formicacid）and mobile phase B is ACN（0.1% formic acid）. Flow rate is 1.5 uL/min and the gradient is 25%-60% B in 6 min.
Serum was spiked with a standard panel of steroids. After protein precipitation 1 uL of supernatant was injected. Data showshigh reproducibility across all runs. Retention time RSD is less than 0.5%, allowing for scheduling MRM’s with a narrow retention
time window. Good linearity was observed for the calibration curves of all analytes with correlation coefficients better than 0.99.
Observed LOQ’s are improved over typically reported LOQ’s in the literature using conventional LC/MS, taking into accountinjected amount of sample.

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 19: Mass Spectrometric Diagnosis

PTu-089

11:10 － 12:20

Accurate Mass LC-MS/MS Profiling of Synthetic Cannabinoids

Benjamin J Figard1, Christopher L Pennington2, Jeff Dahl3, Susan Leonard4, Jorge R Smith1

1
Shimadzu Scientific Instruments, South Central Region, Houston, TX, USA, 2Rice University, Core Mass Spectrometry
Facility, Houston, TX, USA, 3Shimadzu Scientific Instruments, Columbia, MD, 4Shimadzu Scientific Instruments,
Marlborough, MA

Keywords:
Accurate Mass Profiling Cannabinoids
Novel aspects:
Accurate Mass Profiling by LC-MS/MS for Sythetic Cannabinoids and comparison to Neutral Loss and MRM methodolies for LCMS
QQQ.
Abstract:
Designer drugs are typified by substances such as ecstasy, bathsalts, and synthetic cannabinoids. Many designer drugs areperceived as trendy, and cutting edge, with the most successful
ones, such as MDMA, becoming mainstream. Rapidly changing
formulations and the high prevalence of related analogues makes
tracking and stopping designer drug usage problematic for law
enforcement. In many cases the compounds in designer drugs
are structurally related to, and just as potent as, illegal/regulated
compounds, but are themselves,“legal.“In emergency rooms the rapid changing nature of designer drugs
present a problem to clinicians attempting to treat individuals
under their influence. In many cases available screening methods
are not capable of identifying what an individual has consumed.
As a result clinicians are required to rely on the account of thepatients who, in many cases, are too impaired to remember , ordo not know, what they took. In an effort to better understandthe complex, changing nature, of designer drugs LC
MS/MS analysis has been applied to characterize the components of bathsalts and synthetic cannabinoids.

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 19: Mass Spectrometric Diagnosis

PTu-090

13:30 － 14:40

A simple GC/MS procedure for vitamin D metabolite analysis

Anna M Przyborowska1, Graham D Carter2, Julia C Jones2, John M Halket1,2,3
1King’s College London, UK, 2Imperial College Healthcare NHS Trust, London, UK, 3Imperial College, London, UK

Keywords:
Vitamin D metabolites； GC/MS； Isotope-dilution； Serum； Derivatization
Novel aspects:
Simple and relatively inexpensive GC/MS procedure for analysis of important vitamin D metabolites in human serum
Abstract:
The level of 25-hydroxy-vitamin D 3 in serum is universally used as an indicator of vitamin D status, a parameter of increasing
importance in many aspects of health, including osteoporosis, cancer and, recently, multiple sclerosis. Although GC/MS methods
have been developed, they have been considered too complex for routine use as they involve a derivatization［ 1 ］or dehydration［ 2 ］step. Immunoassay, HPLC and LC/MS/MS methods are widespread. In the current work, GC/MS has been revisited and asimplified procedure developed using a relatively inexpensive mid-range GC/MS system as commonly found in clinical laboratories.
With superior chromatography as well as high specificity, the isotope-dilution procedure has potential as a standard reference
method available to DEQAS（http：//deqas.org/）, the external quality assessment scheme for vitamin D metabolites, in order toassess the validity of using the All-Laboratory Trimmed Mean（ALTM）as a surrogate for the“true“ value.
Calibrators, blank, spiked serum and patient serum samples（ 1 ml spiked with d 6 -25-hydroxy-vitamin D 3 as internal standard）
are deproteinized with acetonitrile and purified by a solid phase extraction procedure（OASIS HLB 1 cc）cartridges. After elution
with acetonitrile/methanol, trimethylsilyl ether derivatives are formed by reaction of dry residues with BSTFA/pyridine/acetonitrile.
Samples are analyzed by GC/MS with data acquired in the SIM mode： m/z 439, 413 and 544 for the pyro forms of 25-OH-VitaminD 3 and m/z 445 and 419 for the internal standard.
The trimethylsilyl ether derivatives of 25-OH-Vitamin D 3 and the deuterated internal standard show good gas chromatographic
and mass spectrometric properties. The common interferent in some LC/MS/MS assays, 3 -epi-25-hydroxy-vitamin D3, is well
separated.
The method is linear in the range 0 -75 ng/ml. Accuracy and precision data are reported together with validation data and resultsof spiking experiments as well as data from a range of DEQAS quality assurance samples analyzed. The method currently has ahigher limit of detection for the vitamin D 2 analogue, used as a supplement in some countries. Although it is not suggested as analternative procedure for the determination of vitamin D status in patients, it should prove useful for the rapid investigation of any
questionable results obtained from routine assay procedures.
［ 1 ］I. Bjorkhem, I. Holmberg, Clin. Chim. Acta 68, 1976, 215-221.
［ 2 ］R.D. Coldwell, D.J.H. Trafford, H.L.J. Makin, J. Mass Spectrom. 30, 2005, 348-356.

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 19: Mass Spectrometric Diagnosis

PTu-091

11:10 － 12:20

Highly sensitive method for arginine vasopressin in human plasma
determined by LC-MS/MS

Shin-ichiro Nitta1, Yasuko Tsukazaki1, Naoto Senda1,5, Shigeru Yamada2, Kazunobu Yamamoto2,
Mark M Garner3, Hesham Ghobarah3, Kunihiko Koriyama4, Kinya Kubo5
14

Mitsubishi Chemical Medience Corporation, Tsukuba, Japan, 2AB SCIEX, Tokyo, Japan, 3AB SCIEX, Toronto, Canada,
Shimadzu Corporation, Kyoto, Japan, 5Chromosome Engineering Research Center, Tottori University, Tottori, Japan

Keywords:
Arginine vasopressin, Vasopressin, LC-MS/MS, Hybrid capillary
Novel aspects:
High sensitive determine of arginine vasopressin in plasma was developed by LC-MS/MS equipped with a“metal and fused silica
hybrid capillary“.
Abstract:
Introduction
Arginine vasopressin（AVP）is one of peptide hormones which is synthesized in the hypothalamus and is stored in the posterior
pituitary. Most of them stored in the posterior pituitary is secreted to the blood stream. The regulation of the amount of water inthe body is the most important role of AVP. AVP increases urine osmolarity, increase of urine contents, and decreases theexcretion of water. Furthermore, AVP increases the vasoconstriction of peripheral blood vessel which leads to the arteriopressor.
The reduction of AVP excretion or that of sensitivity on AVP in the kidney leads to the diabetes insipidus. It also shows
hypernatremia（the increase of sodium concentration in plasma）, polyuria（over production of urine）and polydipsia（thirst）. On
the other hand, the hyperexcretion of AVP（syndrome of inappropriate antidiuretic hormone, SIADH）cause hyponatremia. We
developed and validated the analytical method of AVP level in human plasma using the hybrid triple quadrapole LC-MS/MS
system equipped with a“metal and fused silica hybrid capillary“ as electrode.
MethodThe standard solution of AVP（ 1 to 400 pg/mL, 7 points）was prepared with 1 % acetic acid aqueous solution containing 5 %acetonitrile. 400 μL of control serum（CONSERA, NISSUI PHARMACEUTICAL CO., LTD. Tokyo, Japan）was mixed with 40 μL ofeach standard solution to prepare for the calibration samples. Calibration/human plasma samples were extracted with solid
phase extraction（OASIS HLB, Waters Corporation, MA, USA）. After extracted sample was reconstituted by 200 μL of 1 % acetic
acid aqueous solution containing 5 % acetonitrile. LC-MS/MS was couple with conventional HPLC system. A mobile phase were
through a Inertsil Sustain C 8 column（flow rate： 100 μL/min, 100 × 2.1 mm ID, GL Science, Tokyo, Japan）using the gradient
program. Mobile phase was used 0.02% acetic acid aqueous solution as line A and acetonitrile as line B. The electrode of this LCMS/MS system was change from 100 μm diameter to 25 μm diameter.
Preliminary
We developed a high sensitive analytical condition（ 1 pg/mL of LLOQ）on AVP in human plasma using a LC-MS/MS system.
Linearity of the calibration curve was ranged from 1 to 400 pg/mL. In the calibration range, the precision（15.0% and lower）andthe accuracy（±15.0% and lower）were acceptable. Our method was very useful to detect AVP levels in human plasma collectedfrom 6 volunteers except for a part of analytical samples, which were collected from several other volunteers because of lower
than LLOQ（about 0.3 to 0.8 pg/mL）.

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 20: The Ion formation and Dissociation Mechanisms in MALDI

PTu-092

13:30 － 14:40

More and Less Susceptible Amino Acid Residues to In-source Decay ofProtein and Peptide in Matrix-Assisted Laser Desorption/Ionization Mass
Spectrometry

Mitsuo Takayama, Issei Osaka, Motoshi Sakakura, Ryunosuke Iimuro
Yokohama City University, Yokohama, Japan

Keywords:
ISD, MALDI, Susceptibility, Protein, Peptide
Novel aspects:
More and less susceptible amino acid residues to MALDI-ISD of protein and peptide have been determined from the stand pointsof secondary structure of protein.
Abstract:
In-source decay（ISD）coupled with MALDI has been recognized as a top-down approach for obtaining internal amino acidsequence from intact protein without any pre-digestion. Further, MALDI-ISD is a useful method for the analysis of post-translational
modification, because the method results in specific cleavage at N-Ca bond on the peptide backbone without loss or degradationof amino acid side chains and modified groups such as phosphate group. The MALDI-ISD uses hydrogen radical transfer frommatrix to analyte molecules in principle. The hydrogen radicals produced from active hydrogens of hydroxyl or amino groups inaromatic matrix molecules attach to carbonyl oxygen on the backbone, and peptide or protein radical ion species are generated
with protonation or deprotonation. The resulting radical ion species degrade within several ten nanoseconds in the MALDI ion
source, leading to the formation of N-terminal side c-series and C-terminal side z-series ions.
In this report, the susceptibility of the N-Ca bond oo the peptide backbone to specific cleavage by MALDI-ISD has been studied
from the standpoints of amino acid residues and secondary structure of protein. Analytes used for this purpose were horse
cytocrome c（Mr 12360）, horse myoglobin（Mr 16951.4）, bovine serum albumin（Mr 66402.8）, cucumber green mottle mosaic virus
court protein（Mr 17305.3）, and a tetra-phosphorylated model peptide（Mr 3606.6）. In order to determine more and lesssusceptible amino acid residues to the ISD, further MALDI-ISD spectra reported from other research groups so far were used.
From the peak abundance of resulting c-series ions in MALDI-ISD spectra of protein and peptide, it was concluded that more
susceptible amino acid residues to MALDI-ISD than other residues were Xxx-Asp, Xxx-Asn, Gly-Xxx, Val-Xxx and Ile-Xxx bonds,
while less susceptible residues were Xxx-Gly, Xxx-Val and Xxx-Ile bonds. Further, the resulting c-series ions originating from thecleavage at N-Ca bonds in flexible secondary structures such as turn and bend, which are free from intra-molecular hydrogenbonded helix structure, gave relatively intense peaks. This is in agreement with the observation that Gly, Asp and Asn residues are
preferred in turns rather than helix.
It has been reported that amino acid residues have preferences for certain secondary structures such as helix, sheet, turn, bend or
isolated residues. The turn, bend and isolated residue are classified as flexible regions without intra-molecular hydrogen-bonding
as seen with helix and sheet, so that the carbonyl oxygen atoms on the backbone in the flexible regions are able to interact withor bind to matrix molecules and hydrogen radicals. In contrast, helix and sheet structures protect the carbonyl oxygen atoms fromthe interaction with matrix molecules and hydrogen radicals. The amino acid residues Glu, Ala, Leu, Met, Gln and Lys are preferredin the hydrogen-bonded secondary structure of helix, while Pro, Gly, Ser, Cys and Tyr residues rather tend to inhibit formation ofhelix. In contrast, Gly, Asn, Asp, Pro and Ser are rather preferred in the flexible secondary structure of turn. It is expected from thetrend described above that the amino acid residues susceptible to MALDI-ISD, such as Gly-Xxx, Xxx-Asp and Xxx-Asn, arepreferred in turn or other flexible secondary structures, although the N-Ca bond of Xxx-Gly residue is less susceptible.
The results obtained from the MALDI-ISD of proteins and peptides here show a trend whereby relatively flexible regions such as
turn and bend structures are more susceptible to MALDI-ISD than intra-molecular hydrogen-bonded secondary structures such as
helix. This suggests that protein molecules embedded into matrix crystal still maintain the secondary structures as determined by
X-ray crystallography. The mechanism for more susceptible to MALDI-ISD in Gly-Xxx, Xxx-Asp and Xxx-Asn bonds will be discussed.

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 20: The Ion formation and Dissociation Mechanisms in MALDI

PTu-093

11:10 － 12:20

New MALDI matrices for negative-mode metabolomics

Stephan R Fagerer, Simone Nielsen, Alfredo J Ibanez, Renato Zenobi
ETH Zurich, Zurich, Switzerland

Keywords:
MALDI, metabolomics, matrix, negative-mode
Novel aspects:
Discovery of matrices for negative-mode MALDI of low-mass compounds（especially metabolites）with higher sensitivity and/or
better versatility than currently known matrices.
Abstract:
Quantitative detection of a wide range of metabolites is a prerequisite to meaningful metabolomic studies. Matrix-assisted laser
desorption/ionization mass spectrometry（MALDI-MS）has been shown to be a useful method for this task, with certain
advantages over electrospray-based measurements［ 1 ］： high sensitivity, high tolerance for salts and contaminants, and agenerally straightforward sample preparation.
Matrices that are suitable for metabolomics studies must fulfill several requirements, such as low chemical background in themass region of interest（usually 50- 1500 m/z）, high ionization efficiency for target compounds, and good co-crystallization（for aseries of solvents）. Only few matrices exist that fulfill all of these criteria： 9 -aminoacridine（ 9 -AA）
［ 1 ］, 1,8-bis（dimethylamino）
naphthalene（DMAN）［ 2 ］, and dihydroxybenzoic acid（DHB）［ 3 ］. In this study, we find new MALDI matrices for the detectionof low mass compounds in negative mode. We focus on matrices with two- and three-membered heterocycles substituted with 1
to 3 amino groups. Special attention was paid to choosing compounds with a very high（gas-phase）basicity. Their performancein MALDI-MS was screened and compared with known, well-established matrices. Several heteroaromatic amines, such as
ethacridine and 4 -amino- 2 -methylquinoline were found to compare favorably with established matrices, such as 9 AA and DMAN
in negative-mode MALDI. Moreover, they also allowed straightforward detection of all three classes of tested metabolites（aminoacids, nucleotide phosphates, as well as Krebs cycle intermediates）, which is not the case for 9 AA and DMAN.
［ 1 ］Vaidyanathan, S. and R. Goodacre, Rapid Commun. Mass Sp., 2007,21, 2072.
［ 2 ］Cohen, L.H. and A.I. Gusev, Anal. Bioanal. Chem., 2002, 373, 571.
［ 3 ］Shroff, R., et al., P. Natl. Acad. Sci. USA, 2009. 106, 10092.

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 20: The Ion formation and Dissociation Mechanisms in MALDI

PTu-094

13:30 － 14:40

Surface plasmon-induced cluster ion formation from Au, Ag, Pt, and Cu
irradiated by visible laser

Dilshadbek T Usmanov, Lee Chuin Chen, Kenzo Hiraoka
University of Yamanashi, Kofu, Japan

Keywords:
LDI-MS； Clusters； Surface plasmon； Nanoparticles.
Novel aspects:
There are two desorption/ionization mechanisms in LDI for metal samples, i.e., strong plasmon electric-field induced cluster ion
formation and delayed thermal ablation.
Abstract:
Introduction： The laser desorption ionization（LDI）near the threshold region was investigated using visible laser（532nm）for Au,
Ag, Pt, and Cu. It was found that for all the metal samples, only the small-size cluster ions were generated but the formation ofsmall-size neutral clusters was negligible. However, nanoparticles with the size of 10s nm were generated due to ablation. Thissuggests that there are the two desorption processes：（ 1 ）the formation of cluster ions due to the surface plasmon excitation（strong near field）at the moment of laser irradiation and（ 2 ）the nano-size neutral particles formation at the later stage by theablation（heat effect）.
Experimental： In this study, we studied the formation mechanisms of neutral and ionic products for metal samples near thethreshold region of LDI. The visible laser of 532 nm green Nd：YAG laser with the fluence in the range of 0.4-2.6 mJ/cm2 was used.
The metal targets examined were Au, Ag, Pt, and Cu. The targets were irradiated by laser in helium gas with pressure of about4600 Pa. The ions formed by the laser irradiation were sampled through an ion sampling orifice into vacuum and analyzed by atime of flight mass spectrometer（JEOL, AccuTOF）. The LDI source was equipped with the dielectric barrier discharge（DBD）ion
source. The barrier discharge was turned on and off during the laser irradiation on the metal samples. The mass spectra with DBD
on and off were measured.
Results and Discussion： LDI with DBD off, positive and negative metal cluster ions were formed. The cluster ions are composed ofless than 10 atoms. The LDI mass spectra were measured with DBD on and off. To our surprise there were no characteristic
differences in mass spectra with DBD on or off. If neural clusters were formed, the mass spectra should change with DBD on.
Thus, we concluded that LDI does not produce small-size neutral clusters.
After the laser irradiation, SEM images of the metal targets were observed. The surfaces were found to be eroded by laser
irradiation and numerous nano particles were found to be formed around the laser spot. These experimental results suggest thatthe cluster ions are formed by the strong near field at the early stage of laser irradiation, and later on ablation takes place by thetemperature rise caused by the relaxation of laser energy.
When 2,4,6-trinitrotoluene（TNT）that was deposited on the laser-ablated metal surface was irradiated by laser, TNT was detected
as negative ions, TNT-. The limit of detection was found to be lower than that obtained for the original metal substrate before
laser irradiation. This suggests the plasmon-assisted desorption/ionization for TNT.

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 20: The Ion formation and Dissociation Mechanisms in MALDI

PTu-095

11:10 － 12:20

Design and synthesis of new efficient MALDI-MS matrices for lowmolecular weight metabolites in negative ionization mode.

Mitsuru Shindo, Terai Yasuaki, Yukihira Daichi, Miura Saisuke, Fujimura Yoshinori, Matsumoto Kenji,
Wariishi Hiroyuki
Kyushu University, Fukuoka, Japan

Keywords:
MALDI-MS, matrix, 9 -aminoacridine, synthesis, metabolite
Novel aspects:
New efficient matrices, which were designed and synthesized based on 9 -AA, for MALDIMS have been developed for lowmolecular metabolite analysis in negative ionization mode.
Abstract:
Purpose： Recent years, MALDI-MS has been used not only for biopolymer analysis but also for low-molecular-weight metabolite
analysis because it is a highly sensitive, high-throughput, and low sample-consuming technique compared with other conventional
analytical platforms. Although metabolites are predominantly organic acids or phosphorylated moieties, making them anionic overa wide pH range, negative ionization mode has rarely been utilized due to poor sensitivity with commonly used matrices. Some of
the authors（DM, YF, and HW）have recently reported that the MALDI-MS-system with 9 -aminoacridine（ 9 -AA）as a matrix
improves the sensitivity of low-molecular weight metabolite analysis in negative ionization mode.1 However, even by using 9 -AA
as a matrix, there are numerous low-molecular weight metabolites which cannot be ionized. Although development of more
effective matrices has been required, especially for metabolomics. systematic study on the matrix including structure-function
relationship has rarely been reported so far. Herein, we report molecular design, synthesis and evaluation of new matrices for lowmolecular metabolite MALDIMS analysis in negative mode.
Method： More than 40 compounds including N- or C-substituted 9 -AA analogues, anthracene derivatives and quinoline
derivatives were synthesized as MALDI-MS matrix candidates. Using these synthetic matrices, 230 kinds of low-molecular weightmetabolites were measured by MALDI-TOF-MS in negative ionization mode. The obtained spectra were compared with thoseobtained using 9 -AA as a matrix in terms of sensitivity and S/N.
Results and Discussion： Several new synthetic matrices were found to be more efficient than 9 -AA, depending on the metabolites.
For example, while the use of 9 -AA could detect only 3 of the 40 amino acids and 5 of the 40 carboxylic acids,
9 -aminoantracene provided a much better analyte signal, allowing 12 amino acids and 20 carboxylic acids to be detected innegative mode. These results indicated that more potent matrices could be developed with organic synthesis. We will also reportour recent results on the structure function relationship of the matrices.
References： 1 ）Anal. Chem.2010, 87, 9789； Anal. Chem.2010, 82, 4278； Anal. Chem.2010, 82, 498.
Acknowledgements： This work was partially supported by KAKENHI（24659007, Grants-in-Aid for Challenging Exploratory
Research）and the Program for Promotion of Basic and Applied Research for Innovations in the Bio-oriented Industry（BRAIN）.

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 20: The Ion formation and Dissociation Mechanisms in MALDI

PTu-096

13:30 － 14:40

Femtosecond Time-Resolved Mass Spectrometry for Desorption Dynamics
of Tetracene Ion from Tetracene-doped Anthracene Crystals

Minagishi Yuuki, Morimoto Daisuke, Matsumoto Jun, Shiromaru Haruo, Hashimoto Kenro,
Tatsuya Fujino
Tokyo Metropolitan University, Tokyo, Japan

Keywords:
MALDI, Desorption, Femtosecond
Novel aspects:
Desorption dynamics of tetracene ion from teracene-doped anthracene crystals was investigated by means of femtosecond timeresolved mass spectrometry.
Abstract:
Matrix-assisted laser desorption ionization（MALDI）has been widely used as a valuable tool for the analysis in many research
fields since operation and preparation of samples are quite easy compared with other soft ionization methods. However, there areseveral drawbacks at the same time； 1 ）low efficiency for ionization, 2 ）application for low-molecular-weight compounds, 3 ）not applicable to all kinds of compounds. In order to develop a useful technique for MALDI mass spectrometry, it is also importantto fully understand desorption and ionization processes. Much effort has been directed to understand the ionization process bymany researchers. Although knowledge of the ionization process in MALDI has accumulated, the mechanism of the desorption
process remains unclear. In this study, we developed a femtosecond time-resolved mass spectrometer to probe a desorption
process. In conventional MALDI, the roles of matrix and analyte are complicated and not always clear, which sometimes hinder
the comprehensive understanding of the MALDI mechanism. To simplify the problem, tetracene-doped anthracene（TDA）crystals,
in which efficient energy transfer from anthracene to tetracene occurs, are used as a model system and the desorption dynamics
of tetracene ion（［tetracene］+）from the anthracene crystals is investigated［ 1 ］.
Using a 400 nm pump pulse, anthracene molecules in TDA crystals are initially excited to the S1 state. After a certain delay（< 10ps）of pumping, the excitation energy in the S 1 anthracene is transferred to a tetracene molecule to produce the S 1 state.
Tetracene in the S1 state is then ionized by a time-delayed probe pulse（266 nm）and desorbed tetracene ions from the crystals
are monitored with a time-of-flight mass spectrometer. The rise time constant of tetracene ion, which is considered to thedesorption time from the crystals, was determined to be 94.7 ps for TDA crystals with anthracene： tetracene concentration ratiosof 1 ： 0.01. The results showed that the desorption of analyte ions was governed by the electronic relaxation of matrix molecules,
which could lead to excitation of dissociative modes between matrix and analyte.
［ 1 ］Y. Minegishi, D. Morimoto, J. Matsumoto, H. Shiromaru, K. Hashimoto, T. Fujino“Desorption Dynamics of Tetracene Ion from
Tetracene-doped Anthracene Crystals Studied by Femtosecond Time-Resolved Mass Spectrometry“ J. Phys. Chem. C 116,3059~3064（2012）.

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 20: The Ion formation and Dissociation Mechanisms in MALDI

PTu-097

11:10 － 12:20

Matrix-assisted variable wavelength laser desorption ionization of
peptides; Influence of the matrix absorption coefficient on expansion
cooling

Sung Hee Ahn, Yong Jin Bae, Myung Soo Kim
Seoul National University, Seoul, Korea

Keywords:
expansion cooling, absorption coefficient
Novel aspects:
The temperature drop between early and late plume was measured by using the various wavelengths in matrix-assisted laser
desorption ionization. The results support the postulation of the expansion cooling.
Abstract:
The product ion yields in the in- and post-source decays of three model peptide ions,［Y5X + H］+（X = Y（tyrosine）, K（lysine）,
and R（arginine））, generated by matrix-assisted laser desorption ionization（MALDI）were measured at six wavelengths, 307, 317,
327, 337, 347, and 357 nm, using α-cyano- 4 -hydroxycinnamic acid（CHCA）and 2,5-dihydroxybenzoic acid（DHB）as the matrices.
The spot size of the laser was kept the same regardless of the wavelength. The laser pulse energy used was two times thethreshold value determined for each matrix at each wavelength. The temperatures of the early and late plumes generated by
MALDI were estimated via kinetic analysis of the product ion yield data. For both matrices, the temperature drop, i.e. thedifference in the temperature between the early and late plumes, displayed negative correlation with the matrix absorption
coefficient. This was in agreement with the previous qualitative reasoning that deeper laser penetration and larger amount ofmaterial ablation arising from smaller absorption coefficient would result in larger extent of expansion cooling. More importantly,
the results support the postulation of the expansion cooling occurring in the plume presented in a previous study.

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 20: The Ion formation and Dissociation Mechanisms in MALDI

PTu-098

13:30 － 14:40

Accompanying Compounds induced dehydration of Reserpine in Matrixassisted Laser Desorption/Ionization Process

Takehiro Watanabe, Tohru Yamagaki

Suntory institute for bioorganic research, Osaka, Japan

Keywords:
MALDI matrix-effect reserpine dehydrogenation
Novel aspects:
We studied the matrix effects of the dehydrogenation reaction of reserpine. The oxidative dehydrogenation of reserpine in the
MALDI process was influenced by functional groups property of matrices.
Abstract:
A plant alkaloid reserpine, extracted from Rauwolfia serpentina, is used as a neural drug such as antipsychoticity andantihypertensive. The chemical formula is C 33H40N2O9, and the molecular structure contains two types of N atoms； one of theindole type（sp 2 -hybridized）and the other is a tertiary N（sp 3 -hybridized）. It was reported that hydrogen atoms were easily
eliminated from the protonated molecule of reserpine［M + H］+（m/z 608）when the conventional electrospray emitter was used
at low solution flow rate. The dehydrogenation molecule of reserpine was observed in the MALDI-MS spectra. In the samemanner, those molecules would be generated at the MALDI ionization process. Until now, there have been no studies for theoxidation reaction of low molecule compounds in MALDI process with matrices and/or accompanying components. In this work,
we studied the matrix effects of the dehydrogenation reaction of reserpine.
MALDI-MS spectra were obtained using 2,5-dihydroxybenzoic acid（DHB）and
α-cyano- 4 -hydroxycinnamic acid（CHCA）,
5 -nitrosalicylic acid（NSA）, 1,5-diaminonaphthalene（DAN）, 5 -aminosalicylic acid（ASA）, 5 -formylsalicylic acid（FSA）as a
matrix. Yohinbin, 1,2,3,4-tetrahydro- 9 H-pyrido［3,4-b］indole and norharman were used for revealed a dehydrogenation part.
At first, whether matrix molecules influences dehydrogenation of reserpine. MALDI-MS spectra of reserpine（m/z 608）was
observed protonation ion［M + H］+ as a most abundant using hydrogen donating ability group（-OH or NH2）matrices such as DHB
and DAN, ASA, and the dehydrogenation ion［M - 2 H + H］+ abundance was low. On the other hands, dehydrogenation ion［M 2 H + H］+ as a most abundant ion observed using hydrogen acceptability matrix such as NSA and FSA. Therefore, the oxidative
dehydrogenation of reserpine in the MALDI process was influenced by functional groups property of matrix.
Following, yohinbin, 1,2,3,4-tetrahydro- 9 H-pyrido［3,4-b］indole and norharman with a partial structure of reserpine were analyzed
to examine a dehydrogenation part of reserpine in MALDI. The dehydrogenation ion was not detected in only 1,2,3,4-tetrahydro9 H-pyrido［3,4-b］indole without tertiary N atom. Consequently, it was revealed that the dehydrogenation process was caused
by a proton adduct to tertiary amine.

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 20: The Ion formation and Dissociation Mechanisms in MALDI

PTu-100

13:30 － 14:40

Highly-Soft-Ionization of Organic Compounds and Polymers by Using
Semiconductor Nanoparticels Sample Plate

Tetsu Yonezawa1, Hayashi Shinji2, Suganuma Takashi3, Hideya Kawasaki3, Ryuichi Arakawa3

Hokkaido University, Sapporo, Hokkaido, Japan, 2Kobe University, Kobe, Hyogo, Japan, 3Kansai Uniersity, Suita, Osaka,
Japan

1

Keywords:
GaP, SALDI-MS, Soft Ionization, Particle, Color
Novel aspects:
Usually SALDI-MS shows lower soft ionization ability than MALDI-MS but GaP nanoparticles overcome this problem.
Abstract:
MALDI-TOF-MS with organic matrices is a common soft ionization technique that features minimal fragmentation of mass analysis
especially for bio-molecules and polymers. Various UV-absorbing organic acids including 2,5-dihydroxybenzoic acid（DHBA）anda-cyano- 4 -hydroxycinnamic acid（CHCA）were introduced into MALDI system, and used to analyze various compounds. However,
MALDI-MS has some weak points, the most severe one is fragment peaks corresponding to the matrix compounds which areobserved in the mass region less than 500 Da. In this mass range, however, one can find many medicals and toxic compounds. In
order to overcome this problem, various organic-matrix-free systems have been introduced to realize the mass analyses of small
molecules. Among various systems, including DIOS and others, nanoparticle（NP）-based SALDI-MS has been proposed
increasingly during the last decade. Various inorganic NPs including metal oxides, metals, semiconductors, have been applied for
SALDI-MS inorganic matrices.
We have investigated the feasibility of GaP nanoparticles for SALDI-MS. GaP is one of the important semiconductor materials,
especially for light emitting diode（LED）. GaP NPs were prepared gas-evaporation method and show yellow dark red color
according to their sizes. These NPs were directly deposited onto stainless steel sample plates without addition of any stabilizing
reagent. Yellow particles are smaller and red particles are larger. This is attributed to the bandgap of GaP NPs. We tried to obtaina mass spectrum of polymers（PEG）with these GaP NP substrate. The fragmentation behavior strongly depended on the particle
color. Red particles showed no fragmentation but yellow ones showed many fragment peaks at lower mass region. Survival yieldmeasurements also strongly supported these data, which indicated that SALDI-MS with GaP nanoparticles show highly soft
ionization of the organic compounds and the obtained survival yield is same as MALDI-MS systems with organic matrix molecules.

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 20: The Ion formation and Dissociation Mechanisms in MALDI

PTu-101

11:10 － 12:20

Desorption/Ionization efficiency of 20 common amino acids in SALDI-MS
using platinum nanoflowers on silicon substrate

Shuhei Nitta, Hideya Kawasaki, Ryuichi Arakawa
Kansai University, Osaka, Japan

Keywords:
SALDI-MS, amino acids, proton affinity, acidity
Novel aspects:
We found that the Pt Nf-SALDI efficiency strongly depends on the kinds of 20 common amino acids as well as the ion modes（i.e.
negative or positive）.
Abstract:
Matrix assisted laser desorption/ionization（MALDI）is known as one of the soft ionizing methods of mass spectrometry（MS）. (1)However, it is sometimes difficult to analyze small molecule-samples because of the presence of interference-ion peaks fromorganic matrixes in the MALDI mass spectra. In contrast, surface assisted laser desorption/ionization（SALDI-MS）using inorganic
matrixes such metal nanoparticles or nanostructured has some advantages on the detection of small molecule-samples becauseof low interference-ion peaks from inorganic matrixes. (2) Recently, we have reported that the SALDI-MS by using a platinum
nanoflower（Pt Nf）with nano-scale sharp edges.(3) Although the desorption/ionization efficiency strongly depends on the chemical
structures of analytes, the relation between the chemical structure of a sample and the SALDI efficiency is not clear. Thus, wehave examined the relation between the chemical structure of analytes and the SALDI efficiency by using model analytes of 20
common amino acids with various side chains. We found that the Pt Nf-SALDI efficiency strongly depends on the kinds of aminoacids as well as the ion modes（i.e. negative or positive）.
We obtained the relative ion peak intensities（Ir）of 20 common amino acids to that of the angiotensin II as an internal standard.
All amino acids can be detected as a proton adduct form. The value of Ir was largest for arginine in positive ion mode, while thatof Ir was the largest for aspartic acid in negative ion mode. There was a good positive correlation between the proton affinity（PA）
of amino acids in gas phases and the I r in positive ion mode. In negative ion mode, we obtained the correlation between thegaseous phase acidity（GA）an of amino acids in gas phases and the I r. These results suggest that the desorption/ionization
process on Pt Nf-SALDI are similar to those of MALDI-MS using organic matrixes. It should be noted that the dominant detection of
protonated molecular ions in the Pt Nf-SALDI much differs from those of other SALDI-MS, where the dominant detection ofmolecular ions was metal-adduct ions（e.g. sodium adduct ions）.(3) The high efficiency of protonaed ions might be attributed tothe acidity-increases of OH groups on the silicon by the deposition of Pt nanoparticles. (4)References（ 1 ）M. Karas, D. Bachmann, F. Hillenkamp, Anal. Chem, 1985, 57, 2935.
（ 2 ）K. Tanaka et al., Rapid Commun. Mass Spectrom. 1988, 2, 151.
（ 3 ）H Sonderegger et al., Anal Bioanal Chem, 2011, 401, 1963.
（ 4 ）H. Kawasaki et al. Chem, Euro, J., 2010, 16, 10832.

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 20: The Ion formation and Dissociation Mechanisms in MALDI

PTu-102

13:30 － 14:40

Hydrogen Removing and Compound Properties of Aromatic Carboxylic
Acids for Negative-ion Generation in Matrix-assisted Laser Desorption/
Ionization Mass Spectrometry

Tohru Yamagaki, Takehiro Watanabe

Suntory Institute for Bioorganic Research, Osaka, Japan

Keywords:
MALDI, isotope pattern, DHBA
Novel aspects:
Only 2,5-dihydroxy benzoic acid（DHBA）showed a special property highly generating a hydrogen radical among six DHBA isomers.
Abstract:
Analysis of isotope patterns in MS signals are powerful methods for elucidation of chemical formula of analytes. Isotope patterns
are simulated and those are compared with experimental data. Generally, the calculation was performed for one chemical
species. It was important for us to determine the chemical species contributing the isotope patterns of the MS signals if a few
chemical species overlapped. In negative-ion matrix-assisted laser desorption/ionization mass spectrometry（MALDI-MS）,aromatic carboxylic acids have aromatic parts producing a molecular ion［M］- and a negative-charged carboxylic acid part
generating a deprotonated molecule［M - H］- . The observed MS signals were generated both from molecular ion［M］- anddeprotonatd molecule［M - H］- . They differed 1 Da, and overlapped each other. These complex overlapped isotope patterns
could hinder elucidation of the chemical formula of the analyte if it was unknown compounds. Moreover, hydrogen removing fromdeprotonated molecules was observed in the negative-ion mode MALDI-MS of aromatic compounds having phenolitic hydroxyl
groups, which caused the more complex isotope patterns from more than three different chemical species. Here, we studied
ionization tendency of aromatic carboxylic acids and those having hydroxyl groups. It is necessary to translate complex isotope
patterns of aromatic carboxylic acids having hydroxyl groups because those structural properties are in many natural compounds.
In the actual experimental data, benzoic acid was ionized as a deprotonated molecule［M - H］-. In contrast, both of the molecularion［M］- and deprotonated molecule［M - H］- were generated from 1 -naphtoic acid, and the ratio of molecular ion to
deprotonated molecule［M］-/［M - H］- was increased in 1 -anthoracenic acid. Aromaticity of the compounds tends to leadgeneration of molecular ion［M］- . In addition to that, the ratios differed among three anthracitic acid isomers, which made itdifficult to predict the isotope patterns. 2,5-Dihydroxy benzoic acid（2,5-DHBA）showed two deprotonated molecules such as［M H］- and［M - H - H］- which was generated from a hydrogen removing from a phenolictic hydroxyl group. The generation of thethird chemical species hindered translation of the isotope patterns, identification of the compounds, and elucidation of thechemical formula. Only 2,5-DHBA showed the special property highly generating a hydrogen radical among six DHBA isomers.

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 20: The Ion formation and Dissociation Mechanisms in MALDI

PTu-103

11:10 － 12:20

Matrix-assisted Laser Desorption Ionization Mass Spectrometry Using
Cation-Substituted Zeolite Surface

Takashi Asano1, Junya Suzuki2, Tatsuya Fujino2

Metropolitan Police Department, Tokyo, Japan / Hokkaido University, Hokkaido, Japan, 2Tokyo Metropolitan
University, Tokyo, Japan

1

Keywords:
MALDI, Zeolite, Alkali metal cation
Novel aspects:
An efficient matrix which is applicable to the compounds that cannot be detected by conventional MALDI was developed by using
2,4,6-trihydroxyacetophenone（THAP）adsorbed on cation-substituted zeolite surface.
Abstract:
Matrix-assisted laser desorption ionization（MALDI）in combination with time-of-flight（TOF）mass spectrometry（MALDI-MS）has
been widely used in many research fields because it enables observation of analyte ions in terms of their molecular weights.
MALDI-MS offers other advantages as well, including tolerance to contamination, a mass spectrum consisting of mostly singly
charged ions, and simple instrumentation. Of course, MALDI also has drawbacks. In MALDI-MS, usually, a peak of a protonatedanalyte,［M+H］+, is observed； however, the ion yield of the protonated analyte is sometimes influenced by the presence of alkali
metal ions. As biological samples contain an abundance of alkali metal ions intrinsically, the peak intensity of the protonatedanalyte is suppressed by peaks of alkali metal ion adducts. Secondly, MALDI is not suited for the study of low-molecular-weightcompounds. In MALDI-MS measurements, an analyte is mixed with a large amount of matrix molecules. The analyte undergoes
soft ionization for which it does not dissociate； however, the matrix molecules that absorb photons from an excitation laser
dissociate, producing many matrix-related peaks in the low-molecular-weight region less than 500 Da as the molecular weight of atypical MALDI matrix is less than 500 Da. Thirdly, the mechanisms of desorption and ionization are still unclear. Because of lack offundamental knowledge of the desorption and ionization mechanisms, the choice of matrix molecule suited for a particular study
is done by trial and error. This hinders the detection of many kinds of molecules by this method. To solve these problems, many
attempts have been made to use various compounds as co-matrices, and those attempts have produced good results. A matrixfree technique, such as DIOS and the use of a nano-structured surface have been proposed as well. Recently, we used
cyclodextrin and zeolite as host molecules for typical MALDI matrices and found that they suppressed the fragmentation of aguest matrix molecule and increased the peak intensities of the protonated analytes.
In this study, we also used zeolite. Zeolites are crystalline aluminosilicates with nanometer order cages, and usually act as solidacid catalysts for such processes as cracking, isomerization, and alkylation of hydrocarbons. Zeolites have high catalytic acidity
due to the charge imbalance at the Si-O-Al bridging sites, and those sites are compensated by such cations as H+ and Na+. In
particular, hydroxyl（OH）groups having Brönsted acidity exist in H+-exchanged zeolite, and it is well known that the Brönsted
acidity is responsible for various catalytic activities. In this study, we exchanged proton on the zeolite surface with alkali metal
cations. By using 2,4,6-trihydroxyacetophenone（THAP）adsorbed on cation-substituted zeolite as the new matrix（zeolite matrix）,
we succeeded in observing large ion peaks of cation-adducted analytes（low-molecular-weight compounds）. It was also foundthat the zeolite matrix is applicable to compounds such as acethylsalicylic acid, barbital, phenobarbital, colchicine, digoxin,
amygdalin, and so on, which cannot be detected by conventional MALDI. We are fully convinced that the zeolite matrix canfurther improve the applicability of MALDI-MS.

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 20: The Ion formation and Dissociation Mechanisms in MALDI

PTu-104

13:30 － 14:40

Correlation of Ionized Carbohydrate Structure with Peak Intensity of
Negative Ion Mass Spectrometry: First Principles Study

Hiromitsu Takaba1, Ryuji Miura1, Ai Suzuki1, Nozomu Hatakeyama1, Kazuko Hirose2, Takashi Nishikaze2,
Junko Amano2, Akira Miyamoto1
1

Tohoku University, 2The Noguchi Institute

Keywords:
MALDI, Negative ion CID, Carbohydrate, quantum calculation, ionization energy
Novel aspects:
This study would be the first study that investigated the stability of deprotonated structures and the correlation of mass intensitywith its ionization energy for longer oligosaccharides by quantum calculation.
Abstract:
Matrix-assisted laser desorption/ionization multiple-stage mass spectrometry （MS n ） has been essential techniques incharacterization of carbohydrates. In particular, negative ion collision induced dissociation（CID）spectra provides detailed
structural information of oligosaccharides by specific fragmentation patterns. Fragmentation is assumed to occur firstly fromdeprotonation of certain hydroxyl group of a carbohydrate, and then bond cleavages occur according to the patterns of electrontransfer from a deprotonated site to other sites. Original structure could be deduced according to the fragmentation patterns. This
fact means that deprotonation process is of fundamental for MSn spectrum acquisition because fragmentation patterns are mainly
specified by a location of the deprotonation site. Considering deprotonation process at a hydroxyl group, there are many possible
sites for the deprotonation in the carbohydrate. Ordering of frequency of deprotonation would depend on the stability of the
deprotonated structure varying by the interaction between the ionized oxygen atom and the other atoms in the molecule via thehydrogen bonding interaction or by the delocalization effect of electron. The degree of stability of a deprotonation site will show
the correlation with the signal intensity because energetically stable fragment is favorable to forming in CID which resulting in ahigh intensity of the signal. Therefore, understanding of the stability of deprotonated structure of oligosaccharide is important forthe better understanding of fragmentation. However, as long as our knowledge, the nature of deprotonation process of longer
oligosaccharides has not been studied.
In this study, atomistic structure and energetically stability of deprotonated structures for several oligosaccharides, e.g. NA2,
MFLNH and maltopentaose, were investigated by first principles calculations. Then the energy of deprotonated structures wascompared with our experimentally obtained MS n data and correlated with the intensity of the spectrum. We found a good
relationship between the stability of deprotonated structure and the intensity of the spectrum. The origin of stability of the
deprotonated structure was also investigated in the term of the specific interaction such as hydrogen bonding. The results show
the existence of the rule of stability for deprotonated sites. The rule will be used for the theoretical prediction and better
understanding of MSn spectrum. In the presentation the details of results and theoretical method for the prediction of intensity ofspectrum will be presented.
Acknowledgement
This work was supported by JST, Japan Science and Technology Agency（Development of Systems and Technology for Advanced
Measurement and Analysis）.

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 20: The Ion formation and Dissociation Mechanisms in MALDI

PTu-105

11:10 － 12:20

Statistical Investigation of Metabolite Structure to Reveal the Principle forPreferred Ionization in MALDI using 9-Aminoacridine

Daichi Yukihira, Yoshinori Fujimura, Mitsuru Shindo, Daisuke Miura, Hiroyuki Wariishi
Kyushu University, Fukuoka, Japan

Keywords:
MALDI-MS,metabolite analysis,9-aminoacridine,molecular descriptor,ionization
Novel aspects:
The ionizability of metabolites in 9 -AA-MALDI was characterized using molecular descriptors.
Abstract:
Background9 -Aminoacridine（ 9 -AA）is one of the matrices most often used for low-molecular-weight metabolite analysis by negative ion
mode matrix assisted laser desorption/ionization mass spectrometry（MALDI-MS）. To date, 9 -AA has been proven to be capableof ionizing a series of metabolites such as phosphorylated compounds, and utilized for various studies including sensitive
detection of metabolites and metabolite imaging. However, to clarify whether a metabolite is detectable, an experimental trial isneeded. As for metabolites that have not been detected by 9 -AA, we may need to employ drastic strategy such as rational
alteration of matrix to cover them. Although the ionizability of analyte in MALDI decisively depends on matrices, it is still elusive
which factor dominates the efficiency of the ionization. In this study, we investigate the interdependency of the chemical structuresof metabolites and their ionization efficiency assisted by 9 -AA in MALDI-MS. Quantitative structure-property relationship（QSPR）
was applied to associate the structure of metabolites with their ionization efficiency in MALDI. A merit of property modeling is not
only a rapid property estimation of unexamined compounds but also useful information about the structure features that are
relevant to desired properties.
Results
MALDI-MS analysis of metabolite standards. The ionization capability of 9 -AA was evaluated using 233 standard compounds ofmetabolites selected to general importance. Individual standard compound was diluted to graded concentrations and mixed with
9 -AA/MeOH solution（10 mg/mL）by 1 ： 1 （v/v）. The mixture was then subjected to MALDI-TOF-MS analysis to confirm thepossibility of ionization and the limit of detection（LOD）. As the result, 121 out of 233 metabolites were detected as deprotonatedpeaks. LOD varied from 10 fmol/well to 100~ µmol/well. We used the LOD value as an inverse measure of the ionization efficiency
at best on an ordinal scale.
QSPR analysis. A QSPR analysis is constituted of five steps： MALDI-MS data collection, descriptor calculation, variable selection,
predictive model construction through cross validation, and interpretation of the model. The molecular descriptors and fingerprints
were calculated by PaDEL-Descriptor[1] using MDL-molfile formatted files of individual metabolites acquired from KEGG（http：//
www.genome.jp/kegg/）or HMDB（http：//www.hmdb.ca/）. Modeling of interrelationship between the descriptors andionization efficiency of metabolites under 9 -AA-MALDI was conducted using several statistic method including logistic regression,
generalized linear model（GLM）and Random Forests. All the statistical analyses were performed using R（http：//www.r-project.
org/）. First we explored suitable combination of descriptors to predict the ionization capability, expressed as a response variable
categorized as ’ionized’ or ’not ionized’； or ’highly effective’ or ’less effective’, and so on, regarding the value of LOD. Random
Forests was employed for classification to select relevant variables. At this point, atom type electrotopological state indices were
found potentially relevant to ionization capability, and autocorrelation descriptor to the efficiency. As these descriptors are indicesreflecting molecular connectivity and constituting elements, potential rule for preferable ionization may be extracted. In thesession, construction of regression models will be discussed.
Conclusion
This study was primarily intended to lead 9 -AA-MALDI-MS analysis to be placed on a rational and predictive foundation. Relevant
descriptors found in this study can be interpreted as the structural preference specific to 9 -AA. Upcoming question is that, when
the structure of matrix alters, how the preference shall shift. This information will play an indispensable role on strategic control of
MALDI-MS-based studies.
［ 1 ］Yap CW, J. Comput. Chem. 2011, 32, 1466-1474.

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 20: The Ion formation and Dissociation Mechanisms in MALDI

PTu-106

13:30 － 14:40

Matrix clusters distribution from monomer to clusters with the mass atmega Dalton’s region

Szu-Hsueh Lai1,2, Kuang-Hua Chang1, Jung-Lee Lin1, Chung-Hsuan Chen1,2

1
Genomics Research Center, Academia Sinica, Taipei, Taiwan, 2Department of Chemistry, National Taiwan University,
TaiwanKeywords:
MALDI, Matrix Clusters, Sinapic acid
Novel aspects:
We gave the first demonstration for quantitative analysis of matrix clusters distribution from monomer to clusters with mass-tocharge（M/z）ratio reaching 2,000,000.
Abstract:
Introduction
Many publications have shown that matrix clusters play an important role in MALDI process. In this work, we try to demonstrate
a completely matrix clusters distribution from monomer to clusters with the mass at mega Dalton’s region by MALDI-TOF and ourhomemade MALDI- quadrupole ion trap mass spectrometer. This frequency scanned ion-trap system can quantitatively detectclusters signals by charge detector for high mass-to-charge ratio（m/z）from 20,000 to 2,000,000. To our knowledge, this is the firstpresentation for matrix clusters distributing to super-heavy region.
Methods
Compare to commercial quadrupole ion trap, lower frequency（10 kHz - 50 kHz）was used for our homemade QIT to trap ionswith large m/z. In order to quantitate the clusters amount, we fix the q z value scanned from 0.908 to 0.5 keeping the same
condition of potential well. Trapping frequency can be changed to cover the different m/z regions which can be calculated by
qzvalue and RF voltage. In general, a broadband power amplier（TREK, PZD2000A）was used to sustain RF voltage between 10 kHz
to 50 kHz and frequency scan was used to identify the m/z values of the ions.
Preliminary Data
We sketch the sinapic acid（SA）clusters distribution from monomer to large clusters with m/z reaching 2,000,000. The signal
intensity drops about 6 - 7 orders of magnitude from monomer to 5000 mers.

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 20: The Ion formation and Dissociation Mechanisms in MALDI

PTu-107

11:10 － 12:20

The initial ion velocity of MALDI-TOFMS matrix DCTB and affect upon themass accuracy of high molecular weight radical ions.

Mark F Wyatt1, Thorsten Jaskolla2

1
EPSRC Nat. Mass Spec. Service Centre, Swansea University, UK, 2Institute of Medical Physics and Biophysics, Universityof Muenster, Germany

Keywords:
MALDI； DCTB； ion velocity； radical ions； calibration
Novel aspects:
Previously undetermined initial DCTB matrix ion velocity and improved calibration of higher molecular weight radical ions.
Abstract:
One of the early investigations into the fundamentals of the matrix-assisted laser desorption/ionisation（MALDI）mechanism
revealed that the initial ion velocity of the matrix varied between matrices, and that this velocity affected the qualitative calibrationor mass accuracy of the analyte being characterised. The initial ion velocities of all the common MALDI matrices were measured
and incorporated into the calibration software of commercial MALDI-TOFMS instruments, so the analyst would choose the matrix
they were using in the software and so obtain the best possible mass accuracy. However, the applications of MALDI havecontinued to increase and with that have come several new matrices available to the analyst. One of these, 2 -［（ 2 E）- 3 -（ 4 -tertbutylphenyl）- 2 -methylprop- 2 -enylidene］-malononitrile（DCTB）, has proved very useful for characterising aryl, aromatic andmetal-containing analytes via the electron-transfer mechanism to produce radical ions. The initial ion velocities of these new
matrices were often not measured, so the analyst would use the median of known values or try all values with a known analyte
before selecting the best fit for future use. When using the initial ion velocity of another matrix with DCTB analyses, the observed
affect upon mass accuracy may be insignificant for small molecules, but significantly different for mass values of high mass
analytes.
For a range of known medium to high mass analytes, the initial ion velocity of DCTB has been estimated by successive analyses
using known values and comparing the observed mass accuracy. The initial ion velocity of DCTB has also been determined
directly, and the data from both approaches is compared and discussed in terms of the affect upon mass accuracy, and the wider
aspects of the MALDI mechanism.

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 41: Chemistries of Trapped Ions and their Applications to Biological Mass
SpectrometryPTu-108

13:30 － 14:40

Gas-Phase Fragmentation of Reducing Sugar Modified Small
Oligosaccharides

Chiharu Konda1, Brad Bendiak2, Yu Xia1
1

Purdue University, West Lafayette, IN, USA, 2University of Colorado Denver, Aurora, CO, USA

Keywords:
oligosaccharides, modification, ion fragmentation chemistry
Novel aspects:
Detail study of the fragmentation chemistry（the charge location effect and the sidedness of the fragmentation）using different
types of modifications on reducing end of oligosaccharides.
Abstract:
Carbohydrates play important roles in essentially all living organisms. Understanding these roles requires that their structures be
determined with confidence. This involves all levels of structural characterization, including the stereochemistry of each sugar, theanomeric configurations, the linkage positions between connecting sugars, and any modifications of sugars. Tandem mass
spectrometry（MS n ）has been widely applied in structural analysis of carbohydrates for determining the sequence, branching,linkage type, and positions of substituents. Collision-induced dissociation（CID）spectra of carbohydrates typically show very
complicated fragmentations due to the existence of structural isomers. Detail understanding of the fragmentation mechanisms isimportant for the characterization of carbohydrates.
Four types of modifications, such as 18 O, 4 -aminobenzoic acid（ABA）, 3 -amino- 2 -napthoic acid（ANA）, and ethyl-paminobenzoate（ABEE）were used on disaccharides to pentasaccharides. 18 O-labeling at the carbonyl oxygen of the reducing
sugar allows the distinction of the sidedness of the fragmentation pathway and mass discrimination of structural isomers. Sixteen
different disaccharides which are the smallest substructures derived from oligosaccharides that still contain a glycosidic linkage
between two monosaccharides were systematically studied. Comparisons of the MS2 CID spectra between 18O-labeled and nonlabeled disaccharides showed that there were some doublet ion peaks（offset of 2 m/z）indicating the existence of the structural
isomers with or without 18O. These ions were further examined by MS3 CID. The m/z 163 ion was a highly abundant ion that wasobserved beside m/z 161 ion in MS 2 spectra of all 18 O-labeled disaccharides and has the possible structure ofanhydromonosaccharide（reducing sugar）containing 18O. Further CID of this ion showed a distinct CID fragmentation patterns
according only to their linkage positions but did not show any differences between anomeric configurations and stereochemistryof the sugar. ABA, ANA, and ABEE modifications were used to study the effect of charge location. The fragmentation chemistry of
the modified disaccharides and trissacharides will be studied.

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 41: Chemistries of Trapped Ions and their Applications to Biological Mass
SpectrometryPTu-109

11:10 － 12:20

Radiosensitizers investigated using electrospray ionization mass
spectrometry

Linda Feketeova1, Niels Bassler2, Brita S Sørensen2, Michael R Horsman2, Jonathan White1,
Richard A J O’Hair1

School of Chemistry, The University of Melbourne, Parkville, Victoria, Australia, 2Department of Experimental ClinicalOncology, Aarhus University Hospital, Aarhus C, Denmark

1

Keywords:
nimorazole, misonidazole, radiosensitizer, hypoxia, electrospray ionization
Novel aspects:
First investigation of radiosensitizer compounds using mass spectrometry. A key finding is that radical anions M.- are directly
formed via electrospray ionization.
Abstract:
Radiosensitizers are used in radiotherapy（following the DAHANCA 5 clinical trial）to enhance tumour control of radioresistanthypoxic tumours. Radiosensitizers implemented particularly in the treatment of hypoxic cells are called ’electron-affinic’radiosensitizers. A number of nitroimidazoles and related compounds were studied in vitro as well as in vivo, with encouraging
results. However, the detailed mechanism of the actual radiosensitization is still unknown. A working hypothesis is that thesecompounds undergo redox reactions inside the cell, and that the nitroimidazole rings facilitate reduction via the formation ofradical anions.
The work presented here investigates the formation of nimorazole and misonidazole ions via positive and negative electrosprayionization mass spectrometry, and the subsequent fragmentation reactions of the cations and anions. Understanding the ionizationand fragmentation of radiosensitizers is crucial in evaluating the radiosensitization potential and developing new and more
effective drugs, which may improve tumour control probability in hypoxic tumours when using ion beams.
The experiments have been performed using a Finnigan- LTQ-FT mass spectrometer equipped with an electrospray ionisation
source. Collision-induced dissociation（CID）and electron-induced dissociation（EID）have been carried out by mass selecting thedesired ions and subjecting them to activation energy in the linear trap and to free electrons of 25.8 eV in the FT-ICR cell. We haveexamined CID and EID of protonated nimorazole and protonated misonidazole and CID of nimorazole and misonidazole radical
anion.
Electrospray ionisation of nimorazole in the positive ion mode yields protonated nimorazole［M+H］+ , with no radical cation
formation being observed. The major fragment ion in the CID spectrum of protonated nimorazole occurs at m/z 114 and arises
from loss of the nitroimidazole ring. Although this fragment is also observed in the EID spectrum, the dominant fragment in the EID
spectrum is m/z 110, due to the loss of nitroimidazole ring and CH2 group. In both spectra, a minor loss of NO2 radical is present.
In negative ion electrospray ionization, a radical anion of nimorazole M.- can be formed. CID of M.- generates amongst others thenegatively charged nitroimidazole ring at m/z 112 and a peak corresponding to a minor loss of NO radical.
In the case of misonidazole, the CID spectrum of protonated misonidazole shows the major fragment to be also an ion at m/z 114,
but in this case it is the nitroimidazole ring, which holds the charge after CID. This fragment is also the most dominant fragment in
the EID of the protonated misonidazole［M+H］+ .In contrast to CID of the protonated nimorazole, all the CID fragments ofprotonated misonidazole［M+H］+ correspond to fragments, where the positive charge is retained by nitroimidazole ring. Only one
fragment of m/z 89, where the charge is located on the side chain, was observed. Negative ion electrospray ionization ofmisonidazole also readily forms the radical anion M .-.The CID of the radical anion of misonidazole shows much more fragmentation
than in the case of nimorazole. Most of them include the loss of NO and NO2 groups. The negatively charged nitroimidazole ringat m/z 112 is also observed as in the case of nimorazole.
The fragmentation of both radiosensitizers appears to depend on the nature of the charge. This detailed gas-phase study will
unlock their fragmentation mechanism and highlight the similarities and differences in the fragmentation products observed.

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 42: New Developments in Instruments and Detectors

PTu-110

13:30 － 14:40

Expected possibilities of a new type gas-dynamic interface for preliminary
separation and transportation of ions into a mass analyzer

Valeriy V Raznikov, Vladislav V Zelenov

The Branch of Institute for Energy Problems of Chemical Physics, Chernogolovka, Russia

Keywords:
supersonic flow； EI； RFQ； ortho-TOFMS
Novel aspects:
Supersonic gas flow formation inside a capillary is confirmed experimentally. The estimated temperature of Ar flow is below 0.01K.
Two-grid mirror is proposed to extract ions from the flow.
Abstract:
A basic problem of mass spectrometric analysis of samples initially being under atmospheric pressure is an efficient input of either
or both an analyzed gas mixture and ionized species into a vacuum part of the instrument. We have described recently one
possible way to solve this problem［1, 2 ］. Its essence is using relatively long thin capillary at rather low initial pressure of a gas
to form a supersonic gas stream. This stream goes through a high-performance ion source with ionization by electrons with
controlled energy and further along the axis of the radio-frequency quadrupole（RFQ）coupled with time-of-flight mass analyzer
with orthogonal injection of ions（ortho-TOFMS）.
Relatively large gas density in the flow along RFQ axis and fast decreasing this density outside the axial zone are specific featuresof the considered case in comparison to conventional gas-filled RFQ. They provide additional opportunities to accumulate andpreliminary separate the target ions and simultaneously to eliminate preventing ion flows of basic components of investigatedmixture unimportant for analysis. The experimental data of cross-section distributions of recorded Xe ion flow from argon - xenon
gas mixture clearly show ring-like ion distribution around stronger focused beam of argon ions along the RFQ axis. Applying
appropriate axial electric field to stop ions inside RFQ, such ion stratification may be used for selective accumulation and forcollision induced dissociation of target ions. Similar and additional possibilities for ion manipulation may be implemented in caseof absence of the buffer gas ions in RFQ. This situation may be realized by reducing the electron ionization energy or by increasing
the RF focusing voltage, which results in instability of motion of small ions.
Experimental data analysis shows that only very small share of the ions formed inside supersonic stream is recorded by existing
system. Rather high density of this stream at the quadrupole exit does not allow to extract the most part of the ions from thestream and to give them energy sufficient for recording by the ortho-TOFMS. To eliminate these difficulties, one can move the ionsfrom the gas stream orthogonally to its axis by two-grid electrostatic mirror and to couple the ortho-TOFMS orthogonally to theexisting gas-dynamic interface. The carried out computer simulation has shown efficiency of such approach with additional
possibility to separate ions according to their mechanic energy in the vicinity of the mirror. Hopefully, realization of supposed
orthogonal coupling will provide high level of sensitivity and selectivity for different kind of analyses, including determination of
trace impurities in air without preliminary sample preparation. The work is carried out in the framework of the Program 8 forBasic researches of Presidium of Russian Academy of Science.
1. V.V.Raznikov, V.V.Zelenov New Way to Build a High-Performance Gas-Dynamic Interface to Produce and Transport Ions into a
Mass Analyzer. Int.J.Mass Spectrom.（in press）2012.
2. V.V.Raznikov, V.V.Zelenov, E.V.Aparina, A.R.Pikhtelev, A.V.Chudinov, M.O.Raznikova, I.V.Sulimenkov Prospects of Development
and Realization of Some Ideas of V.L.Talroze Mass Spectrometry School. Effective Gas Dynamic Interface. Russian Academy of
Science Izvestia, Energetica, No 3 （2012）p. 3 -18.（in Russian）

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 42: New Developments in Instruments and Detectors

PTu-111

11:10 － 12:20

Dramatically Improved Hydrocarbons Analysis with the 5975-SMB GC-MS
with Cold EI

Alexander B Fialkov, Aviv Amirav, Alexander Gordin
Tel Aviv University, Tel Aviv, Israel

Keywords:
GC-MS, Cold-EI, Wax, Hydrocarbons, Fuel-Oil
Novel aspects:
GC-MS with supersonic molecular beam and cold-EI of vibrationally-cool molecules in fly-through ion source. Improvedhydrocarbons identification and characterization with compounds extended range, enhanced molecular ion, novel isomerabundance analysis.
Abstract:
Hydrocarbon analysis, including fuels, oils and waxes are of vital importance to broad range of applications. However their analysis
by GC-MS is confronted with limitations in the elution of large and low volatility compounds and in the absence of trustworthymolecular ions and representative high mass fragments which consequently often precludes proper identification. In addition, suchanalysis is further exacerbated by the lack of proper LC and/or LC-MS alternatives.
GC-MS with supersonic molecular beams（SMB）and its Cold Electron Ionization（Cold EI）allow to overcome the challenges ofhydrocarbon analysis occurring in standard GC-MS technique. Cold EI with it fly-through ion source eliminates the well-known
conflict between ion source related peak tailing and molecular ion abundance which originates from the conventional ion sourcelayout as will be explained and discussed. In addition, the SMB based approach significantly increases the range of compounds
amenable for GC-MS analysis via lowering the compounds elution temperatures as will be demonstrated.
We used an Aviv Analytical model 5975-SMB GC-MS with Supersonic Molecular Beams（SMB）for hydrocarbons analysis. It is basedon the combination of the SMB technology with the Agilent 5975 GC-MS, forming a new and powerful GC-MS system. The GC
eluting sample compounds are mixed with helium make up gas in a temperature programmable transfer line, expand from asupersonic nozzle into a vacuum chamber, vibrationally cooled, skimmed, and pass a fly-through, electric field free EI ion source
where they are ionized by 70 eV electrons as vibrationally cold molecules（hence the name Cold EI）. Ions originated from thebeam molecules continue their straight flight, exit the ion source, are reflected at right angle towards the quadrupole MS, aremass analyzed and detected.
GC-MS with SMB enables the analysis of large wax compounds up to C74 via the use of short columns with high column flow rates
to ensure their proper elution from the GC, as well as due to the features of fly-through ion source that does not cause any peaktailing of low volatility compounds. GC-MS with Cold EI（using SMB）provides trustworthy and largely enhanced molecular ions to
all analytes as well as enhanced high mass fragments, for their improved identification. Isomer abundance analysis is enabled for
obtaining unique and powerful hydrocarbon mixtures characterization method, that can be used for geochemical information, fueland oil characterization and source location in oil spill and arson investigations.
The analysis of the following samples will be presented and described： polywax, oxygenates in wax, flesh flies wax, flower wax,
beeswax on fruits and vegetables, jet engine oil, Jojoba oil, Nonoxynol- 9 condom spermicide oil, motor oil freezing properties,
triglycerides in biodiesel Diesel, fuel characterization, biodiesel in jet fuel and more.

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 42: New Developments in Instruments and Detectors

PTu-112

13:30 － 14:40

Suitability of dual solid probe FT-ICR-MS system for gas-phase acidity andbasicity measurements of low volatile compounds.

Ivari Kaljurand, Ivo Leito, Ilmar A Koppel
University of Tartu, Tartu, Estonia

Keywords:
gas-phase acidity, gas-phase basicity
Novel aspects:
dual solid probe accessory for introduction of compounds for gas-phase acidity and basicity measurements
Abstract:
FT-ICR-MS is one of the most used methods for determination of gas-phase acid-base properties of compounds. Although there isavailable large data collection of gas-phase acid-base properties1 there is made only small progress during last decade in the fieldof experimental research of superacidic and superbasic compounds. 2 At the same time were designed and by using computational
chemistry tools were shown that considerably stronger superacids and -bases may exist.3 Several of these compounds have beensynthesized and their strength in solution have been verified, but their gas-phase acid-base properties have not been yet
experimentally determined. The main reason is that many of the neutral organic superacids and superbases have high molar
masses and very low volatility. Such compounds are difficult to study experimentally as for relative gas-phase acidity or basicity
measurements volatile reference compounds are needed on common FT-ICR-MS instruments（single heatable solid probe andseveral leak valves）, which have been used for that task.
To overcome these experimental difficulties was designed and built together with Varian（later acquired by Agilent）FT-ICR-MS
team a dual solid probe inlet accessory and fitted it with Varian 930 MS 7 T system. Optimal experimental parameters were
determined for the customized system by using previously studied compounds and performance was tested with some low
volatile compounds.
Work was supported by research grants 8689 and 8162 from Estonian Science Foundation, by the targeted financing project ofMinistry of Education and Science of Estonia SF0180089s08 and by the Estonian Centre of Excellence HIGHTECHMATSLOKT117T
References：
1. http：//webbook.nist.gov/chemistry/； Hunter, E. P. L.； Lias, S. G. J. Phys. Chem.Ref. Data. 1998, 27, 3,413-656.
2. J. Phys. Chem. A 2009, 113,8421-8424； J. Phys. Chem. A 2007, 111, 1245-1250.
J.Phys. Chem. A,2009, 113, 12972-12978； J. Am. Chem. Soc., 2005, 127, 17656-17666.

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 42: New Developments in Instruments and Detectors

PTu-113

11:10 － 12:20

Development of mass spectrometer equipped with ECRIS and its
application to isotopic analysis of Mo

Tsuyoshi Nagamatsu1,2, Masanori Kidera1, Masayoshi Toda3, Tatsuhiko Tanaka2, Kazuya Takahashi1

1
RIKEN, Wako, Japan, 2Tokyo University of Science, Shinjuku-ku, Japan, 3Tokyo University of Marine Science andTechnology, Minato-ku, Japan

Keywords:
ECR-IS, molybdenum, isotope, ion sputtering
Novel aspects:
Our results suggest that ECRIS-MS has great potentiality and that it can be widely applied to isotopic analyses with high sensitivity.
Abstract:[Introduction] The precise measurement of isotope ratios in geological and planetary materials has been expected to give us
significant information on their origin. For example, several elements in meteorites, such as Mo and Sn, have been recognized asimportant for examining the process of nucleosynthesis. Therefore, a development of the mass spectrometer with the potential
ion source has been regarded as one of essential subjects in mass spectrometry. We focused our attention on the highpotentiality of an electron cyclotron resonance ion source（ECRIS）as an ion source for mass spectrometers and attempted tocustomize the mass spectrometer equipped with an ECRIS for application to the analysis of Mo isotopes. The ionization efficiencyof the ECRIS is generally high, and all elements can be fundamentally ionized. In this study, for metal samples, we employed an ion
sputtering system as a sample introduction system.
[Method] The ECRIS consists of the following three parts： permanent magnets for the magnetic mirror field, a high-frequency
power supply（10 GHz）, and a plasma chamber. This ion source was combined with ananalyzing magnet followed by a detection
system（Faraday cup）. Using the above system, we performed the isotopic analysis of Mo assisted by the sputtering method. To
establish ECR plasma, we used Kr or O gas. The sample was sputtered with Kr or O ions produced in the ECR plasma with apotential between 1 and 5 kV. As a sample, we examined several metal materials, such as the SUS 316 standard material, tosimulate the analyses of iron meteorites.
[Preliminary results] We confirmed with a mass spectrometer that the sputtering yields of ions obtained using the Kr and O gases
are sufficient. Furthermore, the observed signal intensity was very stable in each measurement and the signal intensity changed byless than 0.5% in 1 hour. The observed isotope ratios of Mo in SUS 316 were in good agreement with the data calculated fromnatural isotope abundances. However, in the case of sputtering with Kr, the precision of isotope ratios decreased owing to theeffect of the background noise derived from Kr isotopes（m/z 80-86）and several interference peaks observed from m/z 100 to 110.
The mass spectrum pattern of those interference peaks exhibited the isotope abundance of Kr. Therefore, those peaks are
assumed to be derived from a certain cluster of Kr or a similar cluster. To date, the origins of those interference peaks have notbeen elucidated yet. On the other hand, using O gas for the ECR plasma and sputtering source, a fine background condition was
obtained. However, small interference peaks were observed at m/z 100 and 101. Those peaks did not depend on the sputtering
potential and total ion current. This suggests that those peaks are not derived from the sample but from the circumstance of ion
source. It should be necessary to refine the parts of the ion source to decrease those interference peaks. The results of isotope
ratios except 100Mo exhibited a high precision within 0.5% and also accordant ratios with natural isotope abundances. Our results
suggest that ECRIS-MS can be widely applied to isotopic analyses with high sensitivity.

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 42: New Developments in Instruments and Detectors

PTu-114

13:30 － 14:40

Analysis of ion cloud dynamics in diffrent geometry orbitrap massanalyzers using Particle-in-Cell code based computer simulations

Gleb Vladimirov1,2, Eugene N Nikolaev1,2,3

Institute for Energy Problems of Chemical Physics, Russian Academy of Sciences, Moscow, Russia, 2Emanuel Institute of
Biochemical Physics of Russian Academy of Sciences, Moscow, Russia, 3Institute of biomedical chemistry, Russian
Academy of Medical Sciences, Moscow, Russia
1

Keywords:
FTMS, orbitrap, simulation, space charge
Novel aspects:
Deeper understanding of ion clouds behavior in orbitraps of different geometry obtained by supercomputer simulations
Abstract:
Fourier transform mass analyzers based on electrostatic traps technique are attracting the growing interest for their compactness
and high performances. The well known orbitrap is a member of more general class of traps - cassinian traps［ 1 ］, in which it ispossible to make ion motion harmonic and measure ion masses via frequencies of oscillation in harmonic potentials. There isgreat interest to comparative analysis of different geometry variants cassinian traps. In this work we consider the simplest
cassinian trap - orbitraps of different geometry. Analytical performance（resolution, dynamic range）of such mass analyzers ingeneral is determined by trap geometry as well as space charge effects and extend of field deviation from ideal profile. We
address these factors and reports acquired dependencies. To reach high performance in these type of analyzers we need tochoose an appropriate trap geometry, injection ion beam parameters, and create an electrostatic field of acceptable quality. Also,
the influence of space charge effects should be removed or compensated.
We used the 3 -dimensional particle in cell approach［ 2 ］to calculate the net field of the ion trap perturbed by the field fromlarge ion population and to integrate motion of individual charged particles. The effective electric field was calculated as asuperposition of the analytictrap field and ion fields’ calculated via the 3 -dimensional Poisson equation. Four different publishedorbitrap geometries were considered： geometry close to preproduction orbitrap, close to standard orbitrap, close to high fieldorbitrap and close to compact high field orbitrap.
Limits of resolution caused by trapping field non-harmonicity and the magnitude of frequency shift as a function of the number ofcharged particles in ion cloud are determined. Dependences of the measured ion frequency on the number of ions for orbitraps
with different field curvature and different average axial amplitude were obtained.
We also addressed the ion peaks coalescence phenomenon in different orbitraps geometries. It was shown that for a higher fieldcurvature peaks coalescence occurs at a larger number of ions in a cloud.
Influence of the electrostatic field imperfections caused by surface geometry deviation from the ideal profile was analyzed. It was
shown that such geometry deviations cause signal decay and resolution degradation. A critical minimum level of surface deviationfrom the ideal profile was determined. The influence of voltage instability on ion motion was considered as well.
All simulations were performed on“GraphIT!“ supercomputer of Moscow State University. The authors acknowledge the support
from the Russian Foundation of Basic Research（grant 10-04-13306）, from the Russian Federal Program（state contracts
14.740.11.0755, 16.740.11.0369）, and from the Fundamental Sciences for Medicine Program of the Russian Academy of Sciences.
［ 1 ］C.Koster, The concept of electrostatic non-orbital harmonic ion trapping //International journal of mass spectrometry, 287,114-118, 2009［ 2 ］A.Kharchenko,G.Vladimirov, R.M.A.Heeren, E.Nikolaev Performance of Orbitrap mass analyzer at various space charge andnon-ideal field conditions： simulation approach //Journal of The American Society for Mass Spectrometry, Volume 23, Number 5,977-987, DOI 10.1007/s13361-011-0325-3, 2012

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 42: New Developments in Instruments and Detectors

PTu-115

11:10 － 12:20

Characteristics of a laser assisted vacuum-type electrospray droplet beamsourceSatoshi Ninomiya, Lee Chuin Chen, Yuji Sakai, Kenzo Hiraoka
University of Yamanashi, Kofu, Japan

Keywords:
Electrospray, Laser, Massive cluster, SIMS
Novel aspects:
A technique for stable vacuum electrospray of volatile liquids was developed as a high-brightness massive cluster beam source.
Abstract:
One of the most serious problems of secondary ion mass spectrometry（SIMS）is its low sensitivity to organic molecules. The
sensitivity has been increased by using cluster probes such as SF5 +, C60 +, Au3 + and Bi5 +, and improvement of spatial resolution on
imaging mass spectrometry for real world organic samples with these cluster ions has been reported. However, secondary ion
emission yield in SIMS is still not enough for high resolution（< 1 micron）imaging, and there is a pressing need to improve thesensitivity. Gas cluster and charged-droplet beams are good candidates for next generation cluster-SIMS probes. Compact gas
cluster ion guns are now commercially available for etching in surface science. In our laboratory, electrospray droplet impact（EDI）
method based on ambient electrospray technique has been developed as a new massive cluster ion beam source, and it has
been very successful in achieving efficient ionization of organic molecules and etching of polymers and metal oxides with leaving
little damage. However, it currently lacks adequate beam focusing and beam density to achieve imaging with sufficient spatial
resolution and within a reasonable time. To solve these problems, we propose a new method of producing a high-brightness
charged-droplet beam, and have developed a technique for producing stable electrospray of volatile liquids（especially water）
under vacuum. It has been known that vacuum electrospray of volatile liquids is extremely difficult because of two major
problems：（ 1 ）the occurrence of electric discharge under low vacuum conditions and（ 2 ）the freezing of the volatile liquids
introduced into the vacuum by evaporative cooling. To prevent the electric discharge, the experimental chamber pressure wassignificantly improved by using turbomolecular, mechanical booster and rotary pumps. To prevent freezing of the liquids
introduced in vacuum by evaporative cooling, the tip of the electrospray emitter was heated by infrared laser. This vacuum
electrospray technique can be expected to be a high-brightness massive cluster ion beam source. In this paper, the characteristics
of a vacuum-type electrospray droplet beam source as a SIMS probe will be presented.

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 42: New Developments in Instruments and Detectors

PTu-116

13:30 － 14:40

New plasma-based ion source mass spectrometer (ECRIS-MS) and its
application to rapid detection of volatile compounds

Tatsuya Urabe1, Masanori Kidera1, Yasuo Seto2, Kazuya Takahashi1, Michiko Kitagawa1
1

RIKEN, 2National Research Institute of Police Science

Keywords:
ECR, plasma, CWA, volatile, in-situ
Novel aspects:
A new plasma-based MS was customized. Its size is considerably small. Volatile compounds can be readily and stably detected,
which allows the on-site detection of chemical warfare agents.
Abstract:［Purpose］
We have been developed new mass spectrometer with plasma ion-source based on“electron cyclotronresonance ion source“（ECRIS）. Since ECR plasma has high electron density and energy, sample compounds introduced into ECR plasma are efficiently
and stably ionized and then detected as their decomposed forms such as atoms and atomic groups.
In this session, we will show a new version of MS with ECRIS,“miniECRIS-MS“. Unlike other ECRIS typically used in accelerator,
the size of its ion source chamber is considerably small, which allows sample monitoring in-situ. As an example, several volatile
chemical warfare agents（CWA）in air were measured with miniECRIS-MS. And the characteristics and potentials of miniECRIS-MS
for rapid detection of volatile compounds will be discussed.
［Equipment］
The miniECRIS-MS consists of three parts： 1 ）ECR plasma chamber made of permanent magnet（cylindrical shape； 10 cm indiameter and 15 cm in length）, 2 ）quadrupole mass analyzer and 3 ）continuous electron multiplier. ECR plasma was generated
by feeding air occasionally including volatile compounds into plasma chamber under micro-wave radiation.
［Sample measurement］
1. Air： atmospheric air was measured as a background of CWA sample. Pure gases（N2, O2, Ne, Ar, Kr, Xe and mix gas）werealso measured. 2. Simulant： dichloromethane（CH2Cl2：DCM）was volatilized in a glass jacket containing atmospheric air. Then
DCM with air was introduced into plasma chamber through fused silica capillary. 3.CWA： several CWAs, such as sarin（C4H10FO2P）, mustard gas（C4H8Cl2S）and phosgene（COCl2）were measured. The sample introduction into the chamber was
the same as the case of simulant.
For each sample, mass spectra were obtained with varying experimental conditions（pressure of sample gas in the plasmachamber, RF power, RF frequency, etc.）［Results and discussion］
1. Air： many peaks derived from air components were observed in the spectra. Main peaks were from nitrogen molecules（N2 +,N+ and N2+）and oxygen molecules（O2 +,O+and O2+）. Other peaks from carbon dioxide（CO2 +）, argon（Ar+, Ar2+）, water（H2O+,
OH+）were also observed. In addition, a peak not corresponding to air components was observed at m/z 30. Since the same peak
was also observed in N2 -O2 mix gas, the peak is considered to be NO+ generated by plasma radical reaction.
2. Simulant： when RF power was high（10 W）, DCM was almost completely decomposed and detected as single atoms such as
C+, Cl+. This indicates that miniECRIS-MS can be used as elemental analyzer in high RF power mode. With decreasing RF power,
fragment ions（e.g. CH2Cl+m/z 49）and even molecular ion（CH2Cl2 + m/z 84）were observed, showing that miniECRIS-MS can also
provide information on the structure of the analyte.
3. CWA： in this measurement, we focused our attention on the detection of CWA as single atoms. In other words, miniECRIS-MS
was operated in high RF power mode. Characteristic elements underlined in the formula were detected from sarin（C 4H10FO2P）,
phosgene（COCl 2 ）, mustard gas（C 4 H 8 Cl 2 S）and the other CWAs. Each CWA had optimum RF power region required for theefficient decomposition and ionization. For example, Cl+ was observed both from phosgene and mustard gas. However, phosgene
required more RF power than mustard gas. This would reflect the chemical environment around Cl atom in each CWA.
［Conclusion］
A prototype miniECRIS-MS was assembled. Various CWAs were observed within one minute. This indicates the potential ofminiECRIS-MS for the rapid detection of volatile compounds, and leads to the comprehensive and real-time detection of CWAs as
countermeasures against chemical terrorism. We will further improve the equipment for hogher sensitivity and identification
ability. The detail of portable-type miniECRIS-MS shall be introduced in the presentation.

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 42: New Developments in Instruments and Detectors

PTu-117

11:10 － 12:20

Adaptive Noise reduction in MALDI TOF Mass Spectrometers

Ian Brookhouse, Andrew R Bowdler, Ian Sherwood
Kratos Analytical Ltd.,Manchester,England

Keywords:
MALDI-TOF Adaptive Noise Reduction Electronics
Novel aspects:
An adaptive noise reduction technique enabled by advances in high performance electronics utilising quasi concurrent
measurement of noise and time of flight data.
Abstract:
Fundamental to the operation of modern MALDI TOF mass spectrometers is the need for digital and analogue electronics all
running at GHz speeds. In the mass spectrometer and TOF analyser, high-voltage switches capable of switching many kilovolts innanoseconds provide the pulses for pulsed extraction and ion gating. Transient recorders in MALDI TOF instruments are typicallyrequired to digitise the analogue signal from the ion detector at sample rates of 2 GHz or more, up to around a millisecond longwith a resolution of 8 bits and repetition rates around 1 kHz. Furthermore, many transients are usually accumulated in therecorder before the final TOF spectrum is transferred to the computer for analysis and storage. There is also a need for highspeed and precision triggering to fire the laser and to provide the various pulses as mentioned above. These requirements are notsatisfied by commercial digital oscilloscopes, which means that purpose designed electronics have to be produced, often by the
instrument manufacturers themselves. In general the resultant design is a variety of very high speed digital electronics including
the digitiser itself but mostly comprising the transient recorder memory.
A typical MALDI TOF MS（or MS/MS）spectrum will be the summation of many individual laser shots and the resulting TOF
transients in order to average out the random noise in the mass spectrometer. In this way the signal to noise can be maximised
and the smallest peaks in the spectrum detected. However, the accumulation and averaging does not reduce the effect of any
systematic noise and ultimately the signal to noise limit can depend on the size of such artefacts present either in the mass
spectrometer electronics or in the digitiser. For the MALDI TOF MS the potential for systematic electronics noise is very significant.
As well as obvious design measures such as the shielding of electronics and power supplies, there are some simple ways toreduce the effect of systematic noise such as randomizing the start time of the transient in the digitiser memory.
We will present another approach to reducing systematic noise in TOF spectra to the point where the effect of the noise isnegligible. The principle is that the noise data is acquired, ideally during the time that the signal data is acquired and the resultant
noise spectrum is then subtracted from the signal spectrum. The subtraction can be carried out during or after acquisition and by
the electronics or by software. Because the noise acquisition produces essentially the same data but without the MALDI ion
signal, the systematic noise can be reduced to near zero. By repeating this process many times during the data acquisition process
noise that has low frequency variation may be removed in an adaptive manner. Examples will be presented of MALDI TOF MS
data before and after the reduction of different types of systematic noise. In each case both the signal spectra and the noise
spectra will be shown. It will be demonstrated that, by this method the signal to noise ratio can be improved to the point where itis effectively limited by the random electronic or chemical noise in the mass spectrometer.

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 42: New Developments in Instruments and Detectors

PTu-118

13:30 － 14:40

Development of an ion transmission enhanced tandem ion guide system
for triple quadruple mass spectrometer

DAISUKE OKUMURA, MANABU UEDA, TOMOHITO NAKANO
Shimadzu Co., Kyoto, Japan

Keywords:
Triple quadrupole, high speed, high sensitivity, neutral loss scan
Novel aspects:
Development of a higher ion transmission tandem RF ion guide system to enhance scan sensitivity
Abstract:
Introduction
The mass spectrometer coupled to atmospheric pressure ionization source needs to have differential pumping system in order tomaintain high vacuum in analyzer part. Commercial triple quadrupole mass spectrometer typically has from 3 to 4 differential
pumping stages and the RF ion guide was generally utilized as an ion focusing device in higher pressure region of ion path. The
ions generated under atmospheric pressure needs to be efficiently focused to minimize the loss of ion prior to introducing into
quadrupole analyzer by using RF lens system. Using a QqQ which has 4 differential pumping stages, we have investigated fringing
field of RF ion guide installed in second and third differential pumping stage and finally the sensitivity was improved 5 times.
Methods
Three distinct designs of the tandem quadrupole RF ion guide in differential pumping stages have been investigated. In order toexamine the characteristics and performance of these configurations, each RF ion guides were installed in a triple quadrupole
mass spectrometer（LCMS-8030, Shimadzu Corporation, Kyoto, Japan）. Each tandem RF ion guide systems have their inscribedradius a）1.5mm, b）2.0mm and c）2.8mm and the system has one DC lens between RF ion guides, and its diameter is 4 mm.
Preliminary Data
The optimum operating RF voltage of quadrupole ion guides was acquired experimentally. The voltages were 100V, 50V and 27V
for r0= 2.8, 2.0 and 1.5 respectively. Then each pseudo potential is calculated by the following equation,
V*（r）=（ 4 qV2/mΩ 2 r0 4）r2
where, r0 is the radius of the inscribed circle of the ion guide, V is the amplitude of the RF voltage, m is the mass of the ion, and q
is the electric charge. The calculated pseudo potential of each ion guides is almost same at m/z 168. In other words, ion focusing
effect inside of the RF ion guide is almost identical. We have simulated the fringing focusing field of smaller r 0 ion guides wasstronger than that of larger r0. Therefore we have achieved higher sensitivity using smaller r 0 ion guides. We have replaced thetandem quadrupole ion guide（r 0 = 2.0）system with existing tandem octapole ion guide system of an ultra fast TQ mass
spectrometer（LCMS-8030, Shimadzu Corporation, Japan）. We acquired Q 1 scan spectra of pesticides at three different scan
speed with flow injection analysis.（5000, 10000 and 15000 u/sec）. Besides the nature of quadrupole ion guide which is narrower
mass range transmission comparing to other ion guide such as hexapole and octopole, the absolute intensity of Q 1 scan with all
three scan speed was increased 2 to 5 times.

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 42: New Developments in Instruments and Detectors

PTu-119

11:10 － 12:20

The A-Wave principle: new way to design ion guides and similar devices

Alexander S Berdnikov1, Nicolay R Gall2

1
Institute for Analytical Instrunentation, St.Petersburg, Russian Federation, 2Ioffe’s Physical and Technical Institute RAS,
St.Petersburg, Russian Federation

Keywords:
ion guides, A-Wave, T-Wave, RF fields, pseudopotentials
Novel aspects:
A new way to desing the ion guides, etc., with the pseudopotential wave which traps ions and moves them synchronically inspace, is considered.
Abstract:
The modern mass spectrometry widely uses the devices based on radio frequency electric fields, including that where the charged
particles move in gas-filled media： ion traps, quadrupole mass analyzers, gas-filled cooling cells, collision cells, ion guides, ion
funnels, Q-arrays, etc. These and similar devices can be found in nearly each commercial or experimental mass spectrometerswith atmospheric pressure ionization. The effectiveness of these devices influences the effectiveness of the mass spectrometer
greatly.
The movement of charged particles in radio frequency（RF）electric fields is described qualitatively with good accuracy by thepseudopotential theory［ 1 ］. However, this useful theoretical approach deals mainly with the stationary pseudopotentials, thelatter results from the nature of RF voltages used to excite the RF electric fields. General theory［ 2 ］overcomes this limitation andenables to create the devices where the movement of the charged particles is controlled by time-dependent pseudopotentials
with the a priory specified properties. While some particular examples of RF electric fields with time-dependent pseudopotentials
were already used before［ 3 ］, only general theory enables to utilize this tool to full extent.（However, if somebody wants to lookdeeply for the predecessors by priority, these will be Sir Isaac Newton, James Clerk Maxwell, Michael Faraday and Alessandro
Guiseppe Antonio Anastasio Volta, like for any other device relevant to the charged particle optics.）In many cases the flexibility
to control the movement of the charged particles by this new tool enables to expand significantly the properties of thecorresponding mass spectrometric devices［ 5 ］.
This presentation considers a specific case of the RF electric fields with time-dependent pseudopotentials which were called by
the author ’the fields with the Archimedean properties’, or the A-Wave fields. These RF fields are characterized by apseudopotential wave composed from alternating maxima and minima, where the said maxima and minima move along the axisof the device following the predefined law. The charged particles are trapped by local pseudopotential wells corresponding topseudopotential minima, are compressed into spatially separated and isolated packages, are moved synchronically with the said
pseudopotential wave independent of their masses, charges and to some extent the initial velocities. The group velocity of the
packages is totally controlled by the applied RF voltages and can vary between units of Hz to hundreds of kHz. In most cases thecorresponding device is formed by a periodical sequence of the electrodes like that used for the T-WaveTM ion guides［ 4 ］,
however, the power supply is strongly different for the A-Wave transport as compared with the T-WaveTM transport. The usage of
non-periodic A-Wave structures and local manipulations with the individual trapping zones enables us more possibilities tomanipulate the trapped ions while their transportation.
［ 1 ］M.Yavor. Optics of Charged Particle Analyzers. Ser. Advances in Imaging and Electron Physics157, Academic Press, 2009,
chapter 4, p. 141-168.
［ 2 ］A.S.Berdnikov, The time-dependent pseudopotential and its application for description of the averaged motion of thecharged particles, Nauchnoye priborostroenie, vol. 21（2011）No 2, p. 77-89； vol. 21（2011）No 3, p. 83-96； vol. 21（2011）No 4, p. 7585； vol. 21（2011）No 4, p. 86-102（in Russian）.
［ 3 ］N.J.Kirchner, Ion processing： control and analysis, Priority patent US 5,206,506.
［ 4 ］K.Giles, R.H.Bateman, An ion guide supplied with a DC potential which travels along its length, Priority patent GB 2,400,231.
［ 5 ］A.Berdnikov, A.Andreeva, Device for manipulations with charged particles, Utility model patent RU 113611.

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 42: New Developments in Instruments and Detectors

PTu-120

13:30 － 14:40

COUPLING OF AN INERT ION CHROMATOGRAPHIC SYSTEM WITH ICP-QMS FOR ROBUST AND ACCURATE ELEMENTAL SPECIATION

Tomoko Vincent1, Daniel Kutscher1, Lothar Rottmann1, Julian Wills1, Shona McSheehy1, Detlef Jenen2
1

Thermo Fisher Scientific, Bremen, Germany, 2Dionex, Switzerland

Keywords:
iCAP Q, ICP-Q-MS, QCell, Speciation, Dionex,
Novel aspects:
The powerful new Thermo Scientific iCAP Q ICP-Q-MS with innovative QCell technology, coupled with Dionex HPLC, can effectively
remove all kinds of spectral interferences whilst maintaining high detection sensitivity.
Abstract:
Speciation of metals and other trace elements becomes more and more important to accurately address the toxicity or otherpotential hazards of a given sample. Many physicochemical parameters such as toxicity, reactivity and mobility are strongly
dependent of the elemental species, so that detailed information on the chemical form of an element is important rather than the
total concentration of the element itself.
A new package for speciation is used to demonstrate the rapid and accurate analysis of sub-ppb levels of elemental compounds,
including examples like hexavalent chromium, that is under regulation in drinking waters, or arsenic species in food. Especially thelatter application has gained attention lately, since it was reported that ingredients used in toddler formula may contain morethan 100 ng g-1 of As being present predominately in its toxic inorganic species, As（III）and As（V）.
The Dionex ICS-5000 ion chromatography system hyphenated to the Thermo Scientific iCAP Q ICP-Q-MS is a powerful tool for thedetermination of the species distribution of an element within different samples. Due to its completely metal-free solvent path,
instrumental background can be reduced, and also aggressive mobile phases such as acids can be used. A variety of analyticalcolumns with outstanding separation capabilities enable the reduction of analysis time and solvent consumption together withexcellent separation performance. The new iCAP Q ICP-Q-MS with innovative QCell can effectively remove all kinds of spectralinterferences while maintaining high detection sensitivity. Therefore, also low concentrated species can be detected andaccurately quantified.
With this combination unattended routine speciation analysis is possible with simple hardware coupling and by use of a newsoftware platform fully integrated control, dedicated chromatographic integration, data processing and compound specific QC isoffered.

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 42: New Developments in Instruments and Detectors

PTu-121

11:10 － 12:20

A Two-Dimensional Particle Tracking Velocimetry Investigation for
Visualization of Gas Flow inside the Ion Funnel

Diamantis Kounadis1, Dimitris Papanastasiou1, Ioannis Orfanopoulos1, Alexander Lekkas1,
Ioannis Nikolos2, Emmanuel Raptakis1

1
Fasmatech Science and Technology SA, Athens, Greece, 2Technical University of Crete, Department of Production
Engineering and Management, Chania, Crete, Greece

Keywords:
ion funnel, vacuum fluid dynamics, Particle tracking velocimetry
Novel aspects:
A novel method for studying and optimising gas flow dynamics with reliable experimental methods and its application to ion
funnel aerodynamics.
Abstract:
Vacuum interface design and intermediate pressure ion optics are critical aspects for the development of high performance mass
spectrometers. The ion funnel is one of the most recent technological advancements in the field with a significant impact on
instrument sensitivity. Characterization of the ion funnel has been mainly performed experimentally under normal operating
conditions of a mass spectrometer and to some extent by ion optical simulations. Information about the gas dynamical effects
present in the device and the effect on ion transmission has not yet been presented. Here, an experimental investigation of thegas dynamics inside the ion funnel is performed based on the Particle Tracking Velocimetry（PTV）method. A spark discharge
particle source is developed to generate tracer nanoparticles capable of scattering laser light. The nitrogen flow is seeded withthe tracers and a CCD camera is used to capture the light which produces an average 2 D velocity vector field along the plane of
the laser sheet. The spark discharge source designed and constructed for the purposes of the experiment is capable of producing
both mono-disperse and poly-disperse particles and has been extensively characterized by a Differential Mobility Analyzer（DMA）.
A prototype ion funnel assembly was designed using CAD software and constructed from a composite material using 3 D color
printing with a resolution of 300x450 dpi which produced a model with accuracy of less than 0.1 mm. The thickness of theelectrodes is 0.6 mm and an exit aperture of 2.5 mm was used. Inserts are constructed along the length of the funnel and sealedwith glass windows which allow for optical access with a laser and a CCD camera. The model is introduced in the first vacuum
compartment of a vacuum chamber equipped with a capillary inlet with 0.5 mm id. The ion funnel model is mounted on a
supporting base and positioned 20 mm downstream the outlet of the capillary interface. A 33 m3/hr mechanical pump is used toevacuate the first vacuum compartment to 30 mbar. The second vacuum compartment evacuated to 2 mbar using a second
mechanical pump is in direct communication with the first compartment through a 2 mm end aperture positioned at the end of
the funnel. A 1 mm thick laser sheet is introduced through a side slot running along the length of the funnel. An exit slot of equal
length allows for the laser light to exit the device and reduce background noise on the CCD. Laser light scattered on graphite
nanoparticles is observed through optical ports with dimensions of 12x12 mm2 near the entrance while smaller size ports are used
toward the exit of the device. Preliminary results show that the formation of the free jet remains undisturbed at the entrance ofthe funnel and image processing shows particles with velocities exceeding 400 m/s. Jet boundaries are confined to within ± 2 mm
during the first 20-30 mm from the entrance. The velocity of the gas drops to below 300 m/s at 50 mm distance relative to theentrance, a region where the onset of jet instabilities occurs. Gas turbulence is observed further downstream and the average
flow field shows particles moving in all directions at speeds as low as 30 m/s. Experimental results obtained by the PTV method
provide significant insights into the gas dynamical aspects related to the design of the ion funnel design.

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 42: New Developments in Instruments and Detectors

PTu-122

13:30 － 14:40

Source Cleaning in Maldi Mass Spectrometers by UV Laser Desorption

John M Allison, Andrew Eaton
Kratos Analytical Ltd

Keywords:
MALDI, Source Cleaning, UV Laser
Novel aspects:
The same UV laser is used for MALDI and to clean the ion source. Most efficient method for the de-contamination of MALDI ion
source electrodes.
Abstract:
The MALDI process facilitates the vaporization and ionization of biomolecules and large organic molecules. The molecules areembedded in a matrix which absorbs UV light. When a UV laser is fired on the sample spot to initiate the MALDI process a plumeof ionised and neutral analyte and matrix molecules is ejected from the sample plate. The ionised molecules are accelerated
away through apertures in the electrodes by an electric field formed between the electrodes. However, much of the matrix
material, and some of the analyte, is not ionized and this neutral plume continues to expand from the sample spot until it isdeposited on surfaces in the vicinity of the ion source. The contamination builds up over time until at some point an insulating
layer is formed that charges up and adversely affects the operation of the ion source. At this point the source electrodes must be
cleaned.
For many years the principal method of cleaning vacuum MALDI ion sources was to vent and open the source housing to enable
the first electrode to be cleaned in situ or the source removed completely for thorough cleaning. In both cases, in addition to thecleaning time, several hours were required to restore the vacuum once the housing was closed and perform the required HV
conditioning, instrument tuning and mass calibration procedures. In many applications of biochemistry there is a growing
requirement for higher throughput machines, which can now be realised by the introduction of instruments capable of running atrepetition rates of over 1 kHz. This has increased the rate of contamination build up on source electrodes, and the frequency withwhich they must be cleaned, to such an extent that it is no longer practical to vent the instrument every time the source requires
cleaning. Thus there is a requirement to find an effective method to clean the electrodes that does not require the source housing
to be vented.
Methods not requiring the source housing to be vented have been described that heat the electrodes up to temperatures of 250oC
using contact heaters or IR laser radiation. The effectiveness of heating the source electrodes is variable and depends very muchon the matrix used and the amount of contamination present, with thin layers being much easier to remove than relatively thicklayers that can build up even in a relatively short time. Further, some contaminants, particularly polymers, can be very difficult toremove simply by heating.
In this paper, a novel effective method for automatically removing contamination from apertured electrodes will be presented. The
method exploits the fact that most of the contamination on the source electrodes was originally desorbed from the sample spotusing UV laser irradiation and thus will be readily desorbed from the source electrodes if it is again irradiated with UV light ofsufficient energy density. This method uses the MALDI pulsed laser already employed in the instrument with scanning reflective
optics coupling the laser energy directly into the contamination to be removed, which leads to very effective and rapid desorptionof the material from the surface. Crucially the method avoids significant heating of the electrodes, thus avoiding shortcomings
associated with other techniques. The method enables fully automatic, rapid and very efficient source electrode cleaning to be
carried out without the need for source removal, venting or even removal of the sample plate from the instrument. This method
has proven to be effective in cleaning all common matrixes from source electrodes even when an appreciable layer ofcontamination has been allowed to build up.

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 42: New Developments in Instruments and Detectors

PTu-123

11:10 － 12:20

Compensating for Space Charge Effects and Increasing the Mass Accuracy
and Dynamic Range of Quadrupole Ion Trap Mass Spectrometers

Jae Schwartz, Philip M Remes

Thermo Fisher Scientific, San Jose USA/CA

Keywords:
Space Charge Effects, Quadrupole Ion Trap, Dynamic Range
Novel aspects:
A space charge calibration process can increase the mass accuracy and dynamic range of quadrupole ion trap mass
spectrometers.
Abstract:
Ion trap mass spectrometers are well-regarded in the analytical instrument field for their sensitivity； however, their inherent
dynamic range can certainly be improved. While single ions can be detected, the upper limit of useful ion capacity is bound by
ion-ion interactions. These interactions induce shifts in observed mass positions, and ultimately cause these mass spectral peaks
to become degraded（broad and less intense）. Practically, the ion limit is set well below this point, corresponding to somearbitrary maximum value of mass shift. However, such a limit can sacrifice dynamic range for mass accuracy, even thoughsubstantial capacity is available before complete peak degradation. A method has been developed to calibrate the mass shifts in
the range before degradation, extending dynamic range while maintaining mass accuracy.
A modified LTQ Velos linear ion trap mass spectrometer was used for these studies. LTQ Calmix, and polyethylene glycol withaverage molecular weights 200, 400, 600, and 1000 Da were used as standards. The relationship between number of ions and mass
shift was calibrated and used to adjust the observed mass position of subsequent spectra. Numerical simulations were used tosupport the experimental results.
Previous efforts to calibrate space charge mass shifts were useful only in very simple or too-specific instances, but never in thecase of an unknown, arbitrarily complex spectra. The difficulty arises because the influence of other ions on an ion of interest
depends on their proximity in a complex way. However, our results show that the interactions with the other ions accumulate
linearly. Therefore, once the relationship between proximity and mass shift is known, the effects of other ions can be predictedbased on their intensity and proximity to the ion of interest. The total effect of the other ions in the trap on an ion of interest is
the linear combination of their individual influences. A method of selectively accumulating ions of various intensity and proximity
to a calibrant was used to perform a calibration of the space charge effect, which can then be used to adjust the mass
assignments. In preliminary results, the method was able to correct for the space charge shifts in MS/MS spectra of 30,000 total
ions, where product ions were shifted by 0 -0.15 Da. This represents a 3 x increase over the typical target total ion abundance of
10,000 ions. The actual new spectral space charge limit will be explored in more detail. Numerical simulations will also becompared with the experimental results, and a model for the influence versus proximity relationship will be proposed.

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 42: New Developments in Instruments and Detectors

PTu-124

13:30 － 14:40

Further expansion of the MCP ion detector output linearity using a current
buffering concept.

ETSUO IIZUKA, MOTOHIRO SUYAMA, TOSHIYUKI UCHIYAMA, YUYA WASHIYAMA,
KATSUYUKI ISHIGURO
HAMAMATSU PHOTONICS K. K., IWATA, JAPAN

Keywords:
Ion detector, MCP, Linearity, Dynamic range, Low aspect
Novel aspects:
Increase output linearity of MCP ion detector is achieved by low-aspect-ratio current buffering MCP concept.
Abstract:
Microchannel Plate（MCP）ion detectors are widely used for TOF-MS because of their fast time response less than 1 ns, a large
effective diameter up to 77mm, and the flatness within 20 micron. However, the output charge saturation usually occurs at lower
levels compared to the discrete-dynode electron multiplier tubes, which limits its usefulness in some applications. The upper limit
is restricted by the resistance of the MCP, which regulates strip current. When the resistance is larger, the flow of charge isinhibited at higher outputs limiting the output linearity.
We have been studying MCP materials to lower its resistance, as well as an appropriate aspect ratio of MCP channels to achieve
the higher output currents, and found the current buffering concept worked well. In this concept, a new MCP with low-aspectratio（LAR）, length per diameter, of 20 compared to the conventional specification of 40 to 60 is utilized as an additional
multiplication stage to increase the electron output from the two stage MCPs. The low resistance LAR MCP can operate without
melting because it requires half the applied voltage, thus half the power consumption a conventional unit would need. Since thegain of a LAR MCP is low, this is used as a current buffer following the two stage MCPs.
Using the current buffering concept, we fabricated a LAR MCP, where the aspect ratios were 20, using the same process andmaterials to achieve the lower resistance, which was 0.25M ohm. The strip current was 1 mA at the applied voltage of 250V. Under
this condition, the power consumption was 0.25W, and we confirmed stable operation while obtaining a gain of 10. This MCP was
combined with a 2 stage MCP, creating a 3 stage MCP, and tested as an MCP ion detector. This combination resulted in an
upper output current limit of 12uA, which is 2 times higher than our lowest resistance MCP detector, but not as high as our
expectation of 70uA .
We have investigated one limitation of the linearity, and tried to optimize the performance. As a result, we found a potential
difference of 500V between the second and the third（current buffering）MCP helps to increase the linearity up to 30uA. Since thispotential increases the gain of the third MCP, it is suggested that the linearity of the second MCP limited the overall performanceof the former configuration. We are sure that there is another limiting factor, thus we are still carrying out additional research.
In an attempt to optimize our detector design, we are designing a one stage MCP with a high aspect ratio that should obtain thesame gain as our initial two stage MCPs. This should achieve the same results with a simpler configuration. The progress of thisoptimization will be presented in our paper.

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 42: New Developments in Instruments and Detectors

PTu-125

11:10 － 12:20

Sensitivity improvement of MCP-based Ion Detectors for Mass
Spectrometry

Masahiro Hayashi1, Yuya Washiyama1, Tetsuya Matsushita1, Akio Suzuki1, Shigetomo Shiki2,
Masataka Ohkubo2

1
Hamamatsu Photonics K.K., Hamamatsu, Japan, 2National Institute of Advanced Industrial Science and Technology,
Tsukuba, Japan

Keywords:
microchannel plates, effective open-area ratio, ion detection sensitivity, pulse height distributions,
Novel aspects:
The large OAR MCP is effective to increase the detection sensitivity.
Abstract:
Introduction
Microchannel plates（MCPs）are widely used as ion detectors in mass spectrometry because of large effective areas, flat
detection surfaces, and fast response times. The signal generation in MCP is triggered by ion-impact-induced secondary electron
emission at the inner wall of each channel. The secondary electrons are accelerated and impinge on the channel wall again, which
generate additional secondary electrons. This electron cascade multiplys the number of electrons, which are collected at an
anode and generate an output pulse signal. Sensitivity of MCP is related to（a）the ion-electron conversion efficiency, which is the
expected number of secondary electrons generated by the initial impact of an ion on the channel wall, and（b）the effective openarea ratio（OAR）, which is defined as the ratio of the channel opening space to the total MCP surface area. The upper limit of ion
counting rate and pulse height is affected by（c）the detector saturation, which is caused by a large amount of the electron
cascade current that preclude proper operation by the charge accumulation near the channel exit.
We improved the sensitivity of MCPs by such methods as channel surface coating with low work function materials to increase
the ion-electron conversion efficiency, resistance reduction to increase the upper limit of the electron cascade current, and OAR
increase by etching the channel walls on the input side of MCPs. This poster reports on the effect of OAR on ion detection
sensitivity quantitatively.
Experimental
The ion detection sensitivity was studied by a double-focusing mass spectrometer（JEOL JMS-600）. The response of the MCPbased ion detectorss to individual single ion impacts was measured with the ions of Ar and L-cysteine generated by electron
ionization（EI）at 70 eV. These ions were accelerated by a static high voltage of 3 kV. The m/z-specified ions were selected by
the electric and magnetic sectors, and impinged on the MCP surfaces. We prepared two types of MCPs with different OAR
values： 57%（S-MCP）and 75%（N-MCP）. These MCPs were interchangeable in vacuum by manipulating the detector assembly. A
comparison of the ion detection sensitivity between the two MCPs was made by recording pulse height distributions with amultichannel analyzer.
Results and Discussions
The peak channels of pulse height distributions measured for Ar  ions were found to be irrespective of OAR. The ion count rate of
N-MCP for Ar ions was 15% higher than that of S-MCP. This difference of the count rates is almost equal to that of the OAR values.
We also studied the effect of molecular species on pulse height distributions. The peak channel position measured for the
L-cysteine  ions became low. The peak position shift is reasonable with a mass dependence of the number of initial secondary
electrons. Since the lower peak channels resulted in the increase of miscounting at a fixed discrimination level, the difference ofthe count rates between the two MCPs became small： 6 % for L-cysteine ions. For both ions, it is demonstrated that our
fabrication method for the large OAR MCP is effective to increase the detection sensitivity.

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 42: New Developments in Instruments and Detectors

PTu-126

13:30 － 14:40

Round Focusing Magnetic Prisms for Surface Treatment Devices

Valentina A Mikhalevich, Viacheslav D Sachenko

Institute for Analytical Instrumentation of RAS, St.Petersburg, Russia

Keywords:
magnetic prism, sector field, fringing field, round focusing, mass-spectroscopic imaging
Novel aspects:
novel compound magnetic prism geometries and novel design ideas for energy- and/or mass-spectroscopic imaging devices
Abstract:
Static magnetic prisms known in electron optics as magnetic sector fields are important elements of many research devices. As
mass/energy analyzers, or as separators of the opposite-directed beams for the image aberration correction by a mirror, or for the
direct illuminating the specimen, the magnetic prisms are essential part of any modern surface treatment devices intended for astudy and a control of morphology and crystal structure, and chemical analysis of the micro- and nano-scale areas of a solid
surface.
It is well known that optical properties of the magnetic prisms in the radial and axial（vertical）directions are quite different by
nature. In the radial direction, the magnetic homogeneous field of the prism acts like a thin planar lens located at the prismbisector plane. In the vertical direction, the focusing action of the homogeneous field is absent. However, in case of the tilted
boundaries of the prism, the vertical focusing action appears due to the well-known“edge field effect“ which depends on the
inclination angle of each boundary. Appearing here“edge lenses“ act like quadrupole lenses. Therefore, the magnetic prism actsin this case like a multi-quadrupole system producing an axial astigmatism and an image ellipticity when the image beam passes
through the prism.
The key goal of our investigations is to find the reasonable geometries of the magnetic prisms which allow to provide thesimultaneous correction of the astigmatism and the image ellipticity for any positions of the object plane. This special type of the
focusing is called by the authors a round focusing, and this focusing for the magnetic prisms with the planar-parallel active polepieces is considered here. It can be shown the singular prism with the homogeneous field cannot produce the round focusing.
Therefore, the specific magnetic prisms, consisting of three differently excited magnetic segments being geometrically symmetricregarding the prism bisector plane, by the equal excitations of the outer segments, are considered here. That symmetricconstruction, invented by L. Veneklasen, allows to produce the stigmatic focusing（vertical focusing occurs here by passing theimage beam through the fringing fields between the outer and inner magnetic segments）. However, to produce the roundfocusing it is necessary to optimize the geometrical sizes of the outer and inner magnetic segments, the intermediate gaps, andthe excitation ratio between the outer and inner segments.
In the frame of well-known“SCOFF“ model for the fringing fields between the outer and inner segments, it is found the prisms
under consideration can provide the round focusing for the different deflection angles by the excitation ratio of about 3. However,by taking into account the extended fringing field distribution between the magnetic segments, it is found the optimal excitation
ratio depends on the deflection angle to a greater extent, and increases noticeably. Additionally, the 3 rd order vertical aberration
coefficient is varied strongly here. Both these effects are the result of the weak“fringing field lens“ action which could not beanalyzed in the“SCOFF“ model. Using the“extended fringing field“model of the prism, the authors have discovered thereasonable geometries providing the round focusing for the prism deflection angles 90 °and 60 °. One of these geometries wasverified experimentally, and has demonstrated a very satisfactory confirmation of the theoretical predictions.
The results of investigations of the round-focusing magnetic prisms, and the novel design ideas for energy- and massspectroscopic imaging devices are presented.

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 42: New Developments in Instruments and Detectors

PTu-127

11:10 － 12:20

From conventional Proton-Transfer-Reaction Mass Spectrometry (PTR-MS)
to a universal trace gas analyzer

Lukas Märk1, Alfons Jordan1, Achim Edtbauer1, Eugen Hartungen1, Simone Jürschik1, Philipp Sulzer1,
Tilmann D Märk1,2
1
IONICON Analytik GmbH., Innsbruck, Austria, 2Institut für Ionenphysik und Angewandte Physik / Leopold Franzens
Universität Innsbruck, Innsbruck, Austria

Keywords:
PTR-MS, proton transfer reaction mass spectrometry, charge transfer, trace gas analysis, PTR-TOFMS
Novel aspects:
Reagent ions in PTR-MS are extended to Kr+ and Xe+. This has never been done before and allows for the analysis of completely
new substance classes.
Abstract:
Proton-Transfer-Reaction-Mass Spectrometry（PTR-MS）is a well established technology for real-time trace gas analysis in the fields
of environmental research, food and flavor science, medicine, homeland security, etc. However, one limitation of conventional PTRMS is that only compounds possessing a higher proton affinity than water can be ionized. Also recent add-ons, like the so-called
Switchable Reagent Ions（SRI）feature, where O2 +（and NO+）can be utilized to ionize trace gas molecules via charge transfer, arelimited to compounds having ionization energies below 12.1 eV, which still excludes important substances like CO, CO2, SO2, CH4,
etc.
Here we present a novel development avoiding these limitations while preserving all advantages of conventional PTR-MS（fastresponse times, high sensitivity, no sample treatment, etc.）.
The setup of a typical PTR-MS instrument has been discussed extensively in literature. For the present innovation, where we
introduce Kr+ and Xe+ as additional reagent ions（SRI+）, the principal instrumental setup stays the same. This means that even in
the modified version no mass filter is necessary between the ion source, that is generating the reagent ions and the drift tube,
where the ionization of the trace compounds take place. Pure Kr or Xe from an external gas cylinder is introduced into the ion
source where Kr + or Xe + is formed, respectively. These reagent ions are subsequently introduced into the adjacent drift tube
where they can interact with air containing traces of the sample and an inert dilution gas.
Kr has an ionization energy（IE）of 14.0 eV and therefore allows to ionize via charge transfer reactions important molecules like
CO, CO 2 , NO x , N 2 O, SO 2 , CH 4 , etc. We present measurements of several certified gas standards, which include“typical“
calibration gases used frequently in PTR-MS, like e.g. benzene and additionally the above-mentioned compounds, which could not
be ionized and therefore detected with a PTR-MS setup so far. By amending a PTR-TOF 8000 to SRI+ we can additionally takeadvantage of the instrument’s high mass resolution. In cigarette smoke we can clearly distinguish CO + on 27.99 m/z from N 2 +（impurity from the ion source）on 28.01 m/z and from C2H4 + on 28.03 m/z.
CO2 and N2O are isobars with the masses 43.990 and 44.001 m/z, respectively. We analyzed a sample bag containing human breath（CO2 source）enriched with N2O from a gas cylinder. The results prove that the mass resolution is sufficient to identify the twocompounds independently.
Xe has an IE of 12.1 eV, which is very similar to the IE of O2. This means that in areas of application where oxygen cylinders are notallowed, Xe+ can be utilized as a substitute, which is confirmed by our studies.
In summary, with SRI+ PTR-MS is now capable of switching between H3 O + , O 2 + , NO + , Kr + and Xe +； thus there are virtually nolimitations on substance classes and subsequently on fields of application anymore.
We gratefully acknowledge that this work was financially supported by the Austrian Research Promotion Agency FFG, Vienna.
Patent pending（PCT/EP2011/064170）.

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 42: New Developments in Instruments and Detectors

PTu-128

13:30 － 14:40

A Novel Precursor Isolation Method using Digital Ion Trap Mass
Spectrometer

Kei Kodera, Masafumi Jinno, Kiyoshi Watanabe, Makoto Hazama, Masaji Furuta, Sadanori Sekiya,
Kaori Kinoshita, Hidenori Takahashi, Kosuke Hosoi, Toshinori Kobayashi, Shinichi Iwamoto,
Koichi Tanaka
Shimadzu Co., Kyoto, Japan

Keywords:
precursor isolation, MS/MS, Iontrap
Novel aspects:
We developed a novel highly effective precursor isolation method by which the yields of single unit peak were 90 % and 80 % for
singly and doubly charged ions.
Abstract:
Mass spectrometry is an indispensable tool for proteomic research. Matrix-assisted laser desorption/ionization mass spectrometry（MALDI-MS）and electrospray ionization mass spectrometry（ESI-MS）have been used for a comprehensive analysis of proteinssince the 1990s, more recently, the importance of an in-depth analysis of post-translational modifications（PTM）is increasing. MS/
MS and MSn analysis on an ion trap mass spectrometer is a useful function for structural analysis of complex compounds such as
oligosaccharides released from glycoproteins or biantennary glycopeptides.
To provide a more useful technology for MS n , we have been working on the development of digital ion trap（DIT）mass
spectrometer. DIT is driven by rectangular wave high voltages produced by high frequency switching of positive and negative highvoltages whereas the conventional ion trap is driven by sinusoidal wave high voltages.
Since DIT has a higher ion capacity than the conventional ion trap at high mass range, it is well suited for the analysis of large
molecules like PTM peptides. In addition, DIT has a feature that the frequency of trapping voltage can be changed instantly andarbitrarily.
Using this flexibility, digital asymmetric wave isolation（DAWI）method has been reported. DAWI is a unique method to isolateions in DIT by the way of changing duty ratio of positive to negative phase of trapping rectangular high voltage. By using DAWI
method, the ions of any mass ranges can be kept in the ion trap instantaneously with high yield. However, the“window size“ ofmass selection is not narrow enough and high mass cut-off in the window is not sharp, so that DAWI alone is not suitable forprecursor selection for MSn analysis.
In mass isolation analysis using the resonant excitation frequency scan, ion ejection can be provided with both ascending anddescending order mass scan, so-called“forward scan“ and“reverse scan“, respectively.
By the combination of forward and reverse mass scan, it is possible to keep the arbitrary ion peak for the precursor of MS nanalysis in the ion trap in which reported as single unit mass manipulation ion trap（SUMMIT）. However, this method takes longtime to isolate ions and the yield was less than 50 % when a single unit mass ion, which is not the monisotopic ion, is selectedfrom the monovalent ion isotope clusters. In this study, we have investigated the novel ion isolation method to achieve highresolution and high yield simultaneously.
Mass selectivity of filtered noise field（FNF）, resonance excitation, and DAWI were investigated by computer simulations.
Based on the optimal parameters determined by computer simulation, experiments for the single unit mass ion isolation werecarried out using a home-built MALDI-DIT-TOFMS prototype. As a result, the yields of isolated ions with single unit mass weresignificantly improved to about 90 % and 80 % for singly and doubly charged ions, respectively. Here we suggest that this novel
precursor isolation method is considered to be applicable to SUMMIT with high resolution and high yield.

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 42: New Developments in Instruments and Detectors

PTu-129

11:10 － 12:20

A high accuracy FDM field solver for prediction of non-linear resonancesin electrodynamic ion traps.

Matthew C Gill1, Alexander Berdnikov2, Roger Giles1

1
Shimadzu Research Laboratory (Europe) LTD, Manchester, UK, 2Institute for Analytical Instrumentation, Rizhskij pr. 26,
190103, St. Petersburg, Russia.

Keywords:
FDM, SIMION, simulation, quadrupole, multipole
Novel aspects:
A high accuracy FDM field solver can give new insight into non-linear resonances in ion trap devices by simulation.
Abstract:
The finite difference method（FDM）is used widely for calculation of approximate solutions to the Laplace equation governing
electrostatic and magnetic fields within bound systems, frequently using an iterative relaxation approach. FDM is commonly used
due to its simplicity, speed and wide availability [1] . In mass spectrometry, FDM-based ion optical modelling of systems iscommonly employed during instrument design and experimental interpretation.
A high accuracy FDM field solver incorporating accurate treatment of electrode boundaries [2] is described here, and aperformance comparison to a commercial FDM solver is given. The high accuracy FDM algorithm is used to solve several electricfields for which analytical solutions may be calculated, and the accuracy is compared to a commercially available FDM field solver
for several FDM grid densities.
For curvilinear electrodes, this 2 nd order FDM algorithm incorporating accurate treatment of electrode boundaries typically resultsin greater accuracy than that obtained using the 2 nd order FDM solver of a commercial software package, where the electrodeboundaries are rounded to the nearest grid node position（using the same grid density in each case）. The use of a similar 4 thorder FDM algorithm, also incorporating accurate treatment of the electrode boundaries, increases this accuracy by several
orders. Although for the same grid sizes these more sophisticated algorithms require more arithmetical operations per individual
grid node（resulting in greater calculation time）, the size of the grid necessary to obtain some fixed accuracy is much less thanthat for conventional finite difference methods. The ability to employ sparse grids provides several advantages： it allows
simulation of large systems whilst maintaining high accuracy when random access memory restrictions might otherwise limit thesimulation, and provides a considerable reduction in computation time for field solving when solving to some fixed accuracy.
The high accuracy FDM field solver reported here is used for analysis of several example problems. The generation of highaccuracy fields for comparatively sparse grid arrays is described. Fields are calculated for a range of ion trap geometries which
include quadrupole, hexapole and octopole fields（for both rod- and hyperbolic- electrode geometries）. Further fields arecalculated for quadrupole fields with added higher order multipole components such as hexapole and octopole, generated byaltering the geometry of rod-electrode quadrupoles [3] . The multipole components of these fields are analysed. The fields
calculated using the high accuracy FDM field solver are used in combination with ion optical ray tracing software to simulate ion
trapping within these devices. Simulations of non-linear resonances[4] are described, and modelling of the first stability region of
the Mathieu stability diagram [5] is described for several geometries. Non-linear resonance lines are clearly observable in thestability diagrams for several geometries.
The combination of a high accuracy FDM field solver with ion optical ray tracing software gives new insight into non-linear
resonances in ion trap devices, and can be used to inform fine details of instrument design.
［ 1 ］D.A. Dahl, Int. J. Mass Spectrom., 2000, 200, 3.
［ 2 ］J.A. Rouse, Three-Dimensional Computer Modelling of Electron Optical Systems, in： Advances in Optical and Electron
Microscopy, 13, Academic Press, Amsterdam, 1994, pp. 1121.
［ 3 ］N. Konenkov, F. Londry, C. Ding and D.J. Douglas, J. Am. Soc. Mass. Spectrom., 2006, 17, 1063.
［ 4 ］M. Yavor, Optics of Charged Particle Analyzers, in： Advances in Imaging and Electron Physics, 157, Academic Press,
Amsterdam, 2009, pp. 327332.
［ 5 ］R.E. March and R.J. Hughes, Quadrupole Storage Mass Spectrometry, Wiley, New York, 1989.

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 42: New Developments in Instruments and Detectors

PTu-130

13:30 － 14:40

More than one order of magnitude higher sensitivities with ProtonTransfer-Reaction Time-of-Flight Mass Spectrometry (PTR-TOFMS)

Eugen Hartungen2, Alfons Jordan2, Achim Edtbauer2, Akio Shimono1, Simone Jürschik2, Philipp Sulzer2,
Lukas Märk2, Tilmann D Märk2,3

Shoreline Science Research Inc., Tokyo, Japan, 2IONICON Analytik GmbH., Innsbruck, Austria, 3Institut für Ionenphysik
und Angewandte Physik / Universität Innsbruck, Innsbruck, Austria

1

Keywords:
PTR-MS, proton transfer reaction mass spectrometry, time-of-flight, PTR-TOFMS, high sensitivity
Novel aspects:
The sensitivity and the limit of detection of two PTR-TOFMS instruments are improved by more than a factor of 10.
Abstract:
In 2009 we published a paper on the development of a high resolution and high sensitivity Proton-Transfer-Reaction Time-Of-Flight
Mass Spectrometry（PTR-TOFMS）instrument. Although the coupling of a PTR source to a TOF mass analyzer had already beenreported by other groups at that time, our development had an outstanding impact in the PTR-MS community as for the first timea mass resolution of about 7000 m/Δm and a sensitivity of up to 25 cps/ppbv was achieved. Here we report on a number of
instrumental developments, leading to serious improvements in sensitivity and in the detection limit, while keeping the mass
resolution outstandingly high. We present measurements on gas standards illustrating the advances in instrumental development.
A typical PTR-TOFMS instrument consists of an ion source, where water vapor is converted into H 3 O + in a hollow cathodedischarge and an adjacent drift tube, where the actual proton transfer to the trace gas compounds takes place. The protonatedproduct ions are finally analyzed and detected in a TOF mass spectrometer. It is obvious that for such an instrument a virtuallycountless number of parameters contribute to the overall performance（instrumental design, lens voltages, orifice diameters,
differential pressures, etc.）. We consecutively improved and optimized these parameters, which in sum contribute to an increase
in instrumental performance of over one order of magnitude without any decrease in mass resolution.
We utilized two different PTR-TOFMS models for the verification of the instrumental improvements, namely a so-called PTR-TOF
8000（FWHM mass resolution between 5500 and over 8000 m/Δm； increasing with increasing masses）and a PTR-TOF 2000（massresolution between 1600 and 2500 m/Δm）. As a gas standard we chose a certified aromatics mixture consisting of benzene,
chlorobenzene, dichlorobenzene, ethylbenzene, styrene, toluene, trichlorobenzene, trimethylbenzene and xylene（about 100 ppbveach）in nitrogen. At first we connected purified air to the PTR-TOF 8000 and systematically recorded the background signal inorder to subsequently determine the limits of detection. Then we connected the gas standard to the instrument and recorded
mass spectra（ 1 s integration time）for about 1 minute. The calculated sensitivities for the corresponding m/z values start with170 cps/ppbv for the lowest mass 79 m/z（protonated benzene）and increase constantly with increasing mass numbers to 198 cps/
ppbv for 113 m/z（protonated chlorobenzene）, 215 cps/ppbv for 121 m/z（protonated trimethylbenzene）and 276 cps/ppbv for 147
m/z（protonated dichlorobenzene）. Compared to the initial PTR-TOFMS data（published by us in 2009）, which ranged from 13 to26 cps/ppbv, this represents an increase of more than one order of magnitude.
The limits of detection were calculated using the 3 σ method（with σ being the standard deviation of the background signal）for
1 min integration time. The results are 3 pptv for mass 79 m/z down to 1 pptv for 147 m/z.
Currently we are performing similar test measurements on an improved PTR-TOF 2000 which is known to have a higher sensitivity
at the cost of a somewhat lower mass resolution. First preliminary data indicate a sensitivity at the high mass compounds of up to
800 cps/ppbv and a detection limit of several hundred ppqv（parts-per-quadrillion）. These data will also be presented.

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 42: New Developments in Instruments and Detectors

PTu-131

11:10 － 12:20

A curtain-gas filter that widely protects a mass spectrometer from neutral
molecule contaminations

Hermann Wollnik, Gary Eiceman, Alexander Tarassov, Ryan Blase, Stephen Devila
New Mexico State University, Las Cruces, USA

Keywords:
ambient air ion sources, elimination of phosphates or silica.
Novel aspects:
Operation of a mass spectrometer with an ambient pressure ion source over extended periods analyzing molecules dissolved inbuffers that can cause deposits of nonvolatile contaminations
Abstract:
Introduction
A newly developed curtain-gas filter is presented that operates between the inlet to an evacuated mass spectrometer and an
ambient pressure ion source like for instance an electrospray ion source. This curtain-gas filter bars the ion-source buffer gas andall contained neutral molecules from entering the mass spectrometer while it efficiently extracts the ionized molecules from thisbuffer gas and transmits them into a mass spectrometer.
Action of the curtain-gas filter
In a curtain-gas filter the flow of an ion-source buffer gas streams widely turbulant free for a certain distance parallel to a purified
clean gas, while an electric field, that is superimposed over both gas flows, moves ions from the ion-source buffer gas into thepurified clean gas. Exclusively this clean gas, that now contains ions, is then entered into the mass spectrometer, while the ionsource buffer gas with all its contaminants is moved to an exhaust. In detail the two gas flows are arranged to both be annularand concentric to the capillary inlet of a mass spectrometer, while the electric field is radial to both gas flows.
First measurements
The curtain-gas filter allowed us to operate a Shimadzu 2020 LC/MS quadrupole mass spectrometer with an electrospray ion
source for 100 hours without clogging the capillary inlet or any other part of this mass spectrometer, though the electrospray ion
source was fed with a solvent that contained 10mM phosphate. Also after the mentioned 100 hour of continuous flow of phosphate
solutions into the curtain-gas filter, there was no perceptible loss in signal of the 1 ppm papaverine sample. Deposits were formedin the easily cleanable ion source container, but not in the inlet to the mass spectrometer or the spectrometer itself. Additionallyto the elimination of deposits, cluster ions of low masses and high mobilities, often abundantly available in the plume of anelectrospray ion source, are virtually eliminated when the magnitude of the electric field placed over the mentioned two gas flows
is chosen to be high enough that the undesired ions of very high mobility are pushed through the flow of the purified clean gas sothat they impinge on the conductive inlet capillary. Besides the experiments with phosphates we also used solutions of 1000ppm
dissolved silica as used in some cases in flow injection mass spectrometric studies of pharmaceuticals.

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 42: New Developments in Instruments and Detectors

PTu-132

13:30 － 14:40

High Mass Microscope-mode MALDI Imaging Mass Spectrometry

Yuto Yanagihara1, Masahiro Hayashi1, Yasuhide Naito2
1

Hammatsu Photonics K.K., Iwata,Japan, 2GPI,Hamamatsu,Japan

Keywords:
MALDI-Imaging mass spectrometry, microchannel plate, detector,
Novel aspects:
An approach aimed at building the standardization method for detection of high mass proteins for MALDI imaging-ms
Abstract:
Introduction
Matrix-assisted laser desorption / ionization imaging mass spectrometry（MALDI-IMS）developed by Caprioli et al. was originally
aimed at high mass analytes such as proteins in tissue sections, but is widely used for rather low mass analytes such as lipids. Oneof the technical barriers to use MALDI-IMS for high mass applications is ion detector sensitivity, which decreases as mass range
increases. Ion detector hardware and methods for high mass MALDI have been provided by Covalx AG（Zurich, Switzerland）andinstalled in some private companies and few public laboratories. Although some applications of MALDI-IMS have beenaccomplished by those facilities, high mass MALDI-IMS still requires a breakthrough in detector technologies.
We are developing microscope-mode MALDI-IMS, in which trajectories of ions extracted from a point on a sample surface in the
MALDI ion source are converged into a focusing point on a detector surface so that the mass-analyzing ion optics keep the spatial
distributions of analytes at the sample plate. Because the ion detector for microscope-mode MALDI-IMS is required to be time/
position-sensitive, the detector assembly is based on microchannel plates（MCPs）. This study explored the imaging ability of
MCP-based detectors in high mass range. In order to achieve a wide mass range that includes high mass proteins, the ion detector
technology which is typically used for MALDI-IMS must overcome two challenges； saturation of the detector caused by intense
signals from low mass ions and a decrease in detection efficiency for more massive ions. Increasing the detectable mass range of
MALDI-IMS could enable the detection of proteins crucial to biomedical research and other applications. Our main goal is toimprove microscope-mode MALDI-IMS where one can obtain subcellular resolution irrespective of the mass region.
Experimental
An MALDI time of flight mass spectrometer, built in-house, was used to examine essential components of microscope-mode
MALDI-IMS. The detector consisted of MCPs and a position sensitive anode, which was able to acquire both the positions andarrival times of detected ions simultaneously. The design of the detection system was optimized to enhance the detection
efficiency of the MCP-based ion detector so that images of protein distributions could be captured from a model sample.
First, we examined the detection efficiency of MCP-based ion detectors for peptide and protein ions ranging from 1046 to 66430 Da
by using our in-house MALDI linear time-of-flight mass spectrometer. We acquired comparison data between masses and signal
intensities of the ions.
Second, stigmatic ion images were generated from each spot of peptide and protein samples which were artificially patterned by
400 lines/in. fine grid on the sample surfaces.
Results and Discussion
It was evident that the signal intensity obtained from higher mass ions was much smaller than that obtained from lower mass ions.
The gain of electron cascade in MCPs was an important factor in the detection of ions exceeding 10000 Da.
Using a high speed circuit which controlled an MCPs/phosphor screen detector to acquire mass-specific stigmatic ion images
while recording TOF spectra, the mesh-patterned ion images were successfully obtained from ions up to 10000 Da. The signal-tonoise ratio of the stigmatic ion images decreased as the mass increased. When the mass of the proteins exceeds 5000 Da, thelaser power had to be increased to generate stigmatic ion images.

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 42: New Developments in Instruments and Detectors

PTu-133

11:10 － 12:20

Analysis of neurosteroids by GC-APPI-MS/MS

Tina J Suominen, Markus Haapala, Anna Takala, Raimo A Ketola, Risto Kostiainen
Helsinki University, Helsinki, Finland

Keywords:
Neurosteroids, athmospheric pressure photoionization, tandem mass spectrometry, gas chromatography
Novel aspects:
A sensitive GC-APPI-MS/MS method for the analysis of neurosteroids was developed. The method provides efficient separationand ionization of neurosteroids, and an easy way to couple GC to API-MS.
Abstract:
Analysis of neurosteroids by GC-APPI-MS/MS
Tina Suominen1, Markus Haapala1, Anna Takala1, Raimo A. Ketola2, Risto Kostiainen1
1
Division of Pharmaceutical chemistry, Faculty of Pharmacy, P.O. Box 56, FI-00014 University of Helsinki, Finland, 2Hjelt Institute, P.O.
Box 40, FI-00014 University of Helsinki
Neurosteroids are generally classified as steroids with local function in the brain, which are formed either in the brain in situ, or in
the peripheral nervous system regardless of the gonads and adrenal glands. These neuroactive steroids are important concerning
several functions in the brain, e.g. protein synthesis, the activation of genes, and the activity of the brain trough the activation of
gamma-aminobutyric acid（GABA）- and N-methyl-D-aspartate（NMDA）receptors.(  1  ) The levels of neurosteroids are often alteredin the case of neurodegenerative diseases, for example in Alzheimer’s disease and multiple sclerosis the levels of neurosteroids
have been shown to decrease in some areas of the brain.(  2  )(  3  ) The concentrations of neurosteroids in the human brain are oftenvery low（pM-nM）and therefore highly sensitive methods are needed. In this work we present a new method for the analysis ofneurosteroids in urine by gas chromatography-atmospheric pressure photoionization-tandem mass spectrometry（GC-APPI-MS/MS）
The urine sample pretreatment consisted of enzymatic and chemical hydrolysis of the glucuronide and sulfate conjugates,
respectively, followed by liquid-liquid extraction with diethyl ether, and subsequent trimethylsilyl derivatisation of the analytes
with MSTFA/NH4I/DTE. The GC-APPI-MS/MS analyses were performed using a HP 5890 II gas chromatograph coupled to an Agilent
6410 triple quadrupole using a modified version of our earlier presented microchip APPI method.(  4  ) Two columns（Thermo TR- 5 MS,
length 25m, i.d. 0.25mm, 5 % phenyl 95% dimethylpolysiloxane, film thickness 0.25µm, and Thermo TR-50MS, length 15m, i.d. 0.25mm,
50% phenyl 50% methyl-polysiloxane, film thickness 0.25µm）were sequentially connected for the separation of the analytes. The
samples（ 3 µl）were injected with 1 min splitless injection. The analytical run was initiated at 190 °C, after which the oventemperature was raised to 240 °C at 12 °C/min, from 240 to 255°C at 1 °C/min, from 255 to 330 °C at 12 °C/min, and the column
was kept at 330 °C for 2.4 min. The neurosteroids were ionized by photoionisation using a krypton discharge lamp（10 eV）and
chlorobenzene as the dopant.
All the 19 steroids were well separated by the used GC method. The trimethylsilyl derivatives of the steroids produced abundantradical cations, which were selected as the parent ions for selected reaction monitoring. The method showed high sensitivity（LODs 0,002- 1 ng/ml）and the repeatability was acceptable（RSD<20%）, showing its potential in neurosteroid analysis.

（ 1 ）Stoffel-Wagner, B. Ann. N. Y. Acad. Sci.2003, 1007, 6478.
（ 2 ）Weill-Engerer, S. Journal of Clinical Endocrinology & Metabolism2002, 87, 51385143.
（ 3 ）Helmut, L. Medical Hypotheses2010, 75, 229234.
（ 4 ）Haapala, M.； Luosujärvi, L.； Saarela, V.； Kotiaho, T.； Ketola, R. A.； Franssila, S.；Kostiainen, R. Anal. Chem.2007, 79,49944999.

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 42: New Developments in Instruments and Detectors

PTu-134

13:30 － 14:40

Potential of direct probe for drug and pharmaceutical

HARUO HOSODA, NORIYUKI IWASAKI, JOUJI SETA, YOSHIHIKO MORISHITA
Bruker Daltonics.k.k.,Yokohama,Japan

Keywords:
APCI, APPI, DirectProbe, time-of-flight
Novel aspects:
Direct probe can detect a solid sample and TLC spots dilectly without melting and extraction.
Abstract:
Detection and identification for pharmaceuticals or drugs are generally used with chromatography. Compositions are extracted
from complicated samples with chromatography technology, and then identification for the composition are performed by meansof specified color reaction, specified absorption, mass spectrometry and so on.
Therefore, sample preparation and waste time are disadvantage in order to remove obstructions from complicated samples.
Direct Probe is one of accessary for mass spectrometry. This is one of technology which are useful for APCI（Atmospherepressure chemical ionization）or APPI（Atmosphere pressure photo ionization）. In generally, soluble sample are treated with
LCMS, while Direct Probe can treat with not only soluble sample, also solid sample.
This characteristic is that this measurement is simple and quick detection.
This is a solution which solves problems, such as time waste and procurement of a sample.
Additionally, when there is no telling into what kind of solvent a sample melts, you can directly detect solid sample with thisprobe.
In this time, we will introduce easy and high accuracy technology which combined this direct probe to a time-of-fright mass
spectrometry.
Drugs or these metabolites were added to a physiological saline solution, and these compositions could be detected with thissystem easily, and then we will discuss limit of quantification for drugs and these metabolites in physiological saline solution withthis direct probe.
In the case of such complicated sample as traditional medicine and mixture sample,
We use thin layer chromatography（TLC）as preparation and then we will introduce some data for these TLC spots.

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 42: New Developments in Instruments and Detectors

PTu-135

11:10 － 12:20

Development of high-speed data-streaming system for time-of-flight mass
spectrometryKen-ichi Bajo1, Osamu Fujioka2, Shingo Ebata1, Morio Ishihara3, Kiichiro Uchino4, Hisayoshi Yurimoto1
Hokkaido University, Sapporo, Japan, 2National Instruments Japan Corporation, Tokyo, Japan, 3Osaka University,
Toyonaka, Japan, 4Kyushu University, Kasuga, Japan

1

Keywords:
High speed data acqisition, TOF-SNMS, SIMS
Novel aspects:
High speed data acquisition system was developed which is important for imaging. We utilized PXI-platform in order to process alarge amount of data of TOF mass spectra.
Abstract:
Secondary ion mass spectrometry（SIMS）is useful to determine elemental and isotopically abundances of various materials in a
microscopic region. Multi-isotopes can be simultaneously detected by using time-of-flight mass spectrometry（TOF-MS）, which isone of the features for TOF-MS. We are developing a spattered neutral mass spectrometer（Laser Ionization MAss nanoscope：
LIMAS［ 1 ］）. LIMAS is consisted of focused Ga ion beam in order to examine a nanometer-scale region, femtosecond laser which
ionized spattered neutral particles, and multi-turn TOF-MS（MULTUM-II,［ 2 ］）.
Secondary ions of each isotope（or compound）pass through the MULTUM-II. Mass-separated ions are detected by ion detection
system with micro channel plate（MCP）. High-speed digitizer with an analog bandwidth of >GHz is required because a timescaleof the ion detection is a few nanoseconds（ns）. Extracted ions by single spatter are about ten thousand less than those bycontinues beam because pulse width of the primary beam is ~200 ns. The low extraction rate leads directly to a low signal output.
A pulse-counting method in common use can dramatically increase a signal-to-noise ratio and obtain signal intensity quantitatively.
In an ion detection system of LIMAS we established high speed data acquisition and processing procedure with NI PXIe-platform.
Two MCPs and preamp amplify the introduced electrical signal. The input signal is split by a distributer called Wilkinson coupler.
The output signal is recorded by using two NI PXIe-5185s（hereafter 5185）. One is for recording the analog output, another is forpulse-counting. 5185 digitizer makes it possible to obtain mass spectra in high precision by ultra-fast sampling rate（ 8 -bit verticalresolution and 12.5 GS/s）. High data transfer speed（up to 1 GB/s）by using PCI Express bus provides real-time data acquisitionand deconvolution and high sampling rate up to 10 kHz.
The wave forms are recorded for several microseconds by post-trigger. In case of pulse-counting, the recorded array issimultaneously transformed to 1 / 0 one. An algorism of the deconvolution is as follows. When a center value of three arbitrary
consecutive points is higher than the other two ones and a threshold which is previously determined from pulse height
distribution, the value of the center index is“ 1 “. The other cases are“ 0 “. A pulse height distribution is fundamental to thepulse-counting because a pulse from the ion has to be separated from noise. The pulse energy and noise level are distributed to
29 ± 28 mV and 2 ± 3 mV in 3 -sigma, respectively. The ion pulse can be distinguished from noise.
Short dead-time and data streaming can allow the pulse-counting for TOF-MS. A time resolution of this method depends on thesampling rate of 5185（currently 3.125 GS/s）and responsively of the MCP assemblage（full width half maximum ~2.5 ns）.
Consequently, it is about 1 ns which is consistent with the three data points（0.96 ns）. In case of constant recurrence timing thesystem acquires ions from one to 1 × 109 cps. Since there is statistical fluctuation for incident ions, the ions are detected up to 1
×10 8 cps for practical purpose. In case of a high intensity signals（>10 8 cps）the signals can be recorded by another 5185 as
ananalog one. A dynamic range of the system is >1010 because the analog signals can be input up to 1011 cps.
［ 1 ］Ebata et al.（2011）SIA, DOI： 10.1002/sia.4857.
［ 2 ］Okumura et al.（2005）Eur. J. Mass Spectrom., 11, 261.

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 42: New Developments in Instruments and Detectors

PTu-136

13:30 － 14:40

Mass coverage range enhancement of reflectron time-of-flight mass
spectrometers by superconducting nano-stripline detectors

Nobuyuki Zen1, Koji Suzuki1, Shigetomo Shiki1, Masahiro Ukibe1, Masaki Koike1, Alessandro Casaburi2,
Mikkel Ejrnaes2, Roberto Cristiano2, Masataka Ohkubo1
National Institute of Advanced Industrial Science and Technology, Tsukuba, Japan, 2National Research Council,
Pozzuoli, Italy

1

Keywords:
MALDI-TOF MS, reflectron, superconducting nano-stripline detector, SSLD
Novel aspects:
The superconducting nano-stripline detector（SSLD）which has the detection area of 5 × 5 mm 2 with the fast response time ofseveral ns was realized.
Abstract:
Introduction
Superconducting detectors are promising for next-generation ion detectors for time-of-flight mass spectrometry（TOF MS）. They
can achieve a mass-independent detection efficiency even for macromolecules, because output pulses are produced through thedeposited kinetic energy at ion impact instead of secondary electron mission that is the ion detection mechanism of conventional
microchannel plate（MCP）detectors or secondary electron multipliers（SEM）. Among the superconducting detectors,
superconducting nano-stripline detectors（SSLDs）which consist of several hundreds of superconducting lines with a width of afew hundreds nm and a thickness of a few tens of nm have a fast response time less than 1 ns. We succeeded in realizing thedetector size up to 5 × 5 mm2 without response time degradation by using parallel nano-stripline configuration. An SSLD installed
in a reflectron TOF MS enables significant enhancement of the mass coverage range.
Experiments
Generally speaking, the response time of SSLDs is determined by the kinetic inductance of superconducting striplines, and thus
longer striplines result in a slower response time. Our strategy to realize a large detection area（or a long stripline）without
degradation of the response time is to connect striplines in a parallel configuration. In this study, we connected 50 striplies inparallel and the detector size of 5 × 5 mm2 was realized.
The SSLD was installed in a reflectron TOF MS（Voyager DE-STR； AB Sciex Pte. Ltd.）. The SSLD is cooled down by a cryogen-freecryostat, and the temperature of 3.2 K is easily obtained without liquid helium. The biomolecular samples are ionized by matrix
assisted laser desorption/ionization（MALDI）and those ions are accelerated by a high voltage of 25 kV.
Results and Discussions
By measuring Angiotensin I（1,296 Da）, ACTH（2,565 Da）, Lysozyme（14 kDa）, and IgG（146 kDa）with the 5 × 5 mm2 SSLD, wehave confirmed that the rise and fall time is less than 1 ns and less than 3 ns, respectively, and the pulse shape is independent ofmolecular ion species.
In addition to the success in the enlargement of the detection area, another important progress is the refinement of the operation
condition. Usually, the SSLD is driven by the bias current, and the larger bias current is necessary to achieve 100 % detection
efficiency. However, previous SSLDs made of the niobium superconductor were easily latched at the normal state after single ion
bombardment, and the bias current was limited at most 60 % of the critical current. In this study, the width of the jointing part ofstriplines was enlarged up to 10 times wider, which realized the 95 % current biasing without the latching problem.
In order to investigate the performance of our TOF MS, we measured a mixture of Lysozyme and IgG with a sinapic acid matrix.
Their mass peaks were recognized and the wide mass coverage range was achieved in the reflectron TOF MS.

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 42: New Developments in Instruments and Detectors

PTu-137

11:10 － 12:20

An Improvement of MCP Dynamic Range for TOF MS Application

Yusuke Hayase, Uchiyama Toshiyuki, Ishiguro Katsuyuki
Hamamatsu Photonics, Iwata, Japan

Keywords:
MCP, Dynamic Range, Linearity, Ion Detector, Microchannel Plate
Novel aspects:
We have found the best material composition and formation to strike a balance between wide dynamic range and robustness.
Then we have developed the robust and wider dynamic range MCP.
Abstract:
Microchannel Plates（MCP）are widely used for Time-Of-Flight Mass Spectrometry（TOF MS）because they have a fast time
response less than 1 ns, small time jitter, long life time, and a large effective area, which are desirable features for TOF MS.
However, the electron multiplier tube（EMT）is still used in MS applications that require a wider dynamic range since the MCP’sdynamic range is limited. Another limitation regarding MCPs is their propensity to warp and crack easily when stored for long
periods of time, which makes the time jitter worse. In order to solve this big problem, we have already developed a“Robust“
MCP which will not warp and crack as easily during these long storage periods. Now, we have developed a“Robust“ MCP with awider dynamic range that will output as much current as EMT.
Dynamic range is dependent on the MCP strip current, which supplies electrons to the MCP channels. When there is a large
incoming ion signal, electrons are not supplied fast enough from the power supply to match the increase in output current. In
order to solve this problem, an MCP with a lower resistance is required. Since the MCP’s resistance is related to its material
composition, we researched many different materials and found the best material composition and formation to strike a balance
between wide dynamic range and robustness.
We evaluated the dynamic range of an MCP, 20mm dia. active area, 12um channel dia. and 0.48mm thickness, made from theabove material composition and formation. We confirmed the dynamic range of the MCP extends to 35uA, 5 times wider than theoriginal MCP, and expect the future versions will extend to 100uA. That means the developed MCP has the equivalent output
dynamic range to EMT. In terms of robustness, we did an accelerated test under 40 degree Celsius and 93% humidity environment
during 16 days, which corresponded to 1000 days storage under a regular environment. Flatness of a normal, non-robust, MCP wasover 150um or cracked after the storage, but flatness of the robust MCP did not change.
We will employ this technology in larger MCPs like 25mm or 40mm active area, a smaller channel MCP like 6 um and a thinner
MCP like 0.3mm.
The poster will show detailed material composition, formation and evaluation results.

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 44: Ambient Ionization

PTu-138

13:30 － 14:40

Specific analyte ionization using negative atmospheric ions formed in
atmospheric pressure corona discharges

Kanako Sekimoto, Mitsuo Takayama

Yokohama City University, Yokohama, Japan

Keywords:
Corona discharge, Negative atmospheric ion
Novel aspects:
It has been found that various atmospheric negative ions formed in atmospheric pressure negative corona discharges can be
useful for specific analyte ionization.
Abstract:
APCI-like ambient ionization techniques such as DART and ASAP have frequently employed corona discharge devices which lead
to abundant generation of the atmospheric ions utilized as reactant ions for analyte ionization. These techniques, however, havesome problems especially in negative-ion mode, e.g., the complexity of negative-ion mass spectra consisting of not only
deprotonated analyte peaks but also a number of peaks of molecular attached and/or fragment ions and atmospheric negativeions, some of which could not be identified. In order to accurately interpret the negative-ion mass spectra obtained, it is necessary
to understand the influence of atmospheric negative ions on analyte ionization. Here we regulated the formation of atmospheric
negative ions such as HO-, COx - and NOx - in ambient corona discharges, and systematically investigated the interaction between
individual atmospheric ions and various type organic compounds.
The corona discharge ion source used contained a needle, whose tip surface was adequately approximated by a hyperboloid of
revolution, and metallic orifice plate of a TSQ7000 triple-quadrupole mass spectrometer（Thermo Fisher Scientific, San Jose, CA）.
Discharge ionization was performed under atmospheric pressure in laboratory air with relative humidity of 50-70 % at 298 K.
The formation of atmospheric negative ions in ambient corona discharges was regulated by the electric field strength on theneedle tip surface, which is determined by combination of the gap between the point-to-plane electrodes, the DC voltage appliedon the needle, and the local curvature of the needle tip surface. The dominant atmospheric ion formed at the low field strength
below 107 Vm-1 was the superoxide anion O2 -. At medium field strength（107-108 Vm-1）, the negative ions originated from nitrogenand HCO3 -, were generated. The high fielddioxide NO2, oxalic acid（COOH）2 and carbonic acid H2CO3, i.e., NO2 -, COO（COOH）
8-1
strength above 10 Vm led to the formation of abundant NO3 ions and its complexes with HNO3, i.e., NO3（HNO3）
n（n = 1,2）.
The relationship between the field strength and the resulting atmospheric negative ions could be interpreted from the standpointof the field-emitted electron kinetic energy which is the principle factor governing the sequential progress of successive ionmolecule reactions in the formation of individual atmospheric ions.
Atmospheric negative ions described above could serve as reactant ions R- for analyte ionization. The individual atmospheric ionsR-specifically ionized analyte molecules M, consisting of aliphatic and aromatic compounds with several functional groups such as
methanol, 2 -phenylethanol, acetic acid, benzoic acid, phenol, phenylethylamine, 4 -aminobutanoic acid, and amino acids, toatmospheric ion adducts［M+R］-. The adducts of R- with higher proton affinities, i.e., O2 -, HCO3 - and NO2 -, furthermore, resulted in
the production of deprotonated analytes［M-H］- due to the efficient proton abstraction from M by R - . The abundances of theindividual atmospheric ion adducts［M+R］- and resulting［M-H］- formed under certain field strength conditions were in good
agreement with the concentration of R - formed under those conditions. The results obtained suggested that the formation of［M+R］- and［M-H］- under certain discharge conditions is attributed to the chemical affinity between M and R - and theconcentration of R - produced under these conditions. The CID experiments of all the atmospheric ion adducts［M+R］- ,furthermore, showed the characteristic fragmentation behavior for the individual atmospheric ions R- , in which deprotonatedanalytes［M-H］- and/or the atmospheric ion R- were mainly formed depending on the proton affinities of［M-H］- and R-. The CID
data obtained will be useful to determine the molecular mass of unknown compounds measured by ambient ionization techniques
using corona discharges.

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 44: Ambient Ionization

PTu-139

11:10 － 12:20

Probe Electrospray and Nanoelectrospray Ionization for Direct
Biomolecule Analysis and Cancer Diagnostics

Mridul Kanti Mandal1, Lee Chuin Chen2, Kentaro Yoshimura3, Subhrakanti Saha1, Obaidur Rahman1,
Matiur Rahman1, Yasuo Shida1, Satoshi Ninomiya2, Sen Takeda3, Kenzo Hiraoka1

1
Clean Energy Research Center, University of Yamanashi, Japan, 2Interdisciplinary Graduate School of Medicine andEngineering, University of Yamanashi, Japan, 3Department of Anatomy and Cell Biology, Interdisciplinary Graduate
School of Medicine and Engineering, University of Yamanashi, Japan

Keywords:
Ambient Mass Spectrometry, Cancer Diagnostics
Novel aspects:
The novel characteristics of PESI-MS can fulfill the requirements of clinical mass spectrometry based cancer diagnostics. Solid
Probe assisted nanoESI can also be used as a cancer diagnostic tool.
Abstract:［PESI-MS］： Biological samples normally contain high concentration of salts which are needed to remove by purification methods
prior to successful omics analysis with mass spectrometry. In addition, detergents are necessarily used for extraction protocols of
membrane proteins from biological cells or tissues. After extraction, elimination of detergent is indispensable for the better
performance of ESI-MS which is usually tedious and time consuming.
In contrast, PESI-MS developed in our laboratory has been successfully applied to the sample solutions that contain highconcentration salts and detergents with no special sample pretreatment. The mass spectra of PESI were measured as a functionof time for a mixture of several analytes. Intriguingly it was found that the analytes were electrosprayed sequentially in the orderof their surface activities during the period of electrospray. To fulfill the clinical goal and optimize the conditions for moleculardiagnosis of malignant tumors, PESI-MS has also been adopted for human pathology. It is found that PESI-MS has successfully
delineated the differential expression of phospholipids（PCs）and triacylglycerols（TAGs）in non cancerous and cancerous kidney
tissues. Therefore, PESI-MS can be used as a diagnostic tool during surgery or after biopsy.
［Solid probe assisted nanoESI］： Extraction procedures are mandatory for successful nanoelectrospray ionization（nanoESI）of
biomolecules from tissue specimen. To eliminate these procedures, solid probe assisted nanoESI has been newly developed. This
technique could minimize the conventional extraction protocols for analyzing biological tissues and cells.
In this method, 0.3 mm acupuncture needle was used for sample loading from biological tissue directly. The needle was simply
stuck to the biological tissue. After sampling, the needle was inserted from the backside of nanoESI capillary in which 1 µL solvent
was loaded in advance. By the application of high voltage, the extracted samples in solution were nano-electrosprayed. Thismethod is shown to be simple and easy to operate. By this method, diseased and healthy tissues could be clearly discriminated.
Since the direct sampling by acupuncture needle is comparatively much less destructive than conventional hypodermic needlebased biopsies, the solid probe assisted nanoESI would be a useful tool for biological tissue diagnostics with lowest invasiveness.
References:［ 1 ］M. K. Mandal, L. C. Chen, Y. Hashimoto, Z. Yu and K. Hiraoka. Anal. Methods, 2 （2010）1905-1912.
［ 2 ］M. K. Mandal, L. C. Chen, Z. Yu and K. Hiraoka. J. Mass Spectrom. 46（2011）967975.
［ 3 ］M. K. Mandal, L. C. Chen and K. Hiraoka. J. Am. Soc. Mass Spectrom. 22（2011）1493-1500.

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 44: Ambient Ionization

PTu-140

13:30 － 14:40

Development of ultra high sensitive ambient ionization mass spectrometry
using Leidenfrost phenomenon-assisted thermal desorption

Subhrakanti Saha1, Lee Chuin Chen2, Mridul Kanti Mandal1, Kenzo Hiraoka1

1
Clean Energy Research Center, University of Yamanashi, Japan, 2Interdisciplinary Graduate School of Medicine andEngineering, University of Yamanashi, Japan

Keywords:
Ambient MS, Leidenfrost phenomenon
Novel aspects:
The application of Leidenfrost phenomenon to the ultra high sensitive ambient ionization mass spectrometry was made. A varietyof compounds could be detected as molecular ions.
Abstract:
In ambient sampling/ ionization mass spectrometry, desorption of analytes plays a significant role for the high-sensitive detectionof less- or non-volatile compounds. Present work demonstrates a new sample desorption technique based on Leidenfrost
phenomenon. When a liquid droplet is dropped on a high temperature（temperature much higher than the solvent’s boiling point）
solid metallic surface, the liquid immediately vaporizes on the solid surface and the vaporized gas layer prevents the droplet tocontact with the metallic surface, i.e., the droplet levitates above the surface. This phenomenon is known as ’Leidenfrost
phenomenon’ and the temperature above which this phenomenon occurs is called as ’Leidenfrost temperature’. During thisprocess liquid droplet evaporates rather slowly due to the presence of the heat-insulating vapour layer under the droplet. Thus,
the temperature of the droplet is kept below the solvent’s boiling point. Thus, the non-volatile analytes in solution do notevaporate but are retained in solution until the last moment of the total liquid evaporation. At the last moment of droplet
disappearance, desorption of non-volatile compounds takes place accompanied by explosive solvent evaporation. The
evaporated/desorbed analytes were ionized by a dielectric barrier discharge（DBD）ion-source. This method was applied to illicit
and therapeutic drugs, explosives, toxicants, steroids, peptides, etc. and was found to be applicable to all of these compounds
irrespective of their volatilities. The lower limits of detection ranged as low as parts per trillion（PPT）level for drugs, toxicants andsteroids（sample amount fg to pg level）, parts per billion（PPB）level for explosives and 10-9M for some peptides（e.g., Cyclosporin
A）. The high sensitivity of the present method is due to the spontaneous enrichment of non-volatile analytes during the slow andsmooth evaporation of the solvent. As the temperature of the droplet does not rise above the boiling point of the solvent,molecular ions are observed as the major ions. This newly developed desorption technique is simple, rapid and high-throughput.

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 44: Ambient Ionization

PTu-141

11:10 － 12:20

Scanning Probe Electrospray Ionization for Ambient Mass Spectrometry

Yoichi Otsuka1, Sayuri Shide2, Junpei Naito1, Masafumi Kyogaku1, Hiroyuki Hashimoto1,
Ryuichi Arakawa2

1
Frontier Research Center, Canon Inc., Tokyo, Japan, 2Faculty of Chemistry, Materials and Bioengineering, Kansai
University, Osaka, Japan

Keywords:
Ambient sampling ionization, Tissue imaging
Novel aspects:
New atmospheric-pressure sampling / ionization method by single capillary probe. The liquid bridge and electrospray at the sideof the probe makes it possible to map the biological components.
Abstract:
Introduction
Imaging mass spectrometry（IMS）with atmospheric-pressure ionization method has received widespread attention because of thepossibility of rapid diagnosis. Desorption electrospray ionization（DESI）was developed in 2004, and it has received strong
attention, so far. DESI utilizes charged solvents and high-pressure nebulizing gas for the ionization process. Recently, nano-DESI
was developed which utilizes charged solvents and two capillaries aligned in a way to separate the desorption and ionization
steps. This method has two advantages over DESI such as improved detection limits and higher spatial resolution. However, thealignment of capillaries is also needed. We have developed the atmospheric-pressure ionization method named“Scanning Probe
Electrospray Ionization（SPESI）“ which uses a single capillary as the probe for ionization.
Methods
SPESI utilizes the capillary and charged solvents like DESI and nano-DESI, but this method have the advantages of simplicity
compared to these methods； High-pressure gas as well as the alignment of two capillaries are unnecessary. SPESI enables the
local desorption and ionization by using a liquid bridge and electrospray with single probe. A charged solvent is flowing throughthe probe. As the probe and the sample are close-positioned, a liquid bridge is formed between them. Immediately after thecomponents are dissolved by the liquid, they are ionized by electrospray ionization.
Preliminary data
The formation of the liquid bridge and the movement of the probe are observed by high-speed camera. We have found two
different operating modes in SPESI. One is named as“Contact Mode SPESI“ in which the position of the probe is kept close to
the sample surface. In this mode, the liquid bridge and Taylor cone are formed simultaneously. The other is named as“Tapping
Mode SPESI“ in which the probe is vibrating so as to separate the formation of liquid bridge and electrospray ionization. We
found that the control of the fluid motion of charged solvents from liquid bridge to Taylor cone was the key to operate thesemodes stably. One dimensional mapping of solid sample by SPESI was also studied. The position of sample was controlled by thesingle-axis moving stage. Each of reference samples and tissue sample were ionized by these two methods. As reference sample,
insulin（5.8kDa）and bovine serum albumin（BSA, 66kDa）solution was deposited on the PTFE substrate to make small spots.
Ionization along several spots was performed by contact mode SPESI. It was confirmed that the distribution of ion intensity along
the moving direction was corresponding to the position of the spots. Moreover, the formation of multivalent ions shows SPESI is a
soft-ionization method. Tissue samples were studied subsequently. Normal mouse pancreas was cryosectioned and mounted on
the glass substrate. It was dried under atmosphere and used without further treatment. Ionization along several thin tissues was
performed by tapping mode SPESI. The result indicated that various kinds of components were ionized on the expected sites of
the tissue samples. Our preliminary results indicate that SPESI can be used for the ambient sampling and soft ionization of
biomaterials. Moreover, the one-dimensional mapping of both BSA film and mouse tissue shows that SPESI can be used for
imaging mass spectrometry.

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 44: Ambient Ionization

PTu-142

13:30 － 14:40

Simultaneous Ionization of Nonpolar and Polar Compounds by Laser
Ablation Electrospray Ionization with Heated Gas Jet

Anu Vaikkinen1, Bindesh Shrestha2, Akos Vertes2, Risto Kostiainen1, Tiina J Kauppila1
1

University of Helsinki, Helsinki, Finland, 2George Washington University, Washington DC, USA

Keywords:
ambient ionization, laser ablation, electrospray, nonpolar, apolar
Novel aspects:
Simultaneous analysis of polar, nonpolar and low polarity compounds by heat-assisted laser ablation electrospray ionization-mass
spectrometry
Abstract:
Laser ablation electrospray ionization（LAESI）is an ambient mass spectrometric ionization method that can be used for molecularimaging, and the analysis of small cell populations and even single cells. In LAESI, a mid-infrared laser is used to ablate watercontaining samples producing a plume of vapor particulate projectiles. The ablated material is ionized by electrospray, after which
the ions are guided into a mass spectrometer for analysis. The ablation is highly localized with ablation areas in the range of 30 to
300 microns in diameter. LAESI efficiently ionizes polar compounds such as proteins, peptides and phospholipids, while theionization of nonpolar compounds is often less efficient. In this work we show that by assisting the LAESI process by heated gas
flow the ionization of low polarity compounds such as steroids, and even completely apolar compounds, such as polyaromatichydrocarbons（PAHs）can be accomplished.
As in conventional LAESI, the novel set-up used a mid-IR laser running at 2940 nm to ablate analytes from water-containing
samples. Analyte ionization was induced by an orthogonal electrospray of water/methanol（ 1 ：1, v：v）+ 0.1 % HCOOH. The
spray angle was approx. 50-60°respective to the mass spectrometer inlet. To facilitate the ionization of low polarity compounds,
the electrospray plume was intercepted by a heated nitrogen jet（~200 degrees Celsius）that was generated using a heatedmicrochip positioned on-axis to point directly toward the inlet of the mass spectrometer.
The novel heat-assisted LAESI ion source was found to enable the simultaneous ionization of polar and low polarity compounds ina single measurement. As expected for LAESI, polar compounds such as bradykinin fragment 1 - 8 were efficiently ionized without
the heated nitrogen flow. Low polarity compounds, such as estrone, were ionized only when the heated gas jet was applied. The
gas flow rate did not have a significant effect on the peak intensity of the bradykinin fragment 1 -8, whereas the signal of estrone
increased 19-fold when the gas flow rate was raised from 30 to 360 mL/min.
The heat-assisted LAESI method was used to analyze 10 microliter aliquots of 100 micromolar standards in water/methanol（ 1 ：1,v：v） placed on a glass microscope slide. Protonated molecules were observed for, e. g., naphtho［2,3-a］pyrene,
dehydroepiandrosterone, estrone, verapamil, and 1,2-dioleoyl-sn-glycero- 3 -phosphocholine, while bradykinin fragment 1 - 8
showed MH+ as well as［M+ 2 H］2+ ions in the spectrum. Thus the ionization of the tested compounds was somewhat similar toelectrospray ionization, except that apolar compounds with low proton affinities, such as naphtho［2,3-a］pyrene anddehydroepiandrosterone were also detected. Some compounds produced structure-specific fragments, e. g., cholesterol showed［M-OH］+ and 1 -palmitoyl- 2 -oleoyl-sn-glycerol［M-OH］+ and ions formed by loss of single fatty acyl chain.
The sensitivity of the method was explored by determining the limits of detection for bradykinin fragment 1 - 8 and estrone.
Volumes of 0.5 microliters in water/methanol were analyzed on a cooled Peltier stage to avoid rapid sample evaporation.
Bradykinin fragment 1 - 8 and estrone were detected from 2.5 and 6 micromolar solutions, respectively, giving absolute limits ofdetection of 1.3 pmol for bradykinin fragment and 3 pmol for estrone. The LODs were somewhat similar to those obtained withpreviously established techniques, as laser ablation atmospheric pressure photoionization gave LOD of 3 pmol for estrone and
LAESI without the heated gas jet detected 0.65 pmol of bradykinin fragment 1 -8. The novel heat-assisted LAESI method produced
linear response over 3 decades for the signal of verapamil with R2 of 0.99（n = 11）.
The novel technique was applied in lipid profiling of plant（avocado fruit）and animal tissues（mouse brain）. The main lipid
species observed from the brain was cholesterol, whereas avocado showed abundant triglycerides.

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 44: Ambient Ionization

PTu-143

11:10 － 12:20

Desorption atmospheric pressure photoionization-mass spectrometry fordirect analysis of tetrahydrocannabinol from Cannabis sativa samples

Tiina J Kauppila1, Anu Flink1, Ulla-Maija Laakkonen2, Laura Aalberg2, Raimo A Ketola1
1

University of Helsinki, Helsinki, Finland, 2National Bureau of Investigation, Vantaa, Finland

Keywords:
ambient ionization, desorption atmospheric pressure photoionization, drug analysis, Cannabis sativa, molecular ion
Novel aspects:
DAPPI can be used for the rapid and reliable analysis of THC from Cannabis sativa samples due to the specific CID fragmentation
pattern of the molecular ions formed in DAPPIAbstract:
The recent innovation of ambient mass spectrometry techniques has speeded up the analysis of confiscated drugs in forensic
laboratories. These techniques make possible the fast and direct analysis of confiscated drugs in their solid forms, such as tablet,
resin or plant, completely without sample preparation or chromatography. However, a serious problem is related to the analysis
of Cannabis sativa originating drugs： most ambient analysis techniques cannot differentiate between the active ingredienttetrahydrocannabinol（THC）and a non-active ingredient, cannabidiol（CBD）, which have the same molecular masses. This isbecause most ambient techniques rely on the formation of protonated molecules of THC and CBD that have similar CID
fragmentation patterns. However, desorption atmospheric pressure photoionization（DAPPI）can be used to form molecular ionsthat - similarly to the molecular ions formed in EI - have individual fragmentation patterns. Here, we show that this feature of
DAPPI makes possible the fast and specific analysis of THC from Cannabis sativa samples.
The dried Cannabis sativa samples were placed on a moving stage outside the mass spectrometer and analyzed as such, using ahome-made DAPPI source. Hot solvent vapor and nebulizer gas were delivered towards the sample using a heated nebulizer
microchip, causing the thermal desorption of the analytes from the sample surface. Nitrogen was used as the nebulizer gas andtoluene as the spray solvent. A krypton discharge VUV lamp with 10.0 and 10.6 eV photon energy was used to initiate the ionizing
reactions.
A set of 10 Cannabis sativa samples with different, known proportions of THC and CBD was chosen. The DAPPI mass spectra of all
the samples showed intense ions at m/z 314, which can be the M+. of either THC or CBD, formed by charge transfer. The production spectra of m/z 314 were collected and compared visually, and in addition, principal component analysis（PCA）of the MS/MS
spectra was performed using Unscrambler X（Version10.0.1, CAMO Software, Oslo, Norway）. The visual observation of the
product ion spectra showed that the spectra of the samples containing mainly THC were significantly different from thosecontaining mainly CBD. The product ion spectrum of the M+. of THC showed the most intensive product ions at m/z 299, 243, 231,
258, 272, 313, and 221（in the order of decreasing signal）, while the product ion spectrum of CBD showed the most intensive
product ions at m/z 272, 195, 258, 299, 108, 231, 208, 207, 206, 120, and 243. It would thus be possible for a forensic laboratory tovisually differentiate the samples containing high amounts of THC from the samples containing no THC. However, to test whether
it would also be possible to differentiate the samples containing small amounts of THC from those containing none, a PCA analysis
using the relative intensities of the MS/MS product ions as variables was performed. To optimize the PCA components in order tofind the simplest possible model, several combinations of ions were explored. The most simple model was found by choosing therelative intensities of just two variables： the product ions at m/z 272 and 299. This model gave an explained variance of 100 % andmade possible the differentiation of Cannabis sativa samples with high, intermediate and low contents of THC from those that
only contained CBD. The differentiation of THC and CBD containing Cannabis sativa samples is thus possible with DAPPI by using
the developed PCA model as the reference or just by visually comparing the spectra and the proportions of product ions at m/z
299 and 272 in the product ion spectrum of m/z 314.

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 44: Ambient Ionization

PTu-144

13:30 － 14:40

Open Spot Ambient Ionization Technology for Near-Instant Determinationof Sample Characteristics by using DART-MS

Musselman D Brian, Joseph LaPointe, Elizabeth Crawford
IonSense, Inc.

Keywords:
DART, Ambient Ionization, Chemical Identification
Novel aspects:
Rapid ionization with electrical current-based thermal desorption facilitates near-instant ionization
Abstract:
A new direct analysis in real time（DART）source has been developed to facilitate rapid desorption chemical ionization of samples
from wire mesh, OpenSpot Cards, designed to carry optimium volumes of sample. The use of the wire mesh coupled to a highcurrent power supply facilitates a simplified approach to the sampling process associated used in qualitative mass spectrometry
with DART-MS. OpenSpot cards are ideal for loading either solid or liquid samples since it is only necessary to scrape the solid
across the screen surface or dispense a microliter of sample laden solvent in the sample region to facilitate analysis. The simplicity
of using this thermal desorption ionization method facilitates Transmission mode DART ionization. Application of the technology
to rapid identification of products of organic synthesis facilitating with high resolution-MS will highlight the utility of the method forreaction monitoring. Detection of contaminants in products with low resolution compact MS and Principle Component Analysis
will be described as a means to improve quality control while reducing the cost per analysis. Finally, qualitative and quantitative
analysis of pesticides and environmental contaminants in various commercial products by using MS/MS will be featured withemphasis on the use of the method to facilitate screening in advance of full analytical determinations.

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 44: Ambient Ionization

PTu-145

11:10 － 12:20

Plasma pencil atmospheric mass spectrometry for the detection andmeasurement of micronutrients in complex solutions

Lo Edward, Maria J Stein, David G Castner, Buddy D Ratner
University of Washington, Seattle, United States of America

Keywords:
ambient ionization, nutrient measurement
Novel aspects:
This work uses ambient plasma ionization for the separation of analytes in complex solutions based on concentration, and applies
a Bayesian regression model for analyte quantitation.
Abstract:
Malnutrition, particularly in the form of micronutrient deficiencies, is a major contributor to the global burden of disease.
Insufficiencies in any of the key micronutrients, specifically vitamin A, iron, zinc, folic acid and iodine, can cause critical healthproblems. Pregnant women and children are particularly vulnerable. While present in all countries, malnutrition is of higher
concern in less developed countries. This is due in part to a lack in established food fortification procedures. Additional nutritional
epidemiology of these groups would be beneficial to determine which nutrients and quantities are necessary to achieve optimum
health in these regions； however, the high cost and time consuming nature of current micronutrient measurement methods makelarge studies unfeasible. Our goal is to utilize a relatively new technique, plasma pencil atmospheric mass spectrometry（PPAMS）,
as a field-deployable method to simultaneously measure the five micronutrients in blood. A multivariate software model is thenused to quantitatively assess the resulting fragment spectrum.
The PPAMS system was assembled in house by constructing a low temperature plasma probe and coupling it to a Bruker Esquire
LC mass spectrometer（Bruker Daltronics, Billerica, MA）. Sample spectra were collected in the m/z range from 50-1100 m/z, after
ionization with a helium based plasma. Sample matrices were mixed using three physiologically-relevant blood levels（BLC,
healthy, average and critically low）of each of the five micronutrients, doped into a 10% porcine plasma solution. Samples wereanalyzed to determine the sensitivity of our device and the ability of the multivariate technique to separate the nutrient
populations based on concentration. Initial studies have focused on varying three nutrients（iron, zinc and vitamin A）at each of
the three levels while holding iodine and folic acid at the critically low BLC. Future studies will be expanded to include all
nutrients. Samples were kept in liquid form and analyzed directly.
Data analysis using principal component analysis（PCA）of the positive ion spectra was completed by grouping data sets to varya single nutrient while holding the other nutrient levels constant. The resulting scores plots show clear separation at the 95 %confidence level between low, medium and high levels of each nutrient, with separation occurring proportionally to concentration.
From this separation, we can study the loadings plots to determine the specific peaks associated with individual nutrients.
Quantitation is being completed through the use of a Bayesian regression model. Initial samples at high, medium and low BLCs
are being used as a test matrix to form the model, which will be used to analyze additional samples. In this method, discrete m/zdata points are converted into a wavelet function to model the system, which is then applied towards“unknown“ spectra toestimate the nutrient levels in the unknown samples. Future work will combine the two systems to improve data prediction byfocusing on specific peaks that are related to nutrient concentration.
Acknowledgements： We thank the Bill and Melinda Gates Foundation for generous funding. The authors would also like toacknowledge Drs. Martin Sadilek, Chris Barnes and Marvi Matos for their shared expertise and assistance.

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 44: Ambient Ionization

PTu-146

13:30 － 14:40

Rapid screening and quantitation of pesticides in fruit commodities byDirect Analysis in Real Time (DART) ambient ionization mass spectrometry

Elizabeth A Crawford, Brian D Musselman
IonSense, Inc., Saugus, MA, USA

Keywords:
Pesticide Screening, Direct Analysis in Real Time（DART）, Multiple Reaction Monitoring（MRM）, High Resolution Accurate Mass
MS/MS
Novel aspects:
Porous sampling surfaces and automation allow for rapid screening and quantitative analyses of pesticides in juices by DART-MS.
Abstract:
INTRODUCTION
Routine pesticide and fungicide use in the United States, as well as abroad warrants the need for analytical techniques that can
rapidly screen and quantify residues in order to efficiently sample products before reaching the consumer market. The allowable
residue levels of pesticides on fruits and vegetables that are processed into juices are governed in the US by the United States
Environmental Protection Agency（US EPA）Code of Federal Regulations Title 40： Part 180 that sets tolerance levels based on the
produce commodity. Of particular interest in the United States, carbendazim was found in imported orange juice from Brazil,
where the use of the fungicide is legal. The US does not permit the use of carbendazim on any citrus fruits and therefore any
imported citrus juice is held to the same regulation. Ambient ionization offers the ability to screen fruit juice samples directly inseconds and with automated sample introduction quantitative measurements can be assessed using direct analysis in real time
（DART®）mass spectrometry.

EXPERIMENTAL
A fourth generation standardized voltage and pressure DART ionization source（DART-SVP）was fitted with a motorized linear rail
and transmission experiment module. In transmission DART experiments the liquid juices are suspended onto a stainless steel
mesh and the DART ionizing gas passes through the porous sample support efficiently ionizing the analytes. A number of juices（cranberry, grape, orange, lemon and a vegetable mix）were spiked with the following pesticides： diazinon, carbaryl, metalaxyl,
difenoconazole, pyridaben, dimethomorph, azoxystrobin, fenhexamid, pyriproxyfen and spiroxamine. The pesticide mix was spiked
at levels ranging from 10 ppb to 5 ppm with caffeine added as an internal standard at 100 ppb. Carbendazim was also spiked intoorange juice at 1 ppb to 5 ppm and orange juices from India（ 4 ）, Europe（ 5 ）and USA（ 3 ）were screened for carbendazim.
The DART source was interfaced with a QTRAP and quadrupole Orbitrap benchtop instruments.

RESULTSAn API 4000 QTRAP was tuned for MRM conditions for the 10 pesticides in the spiking mixture as well as carbendazim using theTurboV source with ESI probe. The spiked fruit juices were spotted（ 3 µL spots）onto a stainless steel mesh and the spots wereallowed to dry before analysis. The samples were driven through the DART ionization region using the motorized linear rail at a
speed of 0.5 mm/s resulting in a single sample time of 25 seconds. A preliminary set of runs were conducted on the solvent
standard pesticide mix to determine an optimal DART heater temperature of 175 °C for the pesticide mixture and 250 °C fortargeted carbendazim analysis. Several of the pesticides could be detected as low as 10 ppb directly from the juices using thismethod and carbendazim was directly detected out of orange juice as low as 2 ppb with limit of quantitation of 15 ppb.
An identical DART set-up was coupled with a Q Exactive benchtop Orbitrap mass spectrometer. The Q Exactive provided aversatile MS platform to both rapidly screen with low limits of detection and preselect ions of interest generating high resolution
targeted MS/MS data for comparison with the QTRAP MRM data. Limits of detection using the DART Q Exactive quantitative
method were below 10 ppb for the 10 pesticides in the mixture, as well as for carbendazim directly analyzed from fruit juices. Of
all of the orange juices screened for carbendazim three juices all purchased in the EU tested positive at levels ranging from < 5
ppb up to 21 ppb.

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 44: Ambient Ionization

PTu-147

11:10 － 12:20

Study of fragmentaion behaviors for atmospheric negative ion adducts ofphenylalanine homologues using atmospheric pressure corona discharge
ionization mass spectrometry

Natsuki Matsuda, Nami Sugasawa, Chika Mifune, Kanako Sekimoto, Mitsuo Takayama
Yokohama City University, Yokohama, Japan

Keywords:
phenylalanine, radical, fragmentation, negative ion
Novel aspects:
Information about fragmentation behaviors for deprotonated molecule of phenylalalnie homologues was obtained, and areactionof hydrogen abstract induced by radical speces was found.
Abstract:
Interpretation of fragmentation behaviors for both（de）protonated analytes［M±H］± using collision induced dissociaton（CID）
spectra makes it possible for the structual elucidation of analytes M, which is useful to identify unknown analytes in mass
spectrometry. However, it is difcult to obtain systematic information of fragmentation behaviors for［M-H］- compared with thosefor［M+H］+. We have recently established an atmospheric pressure corona discharge ionization mass spectrometry（APCDI-MS）
that results in soft ionizaton in negative-ion mode using various atmospheric negative ions R（e.g., O2 -, COx - and NOx -）to form
atmospheric ion adducts［M+R］-. The adduct ions［M+R］-, furthermore, can be fragmented to deprotonated analytes［M-H］- andother product ions. Here we performed the CID experiments for atmospheric ion adducts of phenylalanine homologues all ofwhich are aromatic compounds having an amino and carboxyl groups, using APCDI-MS. The CID data obtained provided theinformation about the fragmentation behaviors for［M+R］- and［M-H］- for individual phenylalanine homologues.
Mass spectra were acquired on a TSQ7000 triple-quadrupole mass spectrometer（Thermo Fisher Scientic, San Jose, CA）. The
discharge ion source used contained a needle（ca. 1 μm in the tip curvature radius）and a metalic orice plate, which wereseparated by a gap of 3 mm. DC needle voltages and needle angle with respect to the orice axis were varied over the range of-1.9--3.9 kV and 0 -π/ 2 rad, respectively. A micro-ceramic heater was placed between the needle and the orice plate in order tovaporize condensed-phase analytes. Discharge ionization was performed under atmospheric pressure in laboratory air withrelative humidity of 50-70 % at 298 K. Collision gas and energy（lab.）for CID experiments used were argon at 2.0×10-3 torr and 5 -25
eV, respectively.
Gaseous L-phenylalanine（L-Phe）was ionized by various atmospheric negative ions R -（=O 2 - , HCO 3 - , HCOO - , CH 3 COO - and）, and resulting in the formation of atmospheric ion adducts［L-Phe+R］- . The CID of all the adducts［L-Phe+R］COO（COOH）
except for［L-Phe+CH3COO］- brought about the production of deprotonated molecule［L-Phe-H］- and/or the atmosperic ions R-,
formed via the dissociation accompanied by proton abstraction from L-Phe by R - and a simple dissociation of the adduct［L-Phe+R］- , respectively. Which product ion peak can have a higher intensity was dependent on the proton afnities between［L-Phe-H］- and R-. When the proton afnity of R- is higher than that of［L-Phe-H］-, R- can efciently abstract a proton from L-Phe toform dominant product ion［L-Phe-H］-. The adduct［L-Phe+CH3COO］-, in contrast, resulted in the formation of abundant［L-Phe3 H］- and less abundant［L-Phe-H］- and CH3COO-. This result suggests that CH3COO- is a biradical anion species CH3COO-. andcan abstrat not only a proton but also two hydrogen atoms from L-Phe.
The gaseous ionic species R- described above bind to L-Phe homologues M, D-phenylalanine（D-Phe）, L-homophenylalanine（HPA）
and L-phenylglycine（PG）, to form［M+R］-. The fragmentation behaviors of［M+R］- for the fragmetation of［M-H］- and/or R-, or［M3 H］- were the same as that of［L-Phe+R］-.
CID of［L-Phe+R］- and［M+R］- with higher collision energies provided the ionformation about the fragmentation behaviors of
deprotonated analytes（［L-Phe-H］- and［M-H］- ）for individual analytes.［L-/D-Phe-H］- could be fragmented via two different
processes, i.e., successive losses of NH 3 and CO 2 to form C 6 H（CH）
5
2 COO and C 6 H（CH52 ） ions, respectively, and the loss oftoluene for the formation of NH（CH）COO- ion, in which the fragmentation behaviors were independent of L and D bodies. The
precursor ion［HPA-H］- led to the formation of product ion NH2CH2COO- via the loss of styrene, whereas CID of［PG-H］- brought（NH2）.
about the loss of CO2 and resulting product ion C6H5C（H）

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 44: Ambient Ionization

PTu-148

13:30 － 14:40

Unusual radial cation formation of arginine by atmospheric pressure
corona discharge ionization (APCDI)

Atsushi Wada, Kanako Sekimoto, Mitsuo Takayama
Yokohama City University, Yokohama, Japan

Keywords:
Radical cation, arginine, APCDI, hydrogen loss
Novel aspects:
Unusual formation of radical cation of most basic amino acid arginine was observe in APCDI method, and its mechanism（s）was
discussed.
Abstract:
Introduction
It has been well known that ambient ionizations such as DESI, ASAP and any other API methods give protonated molecules［M+H］+ in positive ion mode, although the resulting analyte ions are according to the thermochemical properties of analyte. The
protonated molecules generate via ion/molecule reactions such as proton transfer from reactant ions RH+ to analyte molecules M,
under atmospheric pressure conditions. The proton transfer reactions depend on the proton affinity（PA）or gas-phase basicity（GB）of analytes and reagent gas molecules R. Here we performed the experiments of atmospheric pressure corona discharge
ionization（APCDI）of all the twenty different amino acids. Amino acids commonly have basic and acidic functional groups calledas N- and C-terminal side, respectively. Furthermore, the amino acids have several physicochemical properties such ashydrophobic, hydrophilic, aromatic, sulfur-containing, acidic, basic and neutral, according to the nature of side chains. Although it
would be expected that most basic amino acid arginine（Arg）gives a dominant peak corresponding to protonated molecules［M+H］+ in positive-ion APCDI experiments, the APCDI mass spectrum of gas-phase Arg molecules gave the dominant peakcorresponding to radical cation M +.   at m/z 174 in the APCDI mass spectrum. In the present paper, we discuss about the mechanism（s）of the formation of radical cations of basic analytes, as well as Arg molecules.
Experimental
Mass spectra were acquired on a TSQ-7000 triple-quadrupole mass spectrometer（Thermo Fisher Scientific, San Jose, CA）.
Atmospheric pressure corona discharge ionization（APCDI）source used contained a needle（ca. 1 um in the tip curvature radius）
and the orifice plate of the mass spectrometer, which were separated by a gap of 3 mm. A micro ceramic heater was placed
between the point-to-plane electrodes to rapidly vaporize condensed-phase analytes. DC voltage applied to the needle was +3.4
kV. The orifice temperature was varied in the range of 333-373 K. The discharge experiments were performed in the laboratory
ambient air under atmospheric pressure with relative humidity of 45-65 % at 298K. The analytes used were Arg, aniline, 5 -amino
salicylic acid（ 5 -ASA）, and related compounds.
Results
The dominant analyte ion formed in APCDI mass spectra of organic compounds was generally protonated molecule［M+H］+. The
formation of［M+H］+ is most likely to be attributed to reaction（ 1 ）involved in the oxonium ion H 3 O + which is a major
atmospheric ion species produced in the discharge area. That is, the formation of atmospheric ions adduct［M+H3O］+ is initiallyoccurred, and subsequently the protonated analyte［M+H］+ is generated by the loss of water molecule H 2 O due to proton
abstraction from H3O+ by M.
M + H3O+→［M+H3O］+→［M+H］+ + H2O（ 1 ）Because of ionization processes including proton transfer from H3O+ to M, only the analytes with proton affinities higher than H2O（691 kJ mol-1）can be ionized.
It was found, however, that several analytes, Arg, aniline and 5 -ASA, are ionized as not only［M+H］+ but also radical cations M+..
It has been well-known that M+. can be formed by electron emission due to electronic excitations of M（reaction 2 ）, i.e., electron
ionization（EI）.
M + e-→ M+. + 2 e-（ 2 ）The CID of the M+. for aniline resulted in the formation of the product ion C5H6 +. via the loss of HCN. This fragmentation behavior
was in agreement with M+. obtained with EI, indicative of that M+. formed with APCDI has the same structure as that with EI. On
the other hand, the CID pattern of M+. for arginine formed with APCDI was inconsistent with that by EI.

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 44: Ambient Ionization

PTu-149

11:10 － 12:20

Theoretical and experimental studies of ionization of explosive
compounds using dielectric barrier discharge ionization

Hirotaka Yabushita1, Hirofumi Nagao1, Toshinobu Hondo1, Michisato Toyoda2
1

HORIBA STEC,Co.,Ltd., Kyoto, Japan, 2Osaka University, Osaka, Japan

Keywords:
explosive, dielectric barrier discharge ionization, fragmentation, matrix effect, quantum chemical calculation
Novel aspects:
We investigated effect of additives on dielectric barrier discharge ionization of explosive compounds not only experimentally but
also theoretically by using quantum chemical calculation.
Abstract:
Method development for rapid on-site detection and identification of criminal compounds is important to find scientific evidenceof terrorism and crimes. Minimized sample preparation and high sensitive method are required.
Recently, several researchers have studied plasma discharge atmospheric pressure ionization（API）mass spectrometry［ 1 - 3 ］forhigh sensitive analysis of explosive compounds. In order to apply their method to practical crime scene investigation, effect ofcommon chemical background should also be addressed.
X. Zhao and J. Yinon reported that the electrospray ionization（ESI）and atmospheric pressure chemical ionization（APCI）mass
spectrometry of nitro ester compounds were affected by impurities in the analytical system to form a variety of adduct ion insteadof molecular ion, which made it difficult to interpret the spectrum. To solve this problem, they introduced ammonium chloride topost-column. The addition of ammonium chloride led to formation of stable chlorine adduct ion and improved detection limit ofnitrate ester［ 4 ］.
The addition of the compound reacting with target to form stable ion also should be useful for plasma discharge API method. Inorder to investigate ionization reactions of explosive compounds, we designed dielectric barrier discharge（DBD）ionization
source in house which was attached to FT-ICR mass spectrometer（Apex III 4.7T, Bruker Daltonics Inc. Billerica, MA, U. S.）. Sample
was placed on the glass plate in ambient atmosphere and vaporized by halogen lamp as heater. Vaporized gas was aspirated into
DBD source through 0.8mmI.D. by 50mmL of stainless-steel capillary that maintained 100 degrees Celsius. Helium gas wascontrolled by mass flow controller（D213, HORIBA STEC, Kyoto, Japan）. Dichloromethane（DCM）was introduced through thestainless-steel capillary.
Both meta-dinitrobenzene（m-DNB）and para-dinitrobenzene（p-DNB）gave similar complex mass spectra consisting of several
reacted ions under the condition without DCM. By DCM addition, simple chlorine adduct ion without significant fragmentation
was observed for m-DNB. The identification efficiency and sensitivity were improved. In contrast, the ionization of p-DNB was
drastically suppressed by DCM addition.
This result shows that addition of DCM may improve m-DNB detection performance but cause false negative result for p-DNB
detection. We assume the orientation of nitro-group changes chlorine affinity that improves m-DNB detection performance but
not for p-DNB. We will report further study of chlorine affinity difference by using quantum chemical calculation.
［ 1 ］L. C. Chen et al, Development of ambient sampling chemi/chemical ion source with dielectric barrier discharge, J. Mass.
Spectrom. 45（2010）861［ 2 ］J. F. Garcia-Reyes et al., Detection of Explosives and Related Compounds by Low-Temperature Plasma Ambient Ionization
Mass Spectrometry, Anal. Chem. 83（2011）1084［ 3 ］F. M. Green et al., Ambient mass spectrometry： advances and applications in forensics, Surf. Interface. Anal., 42（2010）347［ 4 ］X. Zhao and J. Yinon, Identification of nitrate ester explosives by liquid chromatography-electrospray ionization andatmospheric pressure chemical ionization
mass spectrometry, J. Chromatogr. A 977（2002）59

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 44: Ambient Ionization

PTu-150

13:30 － 14:40

Quantification of six anti-diabetic drugs in human plasma by indirect spray
FT ICR mass spectrometry

Wan Li, T W Dominic Chan

The University of Hong Kong, Hong Kong, China

Keywords:
Ambient ionization； Indirect spray； Anti-diabetic drugs； Quantification；
Novel aspects:
A a novel ambient ionization technique named indirect spray are used for quantification.
Abstract:
Indirect spray is a novel ESI-based ambient ionization technique. It has similar geometry arrangement as TM-DESI, instead of usinga mesh, membrane funnel plate is used as sample loading substrate. No pneumatic assistance is needed in indirect spray. Whencharged solvent droplets were sprayed onto the membrane funnel and the deposited analytes dissolves in, then a secondary
electrospray i.e. indirect spray is induced. Indirect spray is applicable for direct detection of different types of compounds, fromsmall drug molecules to protein, using a small volume of sample（typically 50 nl）. It has been proved indirect spray is of higher
sensitivity than TM-DESI. In this work, we validate the ability of indirect spray for quantitative analysis. Six chemically diverse antidiabetic drugs, including acarbose, pioglitazone hydrochloride, acetohexamide, repaglinide, insulin and vildagliptin, were
examined. Different drugs have different limit of detection（LOD）ranging from 0.5 fmol to 2 fmol. Dynamic range of this technique
was typically three or four orders of magnitude. Precision and accuracy values for quality control vildagliptin samples range from
1.7 % to 14 % and from 6.5 % to -15 % respectively. Extending the current indirect spray analysis to biological background is inprocessing. SPE sorbent attached membrane funnel will be fabricated to eliminate the matrix effect. Different methods of adding
internal standard（vildagliptin-d 3 ）will be tested. Inter-and intra-day precision and accuracy and LOD will also be investigated forvildagliptin in plasma.

References
1. Hess, C.； Musshoff, F.； Madea, B. Anal Bioanal Chem. 2011, 400, 33-41.
2. Manicke, N. E.； Yang, Q.； Wang, H.； Oradu, S.； Ouyang, Z., Cooks, R. G. Int. J. Mass Spectrom. 2011,300, 123-129.
3. Kennedy, J. H.； Aurand, C.； Shirey, R.； Laughlin, B. C.； Wiseman, J.M. Anal. Chem. 2010, 82, 7502-7508.

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 44: Ambient Ionization

PTu-151

11:10 － 12:20

Physical process hierarchy in ion movement inside a gas dynamic interface
for the mass-spectrometer with ambient ionization.

Nicolay R Gall1, Alexander S Berdnikov2, Lidia N Gall2

Ioffe Physico Technical Inst. of Russian Academt of Sciences, St.Petersburg, Russia,, 2Institute for Analytical
Instrumentation, St.Petersburg, Russia

1

Keywords:
ambient ionization, gas dynamic interface, ion mobility,
Novel aspects:
the model to account simultaneously the effect of gas dynamic, DC and RF electric fields in a gas dynamic interface including theeffect of ion inertia
Abstract:
Ion transport inside the gas dynamic interface of the mass-spectrometer with ambient ionization is one the most important factors
affecting losses in ions and hence, the detection limit and instrument discriminations. One needs to simultaneous account for gas
dynamic forces, DC and RF electric fields to provide correct description of the interface processes. Electrode positions can change
the structure of gas dynamic flows, so generally, one should consider a self-consistent task.

For an empty chamber or for electrode positions affecting poorly on gas flows, the most important parameter is the ratio of thechamber length L to the Mach barrel length LM = 2 / 3 d（P/P0）1/2, where d is the inlet diameter, P and P0 are the static pressuresat the interface and at the inlet respectfully. Pulsations of the shock wave position and macro turbulence are inherent for a ’short’chamber with L< 2 LM； on the contrary, a stable gas jet with the strict position of the shock wave and the automodel shape down
the Mach barrel is typical for L>> LM. Usually, the Mach barrel length LM = 2 6 mm.

The second dimension parameter is important for a ’long’ chamber, the one characterizing the jet divergence. If the automodel
structure is well organized, the jet becomes several times wider along the chamber, gas if looses its velocity, and the region of
micro turbulence appears； it can be observed, e.g., at L~40 mm for P=1000 Pa and d = 0.5 mm. However, the jet is nearly parallel
and the gas velocities are about 400 500 m/s reaching all the way to the skimmer at an interface pressure of 200 Pa.

Accounting the role of electric field, one can try to consider the ion velocity as a vector sum of the gas velocity Vg and electric
velocity in a form V = V g + V E    = V g + K*E, where K is the mobility, and E is the field. This approach works well for theatmospheric region, but it neglects the time necessary for an ion to accelerate or decelerate； one needs to account correctly thistime depending on the ion mass, when working in the intermediate vacuum pressures. This mobility approach limits the spatial
and time gradients of the electric and gas velocity fields, that may be considered. Extention of the validity range may be betteraccounted by a Stocks criterion Sk = tin/t0 , where tin is the ion inertia time, tin = K*M/Z, M/Z is the mass to charge ratio, and t0 =E*（dE/dr）-1 *V-1 + E*（dE/dt）-1 characterizes the changes in field along the ion trajectory.
To account for RF field role requires a more complex approach. We have shown that the effect of pseudo-potential, i.e. ion
repulsion from the spatial gradient of RF filed, appears only due to the phase difference between the momentary value of the ion
velocity and the field； this difference is maximal at high vacuum and decreased with the increase of the gas density. This allows
one to predict and calculate ion repulsion from spatially non-uniform RF fields for the given pressure, frequency, RF magnitude,and electrode structure.

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 44: Ambient Ionization

PTu-152

13:30 － 14:40

Using Thin Layer Chromatography Spray (TLC spray) Ionization Mass
Spectrometry to Analyze Drugs in Whole Blood

Yi-Tzu Cho1, Chu-Nian Cheng2, Hung Su2, Jentaie Shiea*2

1
Yuh-Ing Junior College of Health Care & Management, Kaohsiung, Taiwan, 2National Sun Yat-Sen Univ., Kaohsiung,
TAIWAN

Keywords:
Thin layer chromatography, mass spectrometry, spray ioinization
Novel aspects:
A new TLC/MS technique, TLC spray, was developed for rapidly characterizing the chemical compounds on the TLC plate.
Abstract:
Thin layer chromatography（TLC）is an analytical tool commonly used for sample purification and separation. To characterize theseparated chemical compounds on TLC plate, coupling TLC with mass spectrometry（TLC/MS）seems to be the most efficientapproach. Since the analytes are buried in the stationary phase bed, the development of an efficient sampling method is crucial in
detecting the analytes by TLC/MS. In this study, a new TLC/MS technique TLC spray was developed for rapidly characterizing thechemical compounds using the TLC plate. The drugs spiked in the whole blood were successfully detected by this TLC spray mass
spectrometry approach.
The design of the TLC spray is similar to that of a direct electrospray probe except the fine rods of DEP. The tip end of a TLC
plate（silica gel on aluminum sheet）was cut to a sharp triangle. The small amount of whole blood sample was spotted on the
TLC plate. A syringe pump was used to apply the mobile phase（acidic methanol solution）through a capillary onto the TLC plate
to separate the analytes in the sample spot. After applying high voltage on the TLC sheet, electrospraying was generated at the
tip of the TLC plate. The analytes eluted out of the TLC plate were ionized by electrospray ionization and detected by aquadrupole/time-of-flight mass spectrometer. The TLC spray MS was used to characterize ketamine, methamphetamine, and beta2 -adrenergic receptor agonist spiked in whole blood. Since blood cells and large molecules were too heavy to be carried in themobile phase, they stayed at the original sample spot during development. Only small compounds such as spiked drugs werecarried to the end of the TLC plate. In this preliminary study, the spiked drugs were detected in whole blood and the detection
limit of methamphetamine by TLC spray MS is estimated to be approximately 1 ppm.

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 44: Ambient Ionization

PTu-153

11:10 － 12:20

Ambient surface mass spectrometry of polymers and molecules using
PADI - Optimising parameters, improving repeatability and damage effects

Tara L Salter, Ian S Gilmore

National Physical Laboratory, Teddington, UK

Keywords:
polymers, plasma, depth profiling, PADI
Novel aspects:
Repeatable ambient surface mass spectrometry of polymers, and initial investigation of depth profiling capability.
Abstract:
Reliable and repeatable measurements are essential for the strong uptake of any analytical technique. Ambient surface mass
spectrometries have demonstrated exciting and revolutionary measurement capability but for wider acceptance in industry theyneed a secure metrology foundation. Plasma sources are increasingly being used for ambient mass spectrometry since thedevelopment of DART（direct analysis in real time）in 2005 and subsequent imaging methods such as PADI（plasma assisted
desorption ionisation）and LTP（low temperature plasma）. Here, we commence establishing the essential metrology for PADI,
which has already been shown to be successful at analysing pharmaceuticals［Anal. Chem. 2007； 79, 6094］and personal careproducts［Analyst, 2011； 136, 3274］.
The PADI system is an atmospheric pressure RF generated helium plasma coupled to a Thermo LTQ-Orbitrap Velos mass
spectrometer. We conduct a systematic study of the effects of geometry, RF power and gas flow rate on the relative signal
intensities, sensitivity and repeatability using molecular（valine）as well as polymer（PET, PMMA, PTFE and PLA）surfaces.
We conduct a detailed study of the instrumental contributions to the spectral repeatability, including the intrinsic noise in themass spectrometer. Firstly, we identify and separate out the contributions to the repeatability（relative standard deviation）of the
PADI peak intensities. We show that the Orbitrap has a noise distribution approximately as expected for random noise so that at
intensities of above 108 ’counts’ it is less than 2.2%. The standard deviation of the PADI signal intensity is proportional to the signal
intensity which is clearly not random but related to systematic effects in the source operation. Optimisation of the plasma sourceis essential for ensuring robustness and reliability. We show how the signal varies with geometry, helium flow rate and plasma
power. Thermal imaging of the sample surface shows that the temperature rises approximately linearly with plasma power and at
19 W is 70 oC. To reduce the effects ofdamage for surface sampling, we recommend keeping the surface temperature below 40 °C
by operating at less than 15 W. General guidance is given for practical analysis.
Importantly, PADI can analyse a wide variety polymers. For this, it is essential that the plasma probe is not near a groundedelectrode. Analysis of polymers gives mass spectra with repeating series containing different fragments and adducts. These are
identified using the high-resolution and mass accuracy of the Orbitrap MS. Preliminary studies of sample surfaces after PADI
analysis show that the plasma can change the chemical make up of the surface leading to damage. With the recommended
operating parameters, the plasma erodes the polymer sample at a rate of 0.87 nm s-1（for PMMA）. Quantitative analysis of theeroded crater bottom by x-ray photoelectron spectroscopy（XPS）shows the chemical damage is less than 10%. This shows thepotential for polymer depth profiling. Similarly, depth profiles for molecules are obtained. The potential for PADI for mass
spectrometry imaging for a wide range of applications will be discussed.

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 44: Ambient Ionization

PTu-154

13:30 － 14:40

Thermal Dissociation Atmospheric Chemical Ionization Mass Spectrometry
with a Miniature Source for Selective Trace Detection of Dimethoate in
Fruit Juices

yongzhong ouyang, Tiqiang zhang, Tenggao Zhu, Huanwen Chen
East China Institute of Technology,Nanchang, P.R. China

Keywords:
TDCI； Ionic liquid； dimethoate； without any sample pretreatment
Novel aspects:
Instead of toxic solvents and high-voltage or high electric field used in ambient ionizations, ionic liquid, a“green solvent“, wasemployed to directly generate reagent ions in the TDCI process
Abstract:
A miniature thermal dissociation atmospheric chemical ionization source（TDCI）coupled with a LTQ-MS has been developed for
rapid trace detection of dimethoate in fruit juices. Instead of toxic solvents and high-voltage or high electric field used in ambient
ionizations, ionic liquid, a“green solvent“, was employed to directly generate reagent ions in the TDCI process. The ionization
was occurred by the followed proton or charge transfer reactions with the analytes presented in the matrices of the raw samples
under ambient conditions. Trace amounts of analyte in the fresh orange juices has been quantitatively detected, without any
sample pretreatment or aid of high-pressure gas. A low limit of detection（LOD=8.76×10-11 g/mL）and acceptable relative standard
deviation（RSD=3.5%-8.5%）, as well as reasonable recoveries（90-112%）, were achieved with this method for direct detection ofdimethoate in complex orange juice samples. The average analysis time for each single sample was less than 30 seconds. Theseexperimental results showed that TDCI-LTQ-MS is a powerful tool for the fast trace detection of pesticide residue such as
dimethoate in complex viscous fruit juices, with the advantage of high sensitivity, high speed, ease of operation, and so on.
Because of no organic solvents pollution and invasiveness to the analytes and environments, the technique has promising
applications for inline quality monitoring in the food safety.

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 44: Ambient Ionization

PTu-155

11:10 － 12:20

Mass analysis of the ions generated by corona discharge ionizers inambient air

Kenkichi Nagato

Kochi National College of Technology, Nankoku, Japan

Keywords:
corona discharge, atmospheric pressure ionization mass spectrometer, air ions, cluster ions
Novel aspects:
The evolutions of the postive and negative ions generated by corona discharge ionizer in ambient air were measured using APIMS.
Abstract:
Corona discharge ionizers are widely used to generate ions in air. They are typically installed in commercial electrical appliancessuch as air cleaners and air conditioners because negative ions have been reported to reduce the levels of particulates, airborne
microbes, odors and volatile organic compounds in indoor air. However, the detailed mechanisms by which negative air ionscontribute to improving indoor air quality are not clearly understood.
Information on the ion composition produced by such devices is essential if we are to understand the role of ions in air cleaning
processes. In order to investigate the characteristic compositions of the ions generated by corona discharge ionizers that are usedin commercial air cleaner, mass spectrometric measurements were conducted using an atmospheric pressure ionization mass
spectrometer. The ionizer was placed in front of the sampling orifice of the mass spectrometer. The distance between the ionizer
and the orifice was varied to see the effect of ion reaction time in ambient air.
+++
In positive ion mass spectra, major ions were H3O（H2 O）
n and NH 4（H 2 O）
n . With increasing reaction time, NH 4（H 2 O）
n became+dominant as a result of proton transfer reaction from H3O（H2O）n to NH3. Unidentified ion peaks often appeared in positive ion
mass spectra. Some kinds of VOCs that have large proton affinities are probably responsible for those ions. Negative ions in themeasured mass spectra included O 2（H2 O）
n , NO 2（H 2 O）
n , CO 3（H 2 O）
n , and NO 3（H 2 O）
n . Among these ions, NO 3（H 2 O）
n wereobserved to persist for long time while other ions declined with increasing reaction time. NO3 HNO（H32 O）
n were also observed toform as stable negative ions in the air. The presence of this cluster ion indicates that a large amount of nitric acid is formed bynegative corona discharge in ambient air. The reaction of NO2 with OH leads to the formation of nitric acid. Both NO2 and OH are
byproducts generated by negative corona discharge in air and they play important role in the formation of stable negative ions. In
addition, ion peaks probably due to HCO3 - ions and also to the ions of some organic compound were observed in negative ion
spectra. The relative abundance of ion species varied depending on the condition of the air in the laboratory.

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 44: Ambient Ionization

PTu-156

13:30 － 14:40

Rapid Screening of Camphor Wood by Surface Desorption AtmosphericPressure Chemical Ionization Mass Spectrometry

Jianping Yan, Huanwen Chen

Jiangxi Key Laboratory for Mass Spectrometry and Instrumentation, East China Institute of Technology, Nanchang,
Jiangxi, China

Keywords:
Surface desorption atmospheric pressure chemical ionization, mass spectrometry, principal component analysis, camphor, cluster
analysis
Novel aspects:
Fast, low-cost, reliably method to distinguish camphor wood from fake camphor wood
Abstract:
It is growing interest to distinguish non-camphor wood and authentic camphor wood quickly, cheaply and reliably. Appearance
and smell are used typically, but are unreliable. Although polymerase chain reaction（PCR）is the gold standard, it is a slow
method. In this work, a surface desorption atmospheric pressure chemical ionization（SDAPCI）source is used to obtain the mass
spectra of 5 camphor wood types and one unspecified wood type without pretreatment. The wood types are analyzed by bothprincipal component analysis（PCA）and cluster analysis（CA）. The characteristic ions for camphor and linalool are identified
from the PCA loading plot. The single-scan method distinguishes the three strongest peaks： 1 ）the camphor molecular ion, 2 ）the camphor-water adduct and 3 ）the linalool molecular ion from the wood surface, and the tandem mass scan confirms theidentities. This method classifies 30 samples by category and quality without pretreatment, is sensitive and is fast（less than 3 min
per sample）.

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 44: Ambient Ionization

PTu-157

11:10 － 12:20

Hydrocarbons Analysis by Desorption Atmospheric Pressure Chemical
Ionization

Fred Paul mark Jjunju Jjunju1,2, Badu_Tawiah K Abraham2, Li Anyin2, Roqan S Iman1, Graham R Cooks2
King Abdullah University of Science and Technology (KAUST), Physical Science and Engineering Division, Thuwal
23955-6900, Saudi Arabia, 2Derpartment of Chemistry Purdue University

1

Keywords:
Mass spectrometry, Ambient ionization, DAPCI, Petroleum hydrocarbons
Novel aspects:
DAPCI ionization source coupled with miniaturized mass spectrometers could provide the first hand-held, in-field instrumentation
for non-volatile compound characterization for the petroleum industry.
Abstract:
The advent of ambient ionization techniques has enabled the mass spectrometric analysis of chemical compounds from surfaces
in their native environment. Such methods are particularly useful for in situ analysis since the sample is analyzed without priorpreparation. Among the ambient ionization techniques are the plasma-based methods of which desorption atmospheric chemical
ionization（DAPCI）is an example. In DAPCI, a corona discharge is directed at a surface using a carrier gas at a low flow rate todesorb and ionize analyte（s）. Interestingly, a charge exchange reagent（e.g. naphthalene）can be doped into the gas stream toeffect electron transfer reactions. This mode of DAPCI is useful for samples, such as hydrocarbons, which have low proton
affinities.
Two types of DAPCI experiments are presented based on the mechanism of sample ionization i.e. proton or charge transfer. The
background mass spectrum for DAPCI experimental conditions utilizing only a corona discharge and nitrogen carrier gas showed
predominantly the presence of high abundant water cluster H［H2O］2 +and  H［H2O］3 + ions at m/z 37 and 55 respectively. Analysis
of petroleum constituents such as hydronaphthalene, thiophenes, alkyl subsitituted benzenes, pyridines, fluorenes, and polycyclicaromatic hydrocarbons（PAH）model compounds using the water clusters as DAPCI probe ions produced mass spectra whichwere dominated by protonated molecules［M+H］+ . On the other hand, molecular radical cations were observed when thenitrogen carrier gas was doped with naphthalene as a charge transfer reagent. In this case, the DAPCI probe ions were
naphthalene radical cations, which upon interaction with the analyte present at the surface ionized it through charge exchange.
This mode of sample ionization extended the applicability of DAPCI to other petroleum constituents（e.g. higher molecular PAHs
such as chrysene）which could not be analyzed in the proton transfer mode of operation. The thermochemistry governing theindividual ionization processes are discussed. Aside from the two main chemical reactions（proton vs. electron transfer）observed
under the two different DAPCI modes, certain specific compound classes（e.g. hydronaphthalenevs. thiophenes vs. alkyl
substituted benzenes）of petroleum constituents underwent different reactions. These differences were especially marked under
the DAPCI condition in which no charge exchange reagent was utilized. For example, in addition to analyte protonation, hydrideion elimination was also observed for the hydronaphthalenes compared with oxidation reactions for tetramethyl- andpentamethyl-substituted benzenes. Intact molecular（or pseudomolecular）ions were mostly observed irrespective of the ion form
（i.e.［M+H］+, M.+ or［M-H］+）and the chemistry involved, with the exception of low molecular weight n-alkanes which produced
extensive fragmentation after hydride abstraction.

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 44: Ambient Ionization

PTu-158

13:30 － 14:40

Rapid Detection of Bacterial Lipids, Metabolites and Signalling
Compounds by Liquid Extraction Surface Analysis Mass Spectrometry

Martin R Paine1, Nicole A Pianegonda1, Philip J Barker2, Todd W Mitchell1, Michael J Manefield3,
Scott A Rice3, Stephen J Blanksby1

University of Wollongong, Wollongong, Australia, 2BlueScope Steel Research, Port Kembla, Australia, 3University of
New South Wales, Sydney, Australia

1

Keywords:
Microorganisms, Acylated homoserine lactones（AHLs）, Biofouling, Metabolites, Liquid extraction surface analysis（LESA）.
Novel aspects:
New methods employing liquid extraction surface analysis-mass spectrometry, have detected bacterial metabolites associatedwith biofilm production directly from the cell surface.
Abstract:
Introduction: Microorganism growth resulting in biofouling of coated steel building materials is a growing concern for the multibillion dollar Australian industry. Proliferation of microorganisms on these materials is facilitated by the creation of a protective
extracellular matrix or ’biofilm’ and is associated with a range of problems including sick building syndrome, corrosion of materials,
and discoloration. Despite its importance, very little is known about the process of biofilm development on such surfaces or theorganisms involved. Environmental adaptation and biofilm formation has also been associated with microbial cell-cellcommunication, mediated by N-acyl-homoserine-lactones（AHLs）and interference with AHL signalling has been proposed as amechanism to control microbial fouling. Targeted strategies to combat biofouling in this manner therefore require thedevelopment of rapid methods for detecting microorganisms present on these surfaces and identifying metabolites as well as
signalling compounds associated with biofilm formation.
Method: Filtered methanol extracts of Pseudomonas aeruginosa cultures pipetted（ 2 µL）onto an Omni SlideTM hydrophobic array（Prosolia Inc., Indiannapolis, IN, USA）and Chromobacteriumviolaceum cultured on agar were analysed by liquid extraction
surface analysis（LESA）using an automated, chip-based nanospray source（TriVersa NanoMate®,  Advion, Ithaca NY,USA）coupled
to a triple quadrupole linear ion trap mass spectrometer（QTRAP®5500, AB Sciex, Foster City CA, USA）. Analyst® 1.5.1 software（ABSciex, California, USA）was used for spectral acquisition and typical experimental conditions were； spray voltage（1.4 kV）, N 2
delivery gas（0.3 psi）and MeOH：CHCl3（ 2 ： 1 ）with 0.1 % formic acid（v/v）as the solvent system. A total volume of 2 µL of
solvent was used for each experiment with 1 µL being dispensed 0.6 mm above the surface by an automated pipette tip. The
solvent forms a liquid-surface junction facilitating liquid extraction of analytes into the solvent that is then subjected toelectrospray ionisation through a nanospray chip.
Preliminary Data: Liquid extraction surface analysis-mass spectrometry（LESA-MS）by means of a TriVersa NanoMate® ionisation
source coupled to a QTRAP® 5500 mass spectrometer has proven to be a fast, simple and effective method in the detection ofbacterial metabolites from solvent extracts and directly from a cell culture surface. Unlike other ionisation techniques including
electrospray ionisation or matrix-assisted laser desorption ionisation, LESA-MS is able to directly analyse the bacterial cells in situ
making the technique extremely fast and simple. Utilising this technique we are able to perform lipid profiling that may allow us todistinguish between different species of microorganisms. With this knowledge we can then direct our analyses to known
metabolites for those species. Employing precursor scans targeting the common AHL product ion at m/z 102, positive ion LESA-MS
analyses of P. aeruginosa solvent extracts were able to detect a range of hydrolysed AHLs including 3 -oxo-C 10 -HS and3 -oxo-C 12 -HS. These AHLs are bacterial quorum-sensing molecules associated with biofilm formation. [1] Negative ion LESA-MS
analyses of C.violaceum cells cultured on agar were able to chemically identify the characteristic purple pigment violacein, anantibacterial compound produced in response to the presence of AHLs.[2] Violacein was detected as an［M-H］- ion at m/z 342.1
with MS/MS spectra exhibiting characteristic product ions at m/z 298.1, 209.1, and 157.0.［ 2 ］
1. T. R. I. Cataldi, G. Bianco and S. Abate, J. Mass Spectrom., 2009, 44, 182-192.
2. K. H. McClean, M. K. Winson, L. Fish, A. Taylor, S. R. Chhabra, M. Camara, M. Daykin, J. H. Lamb, S. Swift, B. W. Bycroft, G. S. A. B.
Stewart and P. Williams, Microbiology, 1997, 143, 3703-3711.

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 44: Ambient Ionization

PTu-159

11:10 － 12:20

A hydrodynamically optimized nanoelectrospray source with 100%transmission

Matthias Pauly1, Mario Sroka2, Stephan Rauschenbach1, Julius Reiss2, Gordon Rinke1,
Alyazan Albargash1, Jörn Sesterhenn2, Klaus Kern1
1

Max Planck Institute for Solid State Research, Stuttgart, Germany, 2Technical University Berlin, Berlin, Germany

Keywords:
Atmospheric Pressure Ionization（API）, Electrospray ionization（ESI）, ion transfer in vacuum, MS instrumentation
Novel aspects:
Our new interface shows transmission into vacuum of up to 100 % of the ions generated at a nanoelectrospray emitter.A muchlarger acceptance volume as compared to conventional interfaces is observed.
Abstract:
Ions generated in atmospheric pressure ionization（API）sources, including Electrospray Ionization（ESI）and Atmospheric Pressure
Chemical Ionization（APCI）, have to be transferred into vacuum in order to be analyzed by mass spectrometry（MS）, ion mobility
spectrometry（IMS）or to be deposited on a substrate（soft landing）. The coupling of an API source to a vacuum chamber ismade via an interface, typically a transfer capillary or a pinhole of small diameter, as pumping speed is limited. Present interfaces
are very inefficient, as they only transfer less than 5 % of the generated ions into vacuum. Nano-ESI sources can reachtransmissions up to 10%.
To improve the performance of the vacuum transfer, we optimized the geometry of the transfer capillary, taking all influences on
the ion motion into account： electrostatic forces from applied voltages and space charge forces as well as hydrodynamic dragforces.
Our new interface shows transmission into vacuum of up to 100% of the ions generated at a nanoelectrospray emitter for analyte
concentration below 10-3 M. For high concentrations and high electrospray voltages a threshold current in the range of 2 -40 nA isreached beyond which the transmission decreases. Further, a much larger acceptance volume as compared to conventional
interfaces is observed. Based on computational fluid dynamics calculations of the gas flow within the capillary and ion motion
simulations, we show that hydrodynamic forces of an optimized gas flow collimate the ion cloud, which effectively counteracts thespace charge forces that are the main reason for ion losses.
This ion source represents a major improvement over present nanospray sources in terms of transmission. In particularmeasurements requiring high sensitivity or high ion flux will profit from this development. Further the approach of hydrodynamic
optimization will allow improving other atmospheric pressure ion sources significantly.

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 44: Ambient Ionization

PTu-160

13:30 － 14:40

Direct analysis of asphaltene by DART FT MS

Hugues PREUD’HOMME1, Felipe CARDOSO2,3, Bruno GRASSL4, Brice BOUYSSIERE1, Herve CARRIER2

IPREM / LCABIE, University of Pau, Pau, France, 2LFC-R, UMR 5150, University of Pau, Pau France, 3Centro de Pesquisas
Leopoldo Americo Miquez de Mello, PETROBRAS/CENPES, Rio de Janeiro, Brazil, 4IPREM / ECP, UMR 5254, University ofPau, France

1

Keywords:asphaltenes, DART, FT MS
Novel aspects:
Direct analysis of asphaltene obtained at high pressure and high temperature by DART FT MS
Abstract:
Direct Analysis in Real Time（DART）is an ambient ionization technique undergoing rapid development. With minimal sample
treatment, ionization of analyte molecules outside the mass spectrometry instrument in the ordinary atmosphere is feasible. The
ionization approach relies upon the fundamental principles of atmospheric pressure chemical ionization and here we are able togenerate ions even for saturated hydrocarbon. We discussed here firstly of optimization of DART source and FTMS setting
parameters in order to achieve routinely fingerprint of Petroleum fluids, aggregates or deposition. They are complex mixturescontaining thousand of different compounds among which asphaltenes. Aggregation and deposition of asphaltenes during crude
oil production, transportation and treatment may cause serious problems as reported in numerous papers. This very complex
matrix is not fully identified from a chemical point of view. Indeed asphaltenes are still defined by a solubility class（IP 143 forexample）, being soluble in aromatic solvents（toluene, xylene, ）and insoluble in paraffinic ones（heptanes, pentane, ...）.
However, the solid material extracted from crude oil according to the above mentioned protocol contains the higher
concentration in metals such as nickel and vanadium. In order to better understand asphaltenes behavior and their chemistry, a
DART ion source and FT MS acquisition has been performed.
The experimental set up used to induce asphaltenes flocculation is able to work up to 100 MPa and 473°K. It allows investigations
with recombined oil or live oil fluids. In the work presented here, asphaltenes deposits engendered by pressure drops similar to
those occurring during oil production have been obtained and collected onto stainless steel filters. The solid materiel has been
directly analyzed by DART FT MS .

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 45: Cell Biology / Cellular Pathways

PTu-161

11:10 － 12:20

BRIDGING BETWEEN LIFE SCIENCES AND MATERIAL SCIENCES: MASS
SPECTROMETRIC PROTEIN AND METABOLITE ANALYSIS FOR THE
PRODUCTION OF BIO-BASED CHEMICALSMichiel Akeroyd, Rob van der Hoeven, Rene Verwaal, Stefan Turk, Fredoen Valianpour, Marcel Tilborg,
Maurien Olsthoorn
DSM Biotechnology Center / Delft / The Netherlands

Keywords:
Industrial Biotechnology, Synthetic Biology, Proteomics
Novel aspects:
Mass spectrometry aided development of industrial bio-based chemical production, Fermentative production of bio-chemicalcompounds using synthetic biology, Mass spectrometric intra-cellular pathway monitoring at metabolite and protein level,
Bioinformatic route scouting
Abstract:
DSM is driving to create sustainable high value products through connecting its competences in Life Sciences and Material
Sciences. Recent examples of this strategy are the production of succinic acid in S. cerevisiae and 6 -amino caproic acid（ 6 -ACA）in both C. glutamicum and E. coli using biotechnological processes. The first process enables large scale renewable production ofa natural occurring metabolite as a building block for high value polymers. The second process is the bio-production of a nonnatural compound, the building block for Nylon-6.
Mass Spectrometry was one of the key techniques used for analyses of the metabolic intermediates in the pathways as well as
the enzymes driving the bioprocesses. Targeted intracellular metabolite analyses were performed to monitor the different steps in
the metabolic pathways. LTQ-Orbitrap identification strategies as well as triple quadrupole quantification strategies were applied.
Methods for analysis of di-carboxylic acids and amphoteric metabolites were carried out using polarity switching. A label-freequantitative proteomics workflow was developed using the APEX tool（Lu. et al. 2007）for quantification based on spectral
counting in combination with machine learning. This workflow was applied to verify the results of the genetic engineering and tomonitor the cellular makeup at the protein level. The relative protein abundances of the enzymes involved in the production
pathways were determined using selected reaction monitoring（SRM）methods. Both proteomics approaches were essential for
confirmation of the genetic engineering process and lead generation for significantly improved production.
The obtained MS data on metabolites and proteins was combined with data generated by other techniques such as NMR andenzyme activity assays to generate leads to improve productivity even further. The dynamic interaction of expertises was essential
for the successful development of succinic acid producing S. cerevisiae strains and 6 -ACA producing E. coli and C. glutamicumstrains.

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 45: Cell Biology / Cellular Pathways

PTu-162

13:30 － 14:40

Large Bio-particle Detection Technologies and Their Applications

Chung Hsuan Chen

Genomics Research Center/Academia Sinica, Taipei, Taiwan

Keywords:
Ion acceleration, biomolecular ion detection
Novel aspects:（ 1 ）Developing synchronized pulsed acceleration to achieve a macro ion accelerator（ 2 ）Frequency scanning to trap cell/virus
in the ion trap（ 3 ）Acoustic desorption of micro/nano particles
Abstract:
Recently, we have developed a few novel mass spectrometry technologies which include（ 1 ）single large biomolecular ion
detector（ 2 ）biomolecular ion accelerator and（ 3 ）acoustic desorption micro-/nano- particle mass spectrometer.
It is well known that the detection efficiency is very low for charge amplification detector to measure very large biomolecular ionsdue to the low yield of the secondary electrons. We designed our detector based on the detection of secondary ions instead of
secondary electrons to significantly enhance the detection efficiency. With this approach, a single large biomolecular ion such as
IgM+ was successfully detected. Due to the improvement in detection sensitivity, it can be quite valuable for top-down proteomic
application.
Accelerator has been very broadly used for research in high energy and nuclear physics. During the past two decades, it has been
extended to biomedical research and therapeutic applications. Nevertheless, only electron and atomic ion accelerators areavailable up to now. Few if any molecular ion accelerators have been reported. We developed the first macromolecular ion
accelerator to accelerate very large molecular ions to reach to megavolt region. A series of well-timed pulsed voltages was used
to accelerate very large macromolecular ions. With this approach, ions with mass-to-charge ratio（m/z）reaching 30,000,000 was
successfully detected by a charge amplification detector such as a channeltron or an electromultiplier. We also coupled ourmacromolecular ion accelerator with a dual-ion trap mass spectrometer to make an accelerator-ion trap mass spectrometer forsensitive detections of very large biomolecules.
Up to now, all commercially available mass spectrometers have the limit on mass range. An ion with m/z above 1,000, 000 cannot
be detected. We developed acoustic desorption charge monitoring mass spectrometer for the detection of bio-particles with m/z
higher than 1,000,000. Laser induced acoustic desorption was used to desorb bioparticles from the sample plate into the ion trap. A
frequency scanning ion trap was used to trap ions with very high m/z. The ion detection is based on charge detector. With thisapproach, we demonstrated particles with m/z as high as 1013 and mass at 1016 Da can be easily detected. We applied this mass
spectrometer to measure microparticles, nanoparticles, cells and viruses. In addition, uptakes of various nanoparticles and virions
by different cells have also been pursued. We found a large number of HIV virus is needed for an immune cell infection. This
technology can also be used for quantitative drug measurement when nanoparticle is used as a drug carrier.
We are looking for further development of the above technologies for being able to do a single cell proteomics in the future.

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 45: Cell Biology / Cellular Pathways

PTu-163

11:10 － 12:20

Comparison between Single-cell and traditional MALDI-MS-based
workflows for metabolome studies in Saccharomyces cerevisiae.

Alfredo J Ibanez1, Stephan R Fagerer1, Matthias Heinemann2, Renato Zenobi1
1

ETH Zurich, Zurich, Switzerland, 2University of Groningen, Groningen, The Netherlands

Keywords:
S.cerevisiae, 2 -deoxy-D-glucose, Single-cell Metabolomics, Microarrays
Novel aspects:
For the first time, microarrays for mass spectrometry（a novel single-cell metabolomic platform）is used to study the non-genetic
cell-to-cell heterogeneity observed within theoretically homogenous（i.e. clonal）yeast cell population.
Abstract:
Stochastic-induced phenotypic heterogeneity is one of the sources of cell-to-cell variation within a clonal cell population.1 This
non-genetic heterogeneity can only be studied using single-cell analytical platforms.1-3
Two years ago, our group introduced a novel analytical platform called microarrays for mass spectrometry（MAMS）.4 MAMS iscapable of boosting the performance of commercial matrix-assisted laser desorption/ionization（MALDI）mass spectrometers inorder to achieve single-cell sensitivity, when used as MALDI targets.
Here, we present the first in depth comparison between two MS-based metabolome approaches：（i）a population-level and（ii）
single-cell level, for describing the biological response of a clonal cell culture of Saccharomyces cerevisiae（budding yeast）when
applying the glycolytic inhibitor, 2 -deoxy-D-glucose（ 2 -DG）.
Our results show that the efficiency of 2 -DG as a drug depends on the biological make-up of each individual cell within theisogenic cell culture. One potential explanation, observed using MAMS, for the cell-to-cell variation in the presence of 2 -DG is thedifferent levels of expression of glucose transporter proteins and/or glucose hexokinase enzymes in yeast cells.
Furthermore, we also present possible applications in which single-cell level metabolome studies, using the MAMS platform,
provide a unique insight, such as： monitoring stochastic-induced phenotypic bistability.
References：
1. Heinemann, M. & Zenobi, R. Curr. Opin. Biotechnol.22, 26-31（2011）.
2. Walling, M.,A. & Shepard, J.R.E. Chem. Soc. Rev.40, 4049-4076（2011）.
3. Avery, S.V. Nat. Rev. Microbiol.4（ 8 ）, 577-587（2006）.
4. Urban, P.L., Jefimovs, K., Amantonico, A., Fagerer, S.R., Schmid, T., Mädler, S., Puigmarti-Luis, J., Goedecke, N., Zenobi, R. Lab
Chip10（23）, 3206-3209（2010）.

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 45: Cell Biology / Cellular Pathways

PTu-164

13:30 － 14:40

The Cuticular Structure and Lipid Composition of Arabidopsis thaliana
Organs and Cells Determined by TOF-SIMS

Gregory L Fisher1, Philip C Wong2, Christopher Buschhaus2, John Hammond1, Reinhard Jetter2
1

Physical Electronics, Minnesota, USA, 2University of British Columbia, British Columbia, Canada

Keywords:
botanical, lipids, imaging, TOF-SIMS, topography
Novel aspects:
We have employed TOF-SIMS chemical imaging to uniquely identify the distinct lipid composition of single cells comprising thestomates and trichomes on Arabidopsis organs.
Abstract:
The cuticle, or outer surface, of a plant has many functions including the regulation of growth, controlling the loss of water andthe exchange of gases, and providing a protective barrier against many environmental stresses. The precise function of plant
cuticles can best be understood on the basis of their characteristic chemical composition as well as their biosynthetic origin［ 1 ］.
In the case of Arabidopsis thaliana, approximately 85 genes have been identified as responsible for the synthesis and metabolism
of dermal and cuticular lipid chemistries［ 2 ］. The cuticle lipids are a mixture of acids, aldehydes and alcohols with chain lengths
in the range of C24 - C32. However, observing the cuticle chemistry of wild-type and knock-out varieties has met with challenges.
Chief among these challenges concerns the sampling and isolation of cuticular wax components for chemical characterization. To
date, characterization of the cuticular lipids has relied on manual dissection of the epidermis followed by solvation andchromatographic separation of the wax components. This sampling method reveals the average composition of the entire organ,
but subtle inhomogeneities are overlooked. More importantly, specific differences in cellular chemistry are impossible to detect.
Time-of-flight secondary ion mass spectrometry（TOF-SIMS）is evolving as an important tool for 2 D and 3 D imaging mass
spectrometry of biological specimens due to its unique capability to detect molecular and elemental ions with high sensitivity,
sub-micron spatial resolution and without the sample treatments required by e.g. MALDI or fluorescence microscopy. For many
biological specimens, the ability to garner reliable chemical distributions from samples having a large degree of surface
topography is both desired and necessary； to achieve this, an imaging mass spectrometer must have a large angular acceptanceand a large depth-of-field. These analytical requirements are satisfied in the design of the PHI TRIFT V nanoTOF. The resulting
elemental and molecular images provide important information regarding the composition of biointerfaces, tissues, and cells
without the artifacts presented by topography and morphological variation.
The chemical imaging capability of TOF-SIMS is demonstrated by the characterization of epicuticular wax components present at
the surfaces of Arabidopsis thaliana organs. High spatial resolution images obtained from intact organs and specialized cells
illustrates the capability of TOF-SIMS to image and characterize biological chemistry at a spatial resolution of < 0.3 μm. Total ion
images and molecular ion images of cuticular lipids demonstrate the capability of the TRIFT analyzer to image organ surfaces
without topographically induced artifacts. High mass spectra in both the positive and the negative secondary ion polarities revealthat the epicuticular surface of each Arabidopsis thaliana organ is comprised of distinct lipid compositions. Mass spectra acquired
from specialized cells forming the stomates and trichomes reveal that, even for single cells on the surface of an organ, the lipid
composition may vary. The differences in wax chemistry on each of the interrogated organs and cells of Arabidopsis thaliana will
be presented and discussed. Structural assignments for characteristic lipid components comprising the epicuticular wax will also
be presented.
［ 1 ］M.C. Suh, A.L. Samuels, R. Jetter, L. Kunst, M. Pollard, J. Ohlrogge and F. Beisson, Plant Phys. 139（2005）1649.
［ 2 ］M. Wen and R. Jetter, J. Exp. Bot. 60（2009）1811.

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 45: Cell Biology / Cellular Pathways

PTu-166

13:30 － 14:40

Proteomic Interrogation of MDSC Exosomes by Bottom-up, Middle-out
and Top-down strategies

Catherine Fenselau1, Meghan Burke1, Waeowalee Choksawangarn1, Avantika Dhabaria1,
Joseph Cannon1, Suzanne Ostrand-Rosenberg2
1

University of Maryland, College Park MD, USA, 2University of Maryland, Baltimore County, USA

Keywords:
exosomes, tumor-micro-environment, proteomics, iTRAQ,
Novel aspects:
identification of > 2500 proteins in exosomes allows us to address their origin and their function.
Abstract:
Exosomes are membrane-bound extracellular vesicles that carry messages between cells. We hypothesize that the exosomes
shed by myeloid-derived suppressor cells（MDSC）contribute to suppression of adaptive and innate immunity by communicating
between MDSC, T-cells, natural killer cells, and macrophages in the tumor micro-environment. Pro-inflammatory agents increase
the quantity and suppressive potency of MDSC, and thereby facilitate tumor progression in patients. We are studying themechanisms by which inflammation drives MDSC in a mouse model. We have exploited bottom-up, middle-out and top-down
proteomic strategies to inventory the proteins of exosomes associated with MDSC. In this presentation, relative andcomplementary merits of the three approaches will be discussed. We have also employed an iTRAQ strategy to quantitate
differential protein abundances in exosomes from tumor- bearing mice with varying degrees of inflammation. Inflammation-relatedchanges in exosomal proteins will be presented and compared with inflammation-related protein changes in the parent MDSC.

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 45: Cell Biology / Cellular Pathways

PTu-167

11:10 － 12:20

Identification of novel cytokinin degradation pathway during adventitious
caulogenesis in Pinus pinea L. cotyledons with quadrupole-time of flightmass spectrometry

Ondrej Novak2, Candela Cuesta3, Karel Dolezal1,2, Lucie Szucova2, Lukas Spichal2, Belén Fernández3,
Ana Rodríguez3, Miroslav Strnad2

1
Institute of Experimental Botany AS CR, Czech Republic, 2Laboratory of Growth Regulators, Faculty of Science, Palacky
University & Institute of Experimental Botany AS CR, Slechtitelu 11, Olomouc, CZ-783 71, Czech Republic, 3Área deFisiología Vegetal, Departamento Biología de Organismos y Sistemas, Instituto de Biotecnología de Asturias,
Universidad de Oviedo, Oviedo, E-33071, Spain.

Keywords:
cytokinin, identification, QqTOF
Novel aspects:
Novel cytokinin degradation pathway was identfied during adventitious caulogenesis in Pinus pinea L. cotyledons with quadrupoletime of flight mass spectrometry.
Abstract:
Background
Cytokinins（CKs）are a group of phytohormones which probably regulate the growth, development, and metabolism of all plants.
The aromatic CK benzyladenine（BA）has been widely applied in in vitro culture for inducing shoot organogenesis. Our study ofendogenous cytokinin profiles during the caulogenic process based on mature cotyledons of stone pine（Pinus pinea L.）showed anovel metabolic pathway of aromatic cytokinins based on modification of purine skeleton.
Methods
Three-year-old mature seeds from two half-sibling selected families and open-pollinated trees of P. pinea were used［ 1 ］and thesamples were collected following the Alonso et al.［ 2 ］procedure. Extraction and purification of cytokinins was based on themethod described by Novák et al.［ 3 ］, including modifications published later［ 4 ］. The samples were purified using a
combination of a cation（SCX-cartridge）and anion［DEAE-Sephadex/C18-cartridge］exchangers. Combination of high performanceliquid chromatography（HPLC）with quadrupole-time of flight mass spectrometry（QqTOF）was used for accurate and sensitive
identification and qantification of cytokinins.
Results and Discussion
Using high-resolution MS, the naturally-occurring BA metabolites as well as new BA forms were identified. In comparision withpreviously publihed profiles of the BA metabolite pool［5,6］, the ribosyl and glycosyl forms were quantified as the most abundant
metabolites. Moreover the biological activity of identified BA and its derivatives were compared in various CK bioassays. The
results indicate that BA uptake during the caulogenic process may be possible to regulate not only by known cytokinin pathways.
Finally, the feeding experiment with stable isotope-labelled standard, 15N4-BA, confirmed our indetification of the novel metabolic
CK pathway.
Conclusions
The identification of the novel BA forms demonstrates that the novel cytokinin pathway is used as a control mechanism of BA
uptake from the bud induction medium during adventitious caulogenesis in cotyledons of P. pinea. Our results help to understandthe processes associated with embryo germination in plant tissue culture.
Acknowledgements
This work was supported by the Grant Agency of the Czech Republic（P505/11/1163）and by Ministry of Agriculture of the Czech
Republic（NAZV QI92A247）.
References
1. Cuesta C, Ordas RJ, Fernandez B, Rodriguez A： Clonal micropropagation of six selected half-sibling families of Pinus pinea andsomaclonal variation analysis.Plant Cell Tiss. Org. 2008, 95：125-130.
2. Alonso P, Moncalean P, Fernandez B, Rodriguez A, Centeno ML, Ordas RJ： An improved micropropagation protocol for stonepine (Pinus pinea L.).Ann. Forest Sci. 2006, 63：879-885.
3. Novak O, Tarkowski P, Tarkowska D, Dolezal K, Lenobel R, Strnad M： Quantitative analysis of cytokinins in plants by liquid
chromatography/single-quadrupole mass spectrometry.Anal. Chim. Acta 2003, 480：207-218.

4. Novak O, Hauserova E, Amakorova P, Dolezal K, Strnad M： Cytokinin profiling in plant tissues using ultra-performance liquid
chromatography electrospray tandem mass spectrometry.Phytochem. 2008, 69： 2214-2224.
5. Cortizo M, Cuesta C, Centeno ML, Rodriguez A, Fernandez B, Ordas RJ： Benzyladenine metabolism and temporal capacity ofPinus pinea L. cotyledons to form buds in vitro. J. Plant Physiol. 2009, 166：1069-107.
6. Zhang H, Horgan KJ, Reynolds PHS, Jameson PE： 6-Benzyladenine metabolism during reinvigoration of mature Pinus radiatabuds in vitro.Tree Physiol. 2010, 30：514526.

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 45: Cell Biology / Cellular Pathways

PTu-168

13:30 － 14:40

Activation Kinetics and Dimerization of STAT5 Proteins Followed by
Quantitative Mass Spectrometry

Martin E Boehm, Lorenz Adlung, Marcel Schilling, Ursula Klingmueller, Wolf-Dieter Lehmann
German Cancer Research Center Heidelberg

Keywords:
STAT 5 Isoforms, dimerization, phosphorylation, immunoprecipitation, relative quantification
Novel aspects:
Recognition of the connection between immunoprecipitation by isoform-specific STAT 5 A/B antibodies, phosphorylation-induced
STAT 5 dimerization and STAT 5 phosphorylation degree determination.
Abstract:
The mouse genome contains one gene each for STAT 5 A and STAT 5 B on chromosome 11. Due to possible functional differences
between both STAT 5 isoforms, individual analysis of their abundance, activation and dimerization is of interest. The isoforms A
and B of mouse STAT 5 differ in about 9 % of their amino acidpositions, enabling their isoform-specific analysis by antibodies andmass spectrometry. The variety of single amino acid substitutions generates multiple closely related tryptic peptides for eachisoform. Comparing signal intensities of these similar but unique peptides allows label-free relative quantification of both isoforms.
One such variation（STAT 5 A V691/STAT 5 B A696）allows A- and B-specific phosphorylation analysis of the STAT 5 activation motif
located atTyr694 in STAT 5 A and at Tyr699 for STAT 5 B. Analysis of the dynamics of STAT 5 activation by Epo in the mouse cell line
BaF 3 -EpoR via a combination of immuno precipitation and mass spectrometry revealed a complex interplay between the isoformspecific degree of phosphorylation at the activation motif and relative A/B isoform abundances. This observation was madepossible on the basis of accurate data for the degree of phosphorylation at the activation motif, which were generated by the useof one-source peptide/phosphopeptide standard pairs and LC-MS. Dimerization of phospho-STAT 5 was hypothesized as the main
source of this phenomenon.
To check this hypothesis, additional STAT 5 activation experiments by Epo in BaF 3 -EpoR cells were performed and analyzed
using immunoprecipitation by different STAT 5 antibodies with established STAT 5 A/B specificity. Their isoform-selectivity was
further verified by mass spectrometric sequencing of their corresponding blocking peptides. These experiments revealed that（i）Aand B isoforms of STAT 5 occur at a ratio of 2 ：1, that（ii）both STAT 5 A and STAT 5 B show a highly similar degree ofphosphorylation, and that（iii）phospho-STAT 5 dimers are formed mainly without isoform selectivity.

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 45: Cell Biology / Cellular Pathways

PTu-169

11:10 － 12:20

Lapatinib-resistant gastric cancer cells express cancer stem cell-like cellsurface antigens

Minjueng Kang1, Hwang-Phill Kim2, Young-dong Yoo1, Yong-Tae Kwon1, Tae-You Kim1,2, Eugene C Yi1
Molecular Medicine and Biopharmaceutical Sciences, Seoul National University, Seoul, Korea, 2Cancer ResearchInstitute, Seoul National University Hospital, Seoul, Korea

1

Keywords:
Cancer stem cell, Lapatinib-resistant, Cell surface antigens
Novel aspects:
Global profiling of unique or highly expressed cell surface antigens of cancer stem cell（CSC）-like cells
Abstract:
Lapatinib is a dual inhibitor for HER 2 and epidermal growth factor receptor tyrosine kinases that has shown promising in vitro
results in inhibiting the growth of HER 2 （+）cancer cells. However, some tumors either do not respond or develop resistance tothe agent. To evaluate the properties of drug-resistant cells, we isolated a lapatinib-resistant, HER- 2 positive gastric cancer cell
population by incubating lapatinib-sensitive SNU-216 cells for a prolonged period with lapatinib. The drug-resistant cells werecharacterized using a panel of cell surface markers identified from a cancer stem cell（CSC）-like cell population derived from an
Oct 3 / 4 -controlled MB-453 breast cancer cell line. The panel of cell surface markers was identified using a large-scale proteomic
analysis and included the known CSC markers CD44, CD133, EpCAM, and CD151 as well as newly identified CSC-specific cellsurface antigens. Immunoblot analysis of lapatinib-resistant SNU-216 gastric cells showed that both the known CSC markers andthe newly identified CSC cell surface antigens were significantly upregulated compared to lapatinib-sensitive SNU-216 gastric cells.
These results suggest that the development of resistance to lapatinib is associated with a shift towards a CSC phenotype.

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 45: Cell Biology / Cellular Pathways

PTu-170

13:30 － 14:40

Tissue specific profiling of the Arabidopsis thaliana auxin metabolome

Ondrej Novak1,2, Eva Henykova1,2, Ilkka Sairanen2, Mariusz Kowalczyk2, Tomas Pospisil3, Karin Ljung2

Palacky University & Institute of Experimental Botany AS CR, Olomouc, Czech Republic, 2Umea Plant Science Centre,
Umea, Sweden, 3Centre of the Region Hana for Biotechnological and Agricultural Research, Faculty of Science,
Olomouc, Czech Republic
1

Keywords:
IAA, auxin, metabolite profiling, Arabidopsis
Novel aspects:
A new sensitive and selective method for multiplex quantification of IAA and its precursors and degradation products in
Arabidopsis tissues.
Abstract:
Hormonal profiling methods use modern analytical tools based on fast chromatographic separation and mass spectrometricquantitative analysis. The plant hormone auxin is believed to influence almost every aspect of plant growth and development. We
have developed and validated a method for profiling the majority of known auxin precursors and conjugates/catabolites in small
amounts of Arabidopsis tissue. Our method includes trace analysis of 21 compounds with different polarity, acidity and basicity as
well as stability and abundance in crude plant extracts. We found that a polymer based（Oasis HLB）sorbent was the best tool for
one-step purification, including a new derivatization method to quantify the most labile of the auxin precursors. The process wascompleted by a single chromatographic analysis of auxin metabolites in 12 minutes using an analytical column packed with sub- 2 microne particles. In multiple reaction monitoring mode, the detection limit for most of the analytes ranged from 1.0 to 5.0 fmoland the achieved linear range was at least five orders of magnitude. Finally, we profiled the auxin metabolome in root and shoottissues from different Arabidopsis thaliana ecotypes and auxin overproducing mutant lines, showing substantial differences in themetabolite pattern between the lines and between different tissues. We also observed differences in abundance of several ordersof magnitude between different auxin metabolites, indicating the relative importance of different auxin precursors and conjugates/
catabolites for maintaining auxin homeostasis. We now have a powerful tool to get a better understanding of the regulation of
auxin metabolism during plant development.

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 45: Cell Biology / Cellular Pathways

PTu-171

11:10 － 12:20

In Situ Pressure Probe Picoliter Single-Cell Sap Sampling and Mass
Spectrometry Metabolite Profiling of Living Plants

Yousef Gholipour1, Rosa Erra-Balsells2, Hiroshi Nonami1
1Ehime University, Matsuyama, Japan, 2University of Buenos Aires, Argentina

Keywords:
Intact cell, quantitation, metabolomics, glycomics,
Novel aspects:
Mass spectrometry analysis of picoliter single-cell sap sampled by using the pressure probe is performed. Single-cell sap volume
sucked is controlled and measured. Additional intact-cell properties can be evaluated.
Abstract:

In plants, anatomy of different organs and tissues is not necessarily uniform and cells located at different tissues and depth mayvary significantly in function, morphology and metabolite composition. Therefore, single-cell metabolite profiling to be applied toplant science demands for accurate and fully-managed sampling from different tissues and cell layers. The sampling is morechallenging due to the difficulty in managing femto- to nanoliter volume of sap each plant cell can provide. Additionally, the singlecell sap volume must be measured if natural changes in the abundance of metabolites of cells are to be analyzed. In our model
plant, tulip, parenchyma cells located at inner layers of bulb scale tissue（e.g. 400 μm from the surface of cuticle）containabundant starch, long-chained fructans, underivatized soluble sugars and amino acids. They function as the source of nitrogen andcarbon for growth of new flower stalk. For flowering, bulbs need to be stored at low temperatures（ 4 -10 °C）for about twomonths. We targeted those parenchyma cells in cold-stored bulbs.
We used a cell pressure probe for in situ single-cell sampling followed by shotgun metabolite profiling with UV-MALDI TOF MS（AB SCIEX 5800 system）and nanoESI MS（ThermoFisher Exactive Orbitrap™ mass spectrometer）. The quartz capillary of thepressure probe was filled with silicon oil and air-tightly connected to a pressure transducer. Pressure inside capillary is
manipulated with a rod moved back and forth by a micrometer. Therefore, while the capillary was penetrating to a predetermined tissue depth, the sap of cells located on the way to the target cell was prevented from entering the capillary tip byregulating the oil pressure. The movement of capillary and penetration depth was controlled and measured with a piezomanipulator and with previous anatomy of tulip bulb tissues, depth of penetration to access favorite cells became possible. After
penetration of the capillary into a cell, cell sap enters the capillary tip due to turgor pressure naturally exists in living plant cells；
and a meniscus forms at the interface of the oil and analyte solution inside the capillary. The diameter of parenchyma cells in tulip
bulb scale varies in the range of 50-150 um and we obtained 100-600 pL cell sap from each cell. The tip with cell sap sample inside
was photographed and the volume of the picoliter sample was accurately measured.
For UV-MALDI MS, the picoliter cell sample was injected to a 1 μL water droplet hanging from a pipette tip. The picoliter cellsample（in 1
μL water droplet）was deposited on previously dried matrix spots on stainless steel MALDI plates. We haveexamined several matrixes for single-cell metabolite profiling and in this study we used 2,4,6-trihydroxyacetophenone（THAP）,
2,5-dihydroxybenzoic acid（DHB）and titanium silicon oxide（SiO2）（TiO2）nanoparticles. With（SiO2）（TiO2）nanoparticles good
linearity of signal abundance vs. number of picomoles of standard compounds was yielded. With all matrixes, signals of cell sap
metabolites were detected. Particularly, big neutral oligosaccharides could be easily analyzed with UV-MALDI MS. For nanoESI MS
analyses, cell sap sample was transferred to a 5 μL water droplet and then injected into the electrospray ion source. NanoESI
MS was also successful in metabolite profiling of single-cell sample； however, smaller oligosaccharides could be detected. In
addition to sugars, amino acids, organic acids, tuliposide, putrescine, and GABA could be detected with both MALDI and nanoESI
MS. Relative abundance of sucrose and kestose in parenchyma cells located at the depth of 100-500 μm was determined. The limit
of detection of sucrose was similar（ 2 - 5 pmol）in both techniques but dynamic range of detection was wider in MALDI MS（with
2 -25000 pmol）than nanoESI MS（ 5 -2500 pmol）.

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 45: Cell Biology / Cellular Pathways

PTu-172

13:30 － 14:40

In-situ Analysis of Plant Bioactive Molecules by Live Single-cell Mass
Spectrometry

Shuichi Matsuda1, Sachiko Date2, Hajime Mizuno1, Naohiro Tsuyama1, Takanori Harada1,
Tsutomu Masujima1,2

Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima, Japan, 2Quantitative Biology Center (QBiC),
RIKEN, JAPAN1

Keywords:
Plant, Bioactive Substances, Live Single-cell MS, Photosynthesis
Novel aspects:
Light responding metabolism of glucosinolates in a live plant was directly analyzed at single cell level.
Abstract:
Introduction
Secondary metabolites in plants have been attracted attention by their bioactive property such as antioxidant and antibacterial
effects. Biosynthesis of these compounds is affected by plant’s growing condition. LC-MS studies of plant metabolites have beenreported in which large numbers of cells were homogenized to get averaged data. These processes are time-consuming, andmolecules which are volatile such as IsoThioCyanates（ITCs）or unstable molecules such as glucosinolates, can be lost during
preparation. We applied in-situ Live Single-cell MS method which allows pretreatment-free analysis for detection and identificationof molecules in a certain region of a plant tissue. In this study, the diversity of glucosinolates in plants with different growth
conditions is reported.
Methods
We left radish sprouts in the dark for five days post seeding, and exposed to white or red light for further two days. Then we
observed pith and cortical tissues in a cross-sectional slice of a radish sprout’s stem by a stereomicroscope. A representative cellin every tissue was selected, and cellular content was micro-sucked into a nanospray tip（HUMANIX）. Then, ionization solvent（acetonitrile）was added into the needle followed by mass spectrometric analysis with a LTQ-Orbitrap Velos（Thermo Fisher
Scientific）equipped with nanoESI source at both positive and negative ion mode. At the same time, SIM（selected ion monitoring）
scan mode was used for high-sensitive analysis. Spray voltage was around 1000 V（positive mode）or -800 V（negative mode）.
Detected peaks were identified by MS/MS and exact mass measurement.
Results
At positive ion mode, we detected several ITCs such as 4 -methylthio- 3 -butenyl-isothiocyanate（m/z 160.0249）, isopropyl
isothiocyanate（m/z 102.0372）in a cell on a cross-sectional slice of radish sprout stem. Besides, we used negative ion mode to
detect several glucosinolates such as 4 -methylthio- 3 -butenyl-glucosinolate（m/z418.0295）, glucobrassicin（m/z 447.0527）,
glucoerucin（m/z 420.0451）. This method enabled us to perform direct and quick analysis of ITCs and glucosinolates which arehighly reactive and easy to be degraded during pretreatment for LC-MS. In addition, we could identify restricted localization ofthese compounds. Glucosinolates were detected only in a cell on cortical tissue, whereas ITCs were detected mainly in pith. Theirlocalization was usually lost in a conventional LC-MS. Then, we performed spatio-temporal analysis of these bioactive compoundswith illuminated radish sprouts. Under white light exposure, levels of most glucosinolates were higher than those in the conditionof red light exposure. On the other hand, level of glucobrassicin was exceptionally higher under red light exposure, whereas its
intermediates showed different distributions on a cross-sectional slice of radish sprout stem. Among them distribution of indole- 3 acetaldehydeoxime, precursor of glucobrassicin, was quite different. It was detected mainly in pith under white light exposure, but
in cortical tissue under red light exposure. Consequently, these results showed this method has enabled us to detect theirbioactive substances and analyze quantitative variations of molecular distribution in a plant tissue easily and quickly. Moredetailed studies of light responding metabolism are under progress.

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 45: Cell Biology / Cellular Pathways

PTu-174

13:30 － 14:40

2D-DIGE analysis of the role of S-nitrosoglutathione reductase inlipopolysaccharide-challenged mice

Kentaro Ozawa1, Hiroki Tsumoto1, Wei Wei2, Chi-Hui Tang2, Akira T Komatsubara3, Hiroto Kawafune3,
Kazuharu Shimizu1,4, Limin Liu2, Gozoh Tsujimoto1,3

World-Leading Drug Discovery Research Center, Kyoto University, Kyoto, Japan, 2Department of Microbiology and
Immunology, University of California, San Francisco, USA, 3Department of Genomic Drug Discovery Science, Graduate
School of Pharmaceutical Sciences, Kyoto University, Kyoto, Japan, 4Department of Nanobio Drug Discovery, Graduate
School of Pharmaceutical Sciences, Kyoto University, Kyoto, Japan
1

Keywords:
Oxidative stress, 2 D-DIGE, Endoplasmic reticulum, Cancer, Liver proteins
Novel aspects:
Deficiency of S-nitrosoglutathione reductase compromises expression of the ER stress-related proteins in LPS-treated liver.
Abstract:
Introduction: S-Nitrosoglutathione reductase（GSNOR）is a key regulator of protein S-nitrosylation, the covalent modification ofcysteine residues by nitric oxide that can affect activities of many proteins. We recently discovered that excessive S-nitrosylationfrom GSNOR deficiency in mice under inflammation inactivates the key DNA repair protein O6-alkylguanine-DNA alkyltransferase
and promotes both spontaneous and carcinogen-induced hepatocellular carcinoma. To explore further the mechanism oftumorigenesis due to GSNOR deficiency, we compared the protein expression profiles in the livers of wild-type and GSNORdeficient（GSNOR-/-）mice that were challenged with lipopolysaccharide（LPS）to induce inflammation and expression of inducible
nitric oxide synthase（iNOS）.
Methods: We performed two dimensional fluorescence difference gel electrophoresis（ 2 D-DIGE）to identify differentiallyexpressed proteins in the liver tissues of wild-type and GSNOR -/- mice following LPS-treatment. To identify the differentiallyexpressed proteins by mass spectrometry（MS）and database search, the spots were destained, reduced with dithiothreitol,
alkylated with iodoacetamide and then digested overnight with trypsin. Matrix-assisted laser desorption/ionisation-time of flight（MALDI-TOF）MS and MS/MS analyses were performed using the AXIMA-Performance mass spectrometer（Shimadzu）. alphaCyano- 4 -hydroxycinnamic acid was used as the matrix.
Results and Discussion: 2 D-DIGE analysis identified 38 protein spots of significantly increased intensity and 31 protein spots ofsignificantly decreased intensity in the GSNOR-/- mice compared to those in the wild-type mice. We subsequently identified 19 upregulated and 19 down-regulated proteins in GSNOR-/- mice using MALDI-TOF MS and MS/MS. Immunoblot analysis confirmed in
GSNOR -/- mice a large increase in the expression of the pro-inflammatory mediator, S100A9, a protein previously implicated inhuman liver carcinogenesis. We also found a decrease in the expression of multiple members of the protein disulfide-isomerase（PDI）family and an alteration in the expression pattern of the endoplasmic reticulum（ER）chaperones in GSNOR -/- mice.
Furthermore, altered expression of these proteins from GSNOR deficiency was prevented in mice lacking both GSNOR and iNOS.
In addition we detected S-nitrosylation of two members of the PDI protein family.
Conclusions: These results suggest that S-nitrosylation resulting from GSNOR deficiency may promote carcinogenesis under
inflammatory conditions in part through the disruption of inflammatory and ER stress responses.

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 45: Cell Biology / Cellular Pathways

PTu-175

11:10 － 12:20

Proteomic analysis of low temperature resistant system in bacteria

Kadoya Ryosuke, Kasahara Yasuhiro
Hokkaidou University, Sapporo, Japan

Keywords:
Bacteria, cold shock resistant system, nano LC-MS/MS
Novel aspects:
This proteomic approach is shown to be ideal as a primary analysis for examination of various physiological characteristics inbacterial cells comparably to a DNA array analysis approach.
Abstract:
Under the cold condition, the increased production of cold shock proteins（CSPs）is mainly a consequence of the increased
stability and preferential translation of the corresponding mRNAs. This response, which in general terms in similar in different
mesophilic bacteria, helps to counteract a number of stresses derived from low temperatures such as reduced membrane fluidity,
reduced enzyme activity, and the greater stability of DNA and RNA secondary structure that can diminish the efficiency of
replication, transcription and translation. The bacteria stay long time under low temperature, the growth of cell was stopped.
After stopped growth, temperature returned 4 o C to 37 o C, the cells be able to grow normally. Usually, the knowledge of cold
shock response system was obtained from model-bacteria（Escherichia coli and Bacillus subtilis）and 15 o C. But most modelbacteria cannot grow at under 16 oC. Most studies on the biology of bacteria have been performed on cells grown at 30oC, its
optimum growth temperature. However, when growing in natural habitats, bacteria frequently live much low temperature placethat appears under 10oC and occupies 70% of the earth. Pseudomonas putida F 1 is model bacteria living in soil, this family was
isolated from the iceberg of the south antarctic pole. Because, we think that the genome of this strain was included essential
genes of constant low temperature growth.
The cold shock system is suggested that it is the stress response protein to rescue from inconvenient environment of low
temperature, and the possibility that there is not the protein necessary to normal grow under the constant low temperature. This
present works report proteomic analysis comparing two conditions, the one is 30oC, 16oC and 4 oC, and the second is cold shockstress phase（CSSP）and constant low temperature phase（CLTP）.
To obtain the low temperature resistant system of bacteria, we prepared three kind of the temperature, 30oC is optimum growthtemperature, 4 o C is low temperature that almost model-bacteria cannot grow under this condition, 16 o C is intermediate
temperature previous two conditions. P. putida F 1 was cultured at its optimum growth temperature in minimal salt medium. Aftergrew at exponential phase（O.D.600 = 0.3）, all proteins were extracted from cell. The proteins were separated by SDS-PAGE, andgels were cut into about 60 slices for in-gel digestions by trypsin. Each peptide mixture was analyzed by nano LC-MS/MS（Thermo
Fisher Scientific, USA）for protein identification. The LC-MS/MS data were searched by the P. putida F 1 database in NCBI, and,expression level of each gene were appeared by emPAI value calculating from Mascot program（ver. 2.3.01）.
About 2000 proteins were identified based on more than two unique peptides. The number of proteins expressed under several
temperatures was about 40 % of all proteins（5250）. The overview of expression genes was appeared to compare protein
expression at 30oC. In 16oC, a group of 340 genes displayed a two-fold significant increase at CSSP, and 789 genes displayed a twofold increase under the phase of the CLTP. In 4 oC, the numbers of CSSP genes are 330, and CLTP genes are 1051. The interesting
result in this, almost genes that showed expression more than two-fold were phase-specific. The expression increase genes wereseparated about function, we don’t find the common genes of low temperature（CSSP vs CLTP and 16oC vs 4 oC）. When sigma
factor（RNA polymerase subunit）expression was checked in several conditions, that expression pattern was independently about
each phase. RpoS（the homolog gene of E. coli control CSPs expression）was only induced in CLTP at both temperatures. This
result shows that the mechanism of low temperature resistance is different between the CSSP and the CLTP, 16oC and 4 oC.

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 45: Cell Biology / Cellular Pathways

PTu-176

13:30 － 14:40

IgSF11 is a member of the immunoglobulin superfamily that promotes
both neuronal adhesion and cell proliferation

Erika Hayashi1, Hiroaki Akutsu2, Hiroshi Funakoshi2, Yokichi Hayashi2
1

Hokkaido University,Sapporo,Japan, 2Asahikawa Medical University,Asahikawa, Japan

Keywords:
immunoglobulin super family（IgSF）, neuronal adhesion, cell proliferation,LC-MS/MS
Novel aspects:
IgSF11 is one of cell adhesion molecules ubiquitously expressed in the CNS, however its function remains to be clarified. Here we
present a piece of data to show its functions.
Abstract:
IgSF11 is a member of the immunoglobulin super family（IgSF）that possesses two immunoglobulin-domains, and is expressedubiquitously in the mammalian central nervous system（CNS）. IgSF11 is up-regulated in gastrointestinal and hepatocellular
carcinomas, and its down regulation leads to lower grades of malignancy. In the CNS, while the molecule is regarded as a celladhesion molecule as judged from its structure, it is still unclear whether IgSF11 functions in the neuronal processes ofdevelopment and maturation.
To assess this possibility, we first examined the effects of IgSF11 on the morphological differentiation of cultured neurons. When
culture dishes were pre-coated with mutant IgSF11 proteins consisting of the extracellular domain, hippocampal neurons preparedfrom mouse embryos formed cell aggregates within a few days of being cultured, and their neurites were observed to fasciculate
through the homophilic interaction of IgSF11. Enhanced IgSF11 expression by gene transfer brought similar changes in cultured
neurons. In contrast, a couple of short hairpin RNAs（shRNAs）complementary to IgSF11 showed neurite branching as a result of
the silencing of IgSF11 gene. Furthermore, IgSF11 proteins with various deletions of the cytoplasmic domain also stimulated suchbranching, suggesting that either the PDZ（PSD-95/D 1 h/ZO 1 ）binding region or some unknown regions in the cytoplasmic
domain are requisite sequences needed to regulate both the neurite fasciculation and cell aggregation induced by IgSF11. Amongthe kinase inhibitors we tested, only those inhibitors for Protein Kinase C（PKC）attenuated both branching and cell adhesion.
We next analyzed whether or not IgSF11 affects neuronal differentiation of mouse embryonic carcinoma（P19）cells inducible byretinoic acid treatment. Differentiated neuronal P19 cells showed lowered expression of IgSF11. As expected, forced expression of
IgSF11 led to a decrease in the number of cells undergoing neuronal differentiation, instead allowing cells to proliferate in an
undifferentiated state. A couple of shRNAs, that effectively down-regulate the IgSF11 gene, cause an increase in the numbers ofboth neurons and glial cells, indicating that IgSF11 affects multiple processes during neuronal differentiation. cDNAs encoding
mutant IgSF11, that were devoid of the PDF binding region, failed to stimulate proliferation of undifferentiated P19 cells.
Interestingly, inhibitors of PKC attenuated IgSF11-induced cell proliferation as well. Therefore, we conclude that there may exist
molecules that interact with IgSF11, or are affected by transduction of IgSF11 signal, that are involved in the neuronal differentiationof P19 cells and morphological changes in cultured hippocampal neurons.
To search for molecules that interact with IgSF11,we constructed and isolated cell lines stably expressing IgSF11. We preparedcell extracts after undergoing isotopic protein labeling of the cell lines, immunoprecipitated IgSF11 with its binding proteins fromthe cell extracts, or prepared cell extracts with other methods in order to analyze molecules using LC-MS/MS combined othermethods. As a couple of candidates have been detected to date, we have been extensively examining these to see if they satisfy
criteria as molecules with which IgSF11 exerts its functions.
Although IgSF11 is a small member of the IgSF ubiquitously expressed in the CNS, these data, taken together, strongly suggestthat its expression affects the development and morphological changes of neurons within the CNS, and that there are somemolecules that interact with IgSF11 or mediate its signal transduction.

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 45: Cell Biology / Cellular Pathways

PTu-177

11:10 － 12:20

Live Single-cell Mass Spectrometry for Tracing Metabolic Processes in
Organelle

HAJIME MIZUNO1, NAOHIRO TSUYAMA1, SACHIKO DATE2, TAKANORI HARADA1,TSUTOMU MASUJIMA1,2
1

Hiroshima University, Hiroshima, Japan, 2QBiC, RIKEN, Osaka, Japan

Keywords:
Single cell, Organelle, Isotope labeling, Metabolomics
Novel aspects:
Metabolic pathways even in a live single cell organelle was clarified by the Live Single-cell Mass Spectrometry.
Abstract:
Introduction
We have developed a direct mass spectrometric method called“Live Single-cell Mass Spectrometry.“ This method enables us
rapid metabolic analysis in the visualized organelle even in a live single cell. This method analyzes metabolites and its metabolic
pathways even in an organelle with its behavior and sub-cellular localizations. Furthermore, using stable isotope labeled reagents
we can trace the metabolic processes in a live single cell.
In this study, we applied this method to analyze the localizations of the histidine and histamine metabolites and to trace their
metabolic processes in a live single rat basophilic leukemia（RBL- 2 H 3 ）cell.
Methods
RBL- 2 H 3 cells were cultured in DMEM and incubated at 37°C in CO2 incubator. A cell contents（secretory granule or cytoplasm）
of a live RBL- 2 H 3 cell aimed under view by CCD video camera mounted on an inverted microscope, was micro-sucked into a
nanospray tip（Cellomics Tip, HUMANIX, Japan）with a micro-manipulator. Ionization solvent containing 0.5 % formic acid（forpositive mode）was added to the content in a nanospray tip. Mass spectrometric detection was performed by a LTQ Orbitrap XL
mass spectrometer equipped with nano-ESI ion source. Spray voltage was around 1000 V（for positive mode）. To increase
sensitivity of peak detection, we used the SIM mode for single-cell molecular detection. For tracing the histidine and histamine
metabolic reactions, RBL- 2 H 3 cells were cultured 15 N labeled DMEM which histidine was replaced to 15 N isotope labeledhistidine.
Results and Discussion
In order to clarify micro-sucked granule contents specifically, we used di-protonated Quinacrine ion peak（m/z 200.6111）as a
marker ion peak of granule-sucked sample. The histidine and histamine metabolites ion peaks were detected in a granule and/or
cytoplasm in a single RBL- 2 H 3 cell. A statistic analysis of t-test was performed for these peaks to clarify the sub-cellular
localization between a granule and cytoplasm（more than 5 cell samples for each）. The ion peak at m/z 156.0764 was detected
in granule side and identified as histidine by exact mass number（within 3 ppm error）and MS/MS fragment pattern. Histamine（m/z 112.0868）, one of the histidine metabolites was found in granule specifically. The other histidine metabolites（e.g. imidazole
acetaldehyde（m/z 111.0552）, methylhistamine（m/z 126.1026）, urocanic acid（m/z 139.0501）, 1 -methylhistidine（m/z 170.0921）,
and so on）were also found in granule side. The results show that histidine and histamine metabolic pathways existing in a
granule. From 15N isotope labeled histidine（C6H9 15N3O2）treated RBL- 2 H 3 cell, 15N labeled histidine（m/z 159.0678）and histamine（m/z 115.0779）, and its metabolites, imidazole acetaldehyde（m/z 113.0493）, urocanic acid（m/z 141.0443）and so on weredetected in granule samples. From these results, it was clarified that histidine transport into granule and clarified the histidine
metabolic pathways locate in granule.
This method has enabled us to analyze not only location of metabolic pathways but also metabolic processes of interest
molecules and it can be used in various organelle metabolomics in a live single cell.

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 45: Cell Biology / Cellular Pathways

PTu-178

13:30 － 14:40

Development of Micro-area-specific Extracting Analysis for Molecules in
Tissue Section

Sachiko DATE1, Tsutomu MASUJIMA1,2
1

RIKEN QBiC, Osaka, Japan, 2Hiroshima University, Hiroshima, Japan

Keywords:
direct tissue analysis, micro solvent extraction, nanoESI, micro-area-specific
Novel aspects:
Small molecules exist in specific micro-area of tissue section were analyzed by micro solvent extraction using nano-spray tip.
Abstract:
Introduction
Animal tissue is composed of some aggregations of differentiated cells, and each group play a role and maintain whole function of
the tissue. The local existence of molecules in particular part of tissue is important in order to understand system of tissue, or
effects of drug. The location of molecules has been found by technology of MS imaging such as MALDI or SIMS imaging. However,these methods have problems that small molecules or molecules inside of cell is hardly detected. In this study, new methods bywhich, molecules exited in specific area of about 10μm wide in a frozen tissue section is selectively extracted, trapped andmeasured by mass spectrometry, was developed by applying Live Single-cell MS method.

Methods
A piece of chicken liver was frozen in hexane cooled in liquid nitrogen, and was embedded in Frozen Section Compound. It wassliced into 10 μm thick of tissue sections at -20&#8451； using freezing microtome（Leica CM3500S, Leica Micro Systems）. Sections
were mounted on glasses and dried in room temperature. Head of nanospray tip（Cellomics Tip, HUMANIX）including 1 μL of
solution for extracting（100%MeOH, 50%MeOH, 100%H2O, 100%Acetone）was adjusted to area close to or distant from vessel using
the micro manipulator, And solvent was taken in and out from the tip with pressure in order to extract the molecules under
microscope observation. The tip was added 3 μL of solvent（90%MeOH/9.0%H2O/1.0%formic acid）behind and directly ionizedand detected using nanoESI-MS.

Result and Discussion
Solvent taken in and out of nanospray tip covered circular area of about 10μm wide on liver tissue section. 1.0kV of Ionspray
Voltage was directly added the nanospray tip trapping the extraction of tissue and molecules in liver was directly analyzed by
LTQ Orbitrap Velos（Thermo Fisher Scientific）, at positive mode, and by full scan at a range of m/z 70-2000. Although solvent used
for extraction was 100%MeOH, 50%MeOH, 100%H2O or 100%Acetone, most peaks, including small molecules such as serine（m/z106.049）, cytidine（m/z 244.093）, creatine（m/z 132.076）and fatty acids, phospholipids, were detected, when 100%MeOH was used.
When acetone was used, lipids, which have more than 1000 molecular weight tended to be detected higher than when othersolvent were used. Mass spectrum data was aligned by MarkerView Software（AB SCIEX）and t-test（peaks detected in area close
to vessel vs. distant from vessel）was performed. In the result, 6 peaks were specific in close to vessel, and 47 peaks were specificin distant from vessel（p>0.01）. Identification of these peaks using MS/MS analysis was performed and a fatty acid, which has theformula of C18H30O2, is specific to the area distant from vessel. It is possible that activity of metabolism or transport of the lipid
vary at a different site of the liver. Analysis of the related metabolites to this lipid and identification of specific peaks in each site
are under progress.

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 45: Cell Biology / Cellular Pathways

PTu-179

11:10 － 12:20

In-situ Analysis of Phototropic Molecules in plant by Live Singe-cell Mass
Spectrometry

TAKASHI FUJII1, SACHIKO DATE2, HAJIME MIZUNO1, NAOHIRO TSUYAMA1, TSUTOMU MASUJIMA1,2
1

Hiroshima University, Hiroshima, Japan, 2Quantitative Biology Center (QBiC), RIKEN, JAPAN

Keywords:
live single-cell mass spectrometry, plant metabolite, phototropism
Novel aspects:
Localized metabolites by light exposure was detected using in-situ live single-cell MS
Abstract:
Introduction
Plants have unique characteristics called phototropism that they can curve their stems toward light source to get more lightenergy for their growth. It has been said that increase of intracellular concentration of auxin, a plant hormone related in plant
growth and body development, is involved in this phototropism process. However, molecular mechanisms of phototropism fromthe view point of low molecular weight metabolite have been remained unclear. In order to clarify its spatio-temporal molecularmechanism, it is important to analyze metabolites quantitatively and qualitatively. In this study, we performed live single-cell mass
spectrometry（MS）using radish sprouts to identify the molecules correlated with phototropic reaction by exposing light.
Methods
Radish sprouts were grown in the humidified dark room at 24 ± 1 degrees C for 5 days. Subsequently, some of them were
irradiated with blue light from horizontal direction for 0, 8, 16, 24 hours respectively. Upper part of irradiated sprout stems bent
toward light source whereas lower part of stems kept straight. Then, the stems were cut in longitudinal direction. Surface cells in
4 parts（bent lighted side, bent shaded side, straight lighted side and straight shaded side）were targeted, and their cellular
contents were micro-sucked into nanospray tips（HUMANIX, Japan）under a stereomicroscope. Mass spectrometric detection
was performed by a LTQ Orbitrap Velos Pro（Thermo Fischer Scientific, USA）equipped with nano-ESI source at both positive andnegative ion mode.
Results
Principal component analysis（PCA）on spectral data of stem parts, bent shaded side and lower shaded side, showed similar
trend of spectral pattern because they were located adjacent to each other in 2 D score plot. So, we analyzed difference ofmolecular distribution between lighted side and shaded side in detail. Glucose（［M-H］- , m/z 179.0561）, source of nutrition,
glutamine（［M-H］-, m/z 145.0618）and asparagine（［M-H］-,m/z 131.0462）, source of nitrogen, were increased in the lighted side incomparison with no light exposed shaded side. These results show that those increased molecules were produced by photosynthesis in the lighted side, and in the shaded side those were consumed by the growth without novel production. It is possiblethat the concentration of metabolites were attenuated by cell elongation in shaded side. In addition, a few dozens of metabolites
showed limited existence. Among them, glucosephosphate（［M-H］- , m/z 259.0224）showed unique trend： this metabolite washigher in the lighted side than in shaded side after light exposure, but surprisingly it was much higher before exposure. Now,
further investigation of molecular mechanism based on these results, and comprehensive analysis of metabolites involved inphototropism are under progress.

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 45: Cell Biology / Cellular Pathways

PTu-180

13:30 － 14:40

Analysis of Specific Molecules in Allergic Granule by Live Single-cell Mass
Spectrometry

YUKI YAMAMOTO1, SACHIKO DATE2, HAJIME MIZUNO1, NAOHIRO TSUYAMA1,
TSUTOMU MASUJIMA1,2
1

Hiroshima University, Hiroshima, Japan, 2Quantitative Biology Center (QBiC), RIKEN, JAPAN

Keywords:
live single-cell mass spectrometry, RBL- 2 H 3 cells, granule, orbitrap mass spectrometer
Novel aspects:
By Live Single-cell MS method, specific molecules in a granule and their metabolism were directly clarified.
Abstract:<Introduction>
Intracellular molecules are distributed in sub-cellular organelle as a function of each, and they control vital response by changing
their amount or localization. In allergic response, mast cells secret molecules in granule and then allergic symptoms are triggered.
So, analysis of molecules in granule gives one of the important information to understand the allergic mechanism. In order toclarify allergy-inducing molecules, we analyzed cytoplasm and allergic granules in RBL- 2 H 3 cell line by using the“Live Single-cell
Mass Spectrometry“. This method can trap particular region in a single cell under a video-microscope, and detect molecules
directly by mass spectrometer. Comparing peaks of cytoplasm to those of allergic granule, we analyzed specific molecules of eachorganelle.
<Method>
RBL- 2 H 3 cells were observed under an inverted video microscope, then granule or cytoplasm was trapped into a nanospray tip（Cellomics tip, HUMANIX, Japan）using a micro manipulator. After sampling, ionization solvent（80%methanol, 1 %formic acid）
was added to the nanospray tip, and then high voltage was applied to the tip for generating charged spray droplet. Mass
spectrometric detection was performed by a LTQ Orbitrap（Thermo Fisher Scientific）equipped with nano ESI source. We
optimized measurement condition and SIM（selected ion monitoring）scan mode for highly sensitive analysis. Paired t-test for
extracting specific peaks of granules was performed. Molecular formation of detected ion was identified by exact mass number.
<Results>
We used quinacirne ion peak（m/z 200.6111）as the marker to confirm the successive granule suction in a RBL- 2 H3, because
accumulation of quinacrine into granules was observed by fluorescence microscope. By comparing peaks of granules to those ofcytoplasm, over 50 specific peaks of granule were detected at positive ion mode, such as creatinine（m/z 114.0663）, metabolites
of arginine, and 4 -hydroxyphenylacetate（ m/z 153.0544 ）, metabolites of tyrosine, and so on. In addition, histamine（m/z 112.0869）
and metabolites of histamine such as methylimidazole acetaldehyde（m/z 125.071）and methylimidazole acetic acid（m/z 141.066）
were also detected. These results indicated that histamine metabolic pathway exists in granule. On the other hand, taurine（m/z148.0039）and tryptophan metabolites such as formylkynurenine（m/z 237.0873）, hydroxyindoleacetate（m/z 192.0655）were
detected from cytoplasm specifically. Identification of other candidate molecules by MS/MS analysis is under progress. In addition,
we applied stimulus to RBL- 2 H 3 by calcium ionophore A23187. As a consequence, the intensity of histamine metabolites
decreased by administration of A23187, whereas the intensity of histamine itself stayed about the same. This result suggest thatmetabolism of histamine is suppressed by the stimulus.

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 45: Cell Biology / Cellular Pathways

PTu-181

11:10 － 12:20

Time Dependent Changes of Intracellular-Signal-Relating Small
Compounds in Allergy Model Cell Line, RBL-2H3

Toshiya Shinasue1, Yui Okamura1, Hajime Mizuno1, Naohiro Tsuyama1, Sachiko Date2,
Tsutomu Masujima1,2

Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima, Japan, 2Quantitative Biology Center, RIKEN,
Japan

1

Keywords:
Orbitrap, Allergy, RBL- 2 H3, Intercellular-Signaling
Novel aspects:
Stimulated-cell-specific compounds and their time dependent changes were clarified in an allergy model cell line, RBL- 2 H3.
Abstract:
PURPOSES
There are enormous small compounds and their behavior in an intracellular signaling remains unclear, while large compound suchas protein participated have been studied in detail. However small compounds are closely related to an intracellular signaling.
Therefore it is important to investigate their intracellular localization and time dependent changes of small compounds in clarifying
the perspective of signaling. In this study, using the Rat basophilic leukemia（RBL- 2 H 3 ）cells as an allergy model cell line, we
compared small compounds between in stimulated and not stimulated cells and analyzed time dependent changes after
stimulation in the signaling of allergic reaction.
METHODSRat basophilic leukemia（RBL- 2 H 3 ）cells were stimulated with 1 µM calcium ionophore A23187 and extracted by methanol for 5,
10 and 30 minutes. Cell extracts were applied a LC-MS. We used L-column 2 C 8 （1.5 x 150 mm, 5 µM, CERI）connected to LC（LC20AD, SHIMADZU）. The mobile phase was 10mM ammonium formate in 90% methanol with 10mM ammonium formate used at
the flow rate of 60 µL/min. Mass spectrometric detection was performed by an Orbitrap Velos Pro（Thermo Fisher Scientific）at
the resolution power of 100,000 with ESI at negative ion mode. Peaks were extracted from LC-MS spectra using MZmine-2.6, and
we identified peaks by comparing with database（kegg http：//www.kegg.jp/kegg/, LIPID MAPS http：//www.lipidmaps.org/ ）.
RESULTS AND DISCUSSIONSFirst, we compared the extract of cells stimulated for 30 minutes and not stimulated. About 20000 peaks were obtained from thecellular extract, and t-test was performed for these peaks to clarify compounds increased or decreased in stimulated cells.
Compounds, for example lipids and saccharoids, differed significantly were identified by database search using exact mass. Peaks
such as m/z 341.1098（C12H22O11, disaccharide）and m/z 508.4735（C32H63NO3, ceramide）decreased in stimulated cells, and peaks
such as m/z 238.0946（C 9 H13N 5 O3, dihydrobiopterin）and m/z 508.4735（C43H81O13P, phosphatidylinositol）increased. Especially
we picked up the peak m/z 335.2228（C20H32O4, a compound in arachidonate cascade）, it increased in stimulated cells. It is
known that lipid mediators such as prostaglandins and leucotrienes are released after degranulation in mast cells. Therefore it
seems that this result reflects increase of prostaglandin and leukotriene-synthesis.
Next, in order to investigate time dependent change in detail, we measured the extract of cells stimulated for 5 minutes or 10
minutes. As a result, it was shown that arachidonic acid peak m/z 303.2330 increased in 5 minutes samples and 10 minutes. It
suggests that arachidonic acid-synthesis increased at 5 minutes, and its synthesis and its consumption in arachidonate cascade
are in equilibrium around 5 minutes to 10 minutes. In addition, phospholipid peaks changed such as m/z 834.5291（C46H77O10NP,
phosphatidylserine, increased）and m/z 744.5549（C41H79NO 8 P, phosphatidylethanolamine, decreased）after stimulation.
Identification of compounds indicated above by MS/MS and more detailed analysis are in progress.

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 45: Cell Biology / Cellular Pathways

PTu-182

13:30 － 14:40

Brain proteomics reveals early molecular signature of pathology in presymptomatic mouse model of Alzheimer’s disease

Hongqian Yang1, Jessica L Wittnam2, Roman A Zubarev1,3, Thomas A Bayer2

Karolinska Institute, Stockholm, Sweden, 2University Medicine Göttingen,Göttingen, Germany, 3Science for Life
Laboratory, Stockholm, Sweden

1

Keywords:
shotgun proteomics, mass spectrometry, LC/MS, amyloid-beta peptides
Novel aspects:
These data confirm that AD-typical molecular pathways can be detected already in pre-symptomatic mice expressing one of the
major amyloid-beta peptides.
Abstract:
Alzheimer’s disease（AD）is a progressive neurodegenerative disorder characterized by the presence of extracellular amyloid
plaques composed of amyloid-β（Aβ）peptides. Aβ aggregation could be triggered by naturally occurring abnormal forms of Aβ
peptide, including N-truncated forms, as well as overproduction of Aβ peptides in brain. Analysis of amyloid deposits in AD brains
revealed various N and C terminal variants. Among them, truncated Aβ peptides with pyroglutamate at third amino acid position（Aβp E3-42 ）are gaining considerable attention as potential key players in the pathogenesis of AD. Aβp E3-42 is abundant in AD
brain and has a high aggregation propensity, stability and cellular toxicity. A novel AD mouse model TBA42 that expresses soluble
AβpE3-42 leading to an age-dependent neurological phenotype without aggregating in plaques has been generated. This model iswell suited to study the role of soluble AβpE3-42 without possible interference of other toxic Aβ peptides, either full-length or Nand C-variants.
Since early events are critical in the development of AD-related pathology, it is important to understand the molecular mechanism
behind these events. Here we studied the early, pre-symptomatic proteome changes in the brain of 4 -month old TBA42 female
mice compared to the wild-type（WT）controls. We complemented the in-gel tryptic digestion protocol with the filter-aided
sample preparation（FASP）method. The proteins found significantly changed in both types of sample preparation represent themost reliable group. To put the results in the context of known signaling pathways, we performed quantitative pathway analysis
using the pathway search engine approach developed by us earlier.
Label-free proteomics was used to identify and quantify 858 proteins using in-gel digestion and 521 proteins using FASP digestion.
Several proteins were found to be significantly regulated in both in-gel and in-solution analysis including Homer proteins involvedin scaffolding, organizing proteins at the synapse and regulating intracellular calcium within the neuron. Moreover, analyzing key
nodes, which are regulatory molecules found on pathway intersections, we identified Rho-kinase（ROCK）, a serine/threonine
kinase and one of the major downstream effectors of GTPase Rho, as well as three key nodes of the mTOR/p70S 6 K signaling
pathway previously implicated in multiple fundamental biological processes including synaptic plasticity and up-regulated in AD.

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 45: Cell Biology / Cellular Pathways

PTu-184

13:30 － 14:40

Realtime molecular analysis of allergenic response in single mast cell byfluorescence probe-assisted Live Single-cell MS

Yosuke Kawai1, Naohiro Tsuyama1, Hajime Mizuno1, Sachiko Date2, Tsutomu Masujima1,2

Hiroshima University Graduate School of Biomedical & Health Sciences, Hiroshima, Japan, 2Quantitative Biology Center(QBiC), RIKEN, Osaka, Japan

1

Keywords:
Single cell, nanospray, RBL- 2 H3, Fluorescent probe, Direct analysis
Novel aspects:
Specific peaks of allergy reaction-linked molecular were clarified by direct and realtime analyses in a live single-cell.
Abstract:< Introduction >Biological studies usually use plenty amount of samples although cell populations are almost heterogeneous and asynchronouseven after they are added with some stimuli. When extracellular stimuli are added on cell surface such as antigen binding to its
receptor, sequential response occurs in/around cellular membrane assumed by certain proteins and small molecules to induce
secondary response inside the cytoplasm such as calcium efflux from endoplasmic reticulum. These responses sometimes
terminate in a short time, and begin and last differently among cells. Thus, analyses using large numbers of cells in these sharp
signaling responses sometimes give ambiguous results, whereas single cell analyses prove this problem. Live Single-cell MS allows
realtime recovery and direct mass spectrometric analysis of a targeted cell under a microscope using a nanospray tip. We applied
this method to follow signal transduction monitored by fluorescent probe for second messenger.
< Methods >We used rat basophilic leukemia cell line, RBL- 2 H3, to monitor stimuli-induced molecular change. First, RBL- 2 H 3 cells were
inoculated onto feeder cells（fibroblasts ceased proliferation by cytotoxic agent）to induce cellular maturation. Before inoculation,
cells were stained with a lipophilic fluorescent probe to distinguish from feeder cells. After inoculation, maturated cells were prestained with fluorescent indicator such as Fluo 4 to monitor secondary intracellular response. Cells were inoculated with antidinitrophenol（DNP）antibody to produce antigen receptor. Then, the cells were stimulated with DNP to start the antigen siglal. In
some case, cells were stimulated with 1 mM of calcium ionophore A23187 or 100 ng/mL 12-O-Tetradecanoylphorbol 13-acetate（TPA）. Stimulated cells were observed under a fluorescent microscope（IX-70, OLYMPUS）equipped with excitation andemmition filters for green（Fluo 4 ）and orange fluorescence（lipophilic RBL- 2 H 3 indicator）. Fluo 4 fluorescence wascontinuously observed and a fluorescence-increased（stimulus-responded）cell was sucked with a nanospray tip of 3 µm bore
（Cellomix tip, HUMANIX）followed by addition of ionizing solvent from the backend and analyzed by a LTQ Orbitrap XL（Thermo
Fischer Scientific）. Peaks obtained with full scan or selected ion monitoring（SIM）mode were extracted and evaluated.

< Results and Discussion >We continuously observed Fluo 4 fluorescence under a fluorescent microscope after stimuli and the fluorescence increased insome cells. The fluorescence-increased cell was sucked with a nanospray tip. Then, ionizing solvent（ 1 % NH3, 9 % water, 90 %acetonitrile）was added from the back-end of the tip and the tip was enforced to nano-ESI analysis at negative ion mode of -800V
spray voltage. The result showed that peaks derived from intracellular Fluo 4 such as m/z 1023.2488（C48H45F 2 N 2 O21）, m/z
807.1854（C36H29F 2 N 2 O13）were detected. We used these peaks as a maker to confirm that content in a cell were micro-sucked
successfully, because accumulation of Fluo 4 was observed under a fluorescence microscope. In addition, peaks such as m/z
508.4765（C32H63NO3, Cermides）and m/z 835.5342（C43H81O13P, Phosphatidylinositol）were also detected. The former decreased,
and the latter increased after stimuli. These results provide that the change of intracellular molecules by extracellular stimuli could
be analyzed in realtime in responded cell by monitoring with fluorescent indicators in intracellular signal transduction of allergic
reaction in a live single RBL- 2 H 3 cell. For detection of specific peaks after stimulation, the negative ion analysis in a single
stimulated RBL- 2 H 3 cell was now in progress.

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 45: Cell Biology / Cellular Pathways

PTu-185

11:10 － 12:20

A host-pathogen interaction network study of Shigella flexneri infection ofhuman cells targeting key metabolic enzymes

Asa Wahlander1, Bernd Roschitzki1, Paolo Nanni1, Claudia Fortes1, Christian Trachsel1, Nicole Freed2,
Simon Barkow-Oesterreicher1, Christian Panse1, Jonas Grossmann1, Petra Tienz2, Dirk Bumann2,
Ralph Schlapbach1
1

Functional Genomics Center Zurich, FGCZ, 2Infection Biology Biozentrum, University Basel, Basel, Switzerland

Keywords:
srm, pathway, host-pathogen interaction network
Novel aspects:
Identification and quantification of key metabolic enzymes in host pathogen interaction
Abstract:
Infectious diseases are a major cause of morbidity and mortality worldwide. Growing resistance to currentant microbials and leanpipelines for novel therapeutics increasingly limit treatment options. However as infectious diseases are usually the consequenceof a fight between two networks of hundreds to thousands of individual factors and not just a single mechanism, interfering withany single factor often has insufficient effects on infection outcome and an integrated system-level analysis is thus required forrational development of novel control strategies. This project focuses on metabolism as a subsystem of the HeLa-Shigella flexneri
interaction network, aiming to quantify as many and specific changes in abundance in the metabolic proteins upon various
conditions of infection.
The discovery workflow is adjusted to enable the maximum number of proteins to be identified with a standard LC-MS/MS setup.
A quicker approach for obtaining approximate quantities of large subsets of proteins across several cell states has been
established. To allow for more accurate quantitative data and absolute quantification, SRM with stable isotope labeling is applied
to sets of target enzymes. These techniques are implemented on samples from various stages of infection, i.e. time-course studies,
as well as on different mutants of the human HeLA cell line and Shigella siRNA knockdown samples.

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 45: Cell Biology / Cellular Pathways

PTu-186

13:30 － 14:40

A highly specific and sensitive determination of the sterols in silkworm
larva by HPLC/APCI-MS/MS for the investigation to insect steroidogenesis.

Fumihiko Igarashi, Juri Hikiba, Takayoshi Nakaoka, Minoru Suzuki, Hiroshi Kataoka

Department of Integrated Biosciences, Graduate School of Frontier Sciences, The University of Tokyo, Kashiwa, Japan.

Keywords:
HPLC/APCI-MS/MS, Cholesterol, Phytosterol, Silkworm, Ecdysone
Novel aspects:
APCI-MS/MS was able to ionize intact cholesterol and phytosterols, allowing simple and rapid sample preparation andsimultaneously quantified the sterols in the silkworm larval tissues in the femtomole range.
Abstract:
The biochemical quantification of sterols in insects has been difficult because only small amounts of tissues can be obtained from
insect bodies and because sterol metabolites are structurally related. We have developed a highly specific and sensitive
quantitative method for determining of the concentrations of seven sterols- 7 -dehydrocholesterol, desmosterol, cholesterol,ergosterol, campesterol, stigmasterol, and β-sitosterol -using cholesterol-3,4-13C 2 as an internal standard. The sterols wereextracted from silkworm larval tissues using Bligh and Dyer method and were analyzed using a high performance liquid
chromatography-atmospheric pressure chemical ionization-tandem mass spectrometry（HPLC/APCI-MS/MS）. APCI was able toionize intact sterols at atmospheric pressure, allowing more rapid and simple sample preparation. Tandem mass spectrometry
using a triple quadrupole mass spectrometer was performed by selected reaction monitoring（SRM）, detecting m/z 145 or 147
fragment ion which includes the A and B rings of the dehydrated ion of each sterol. The method achieved simultaneous analysis of
most of the sterols with femtomole sensitivity.
The newly acquired method can be utilized for the investigation to the molecular mechanism of steroid hormone biosynthesis in
insects. In this report, we focused on the cholesterol uptake mechanism in steroidogenesis organ, prothoracic gland. The
cholesterol is a starting material for the steroid hormone, ecdysone which regulates the timing of insect molting. However, themolecular identity of the cholesterol supply mechanism has remained elusive. Because insects cannot synthesize sterols de novo
from acetate, each type of insect could have developed abilities to absorb sterols at the cellular levels to maintain optimum
ecdysteroid biosynthesis. We first analyzed the sterol concentration changes in prothoracic glands based on presence or absenceof ecdysone secretion. The data revealed free cholesterol and dietary phytosterols accumulation in the ecdysone biosynthesis
stages. Second, we analyzed the function of LpR, one of the transporters expressed in the prothoracic glands, and revealed that
LpR could uptake the sterols from the plasma lipoprotein. In the future work, we will try to analyze the transporter function morein detailed and search its regulator with utilizing quantitative performance of the HPLC/APCI-MS/MS system, elucidating the natureof the cholesterol uptake mechanism in the insect steroidogenesis organ.
A simple, sensitive and specific method was successfully developed for the quantification of the sterols concentrations in eachtissue of an individual silkworm larva. The method was also a useful tool for the investigation of the cholesterol transporter
function. This method will be a useful for further investigation to molecular basis of sterol physiology in insects, facilitating thequantification of femtomole quantities of sterols in biological samples.

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Polymer sciences

PTu-187

11:10 － 12:20

Evolved gas analysis of a Japanese lacquer film

Noriyasu Niimura
JEOL, Tokyo, Japan

Keywords:
Japanese lacquer, urushiol, field ionization
Novel aspects:
It is the first time that field ionization mass spectrometry has been applied to the evolved gas analysis of a Japanese lacquer film
for the structural study.
Abstract:
Japanese lacquer is sap of lacquer trees. The sap is latex composed of urushiol, water, plant gum, glycoprotein and laccase
enzyme. It dries into a tough film, so it is used as a coating material for wood and pottery. The lacquer film, which is urushiol
polymer, is insoluble. Therefore, analytical methods are limited. The evolved gas analysis is one of the methods. Field ionization（FI）mass spectrometry is an effective method for evolved gas analysis, because it preferentially produces molecular ions and, insome cases, protonated molecular ions. There is less fragmentation in FI than in electron ionization（EI）, therefore, FI mass
spectrum of evolved gas is simpler than EI mass spectrum.
Using EI and FI mass spectrometry, the evolved gas from a Japanese lacquer film at 320 &#8451； was analyzed and two results
were compared. We previously reported that the thermogravimetry（TG）curve of the lacquer film shows the weight decrease in
two stages, i.e., the thermal degradation gradually begins at around 200 &#8451；, and the degradation rate becomes fastest at
400-500 &#8451；. The derivative thermogravimetry（DTG）curve shows two peaks at 320 &#8451； and 470 &#8451；. The evolved
gas at 320 &#8451； contains the thermal degradation product from the surface of the lacquer film.
The EI mass spectrum was complicated because it was consisted of peaks due to the various kinds of fragment ions as well as
molecular ions of the thermal degradation products. We compared the observed spectrum to that of the evolved gas from3 -pentadecylcatechol, which is a component of urushiol. The molecular ion was detected at m/z 320, and the typical fragment ion
was detected at m/z 123 as the base peak in the mass spectrum. The molecular ion gives rise to the fragment ion at m/z 123 bypreferential cleavage at β to the aromatic ring. Both the molecular ion and the fragment ion were also detected in the mass
spectrum of the lacquer film. These ions are attributed to 3 -pentadecylcatechol, which is a thermal degradation product of thelacquer film. The lacquer was reported to be hardened by laccase-catalyzed oxidative coupling of urushiol and autoxidative crosslinkage of the side chains. In these reactions, saturated side-chains serve as terminal groups of the polymer, whereas active
unsaturated side-chains like alkenylcatechol form polymer skeletons. Therefore, 3 -pentadecylcatechol is preferentially dissociatedout by the thermal degradation from the terminal groups at the lower temperature around 320 &#8451；.
In the FI mass spectrum of the evolved gas from the lacquer film, the molecular ion of 3 -pentadecylcatechol was also detected
as the base peak. This result indicates that the gas consists of 3 -pentadecylcatechol, which supports the result obtained using
the EI mass spectrometry. Several peaks were also detected at the lower m/z. These peaks could not be attributed to
3 -pentadecylcatechol, because any of these peaks were not detected as fragment peaks in the FI mass spectrum of the evolved
gas from 3 -pentadecylcatechol. Two of these peaks were identified as the molecular ion of hydroxymethylfurfuraldehyde andprotonated molecular ion of anhydrosugar. The lacquer film is composed of plant gum. The constituent of the plant gum ispolysaccharide and its principal constituent sugar is galactose. It has been reported that galactose is thermally degraded tohydroxymethylfurfuraldehyde and anhydrosugar. Therefore, the observed hydroxymethylfurfuraldehyde and anhydrosugar werereasonably identified as thermal degradation products of plant gum. It is concluded that the plant gum is concentrated in thesurface of the lacquer film and it is thermally degraded at the lower temperature around 320 &#8451；.

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Polymer sciences

PTu-188

13:30 － 14:40

Structural characterization of ethylene-vinyl acetate copolymers using
high-resolution MALDI-TOFMS with a spiral ion trajectory

Hiroaki Sato1, Hidenobu Aizawa1, Sayaka Nakamura1, Takafumi Sato2
1

National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Japan, 2JEOL Ltd., Akishima, Japan

Keywords:
EVA, Spiral-TOFMS, polymer characterization
Novel aspects:
Detailed structural characterization of ethylene-vinyl acetate copolymer（EVA）could be performed by MALDI Spiral-TOFMS. The
high-resolution mass spectra enabled us to evaluate the profiles of thermo-oxidative degradation of EVA.
Abstract:
Ethylene-vinyl acetate copolymer（EVA）is a typical polymer encapsulant of a solar photovoltaic（PV）module. Because the EVA
encapsulant serves as a sealant protecting silicon solar cell from moisture, degradation of EVA strongly affects the lifetime of PV
modules. In general, degradation of EVA is initiated by deacetylation at vinyl acetate（VA）units, forming polyunsaturatedhydrocarbon chains. Under an oxidative condition, the formation of ketones is also possible. Although degradation profiles andmechanisms of EVA have mainly been investigated by means of thermal analysis and Fourier transform infra-red absorption
spectroscopy（FT-IR）, these techniques give less information on the chemical structure of each polymer chain.
Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry（MALDI-TOFMS）is commonly used as a techniquefor polymer characterization. However, the structural characterization of EVA is a challenging task by using a conventional TOFMS,
since the random nature of comonomer distribution generates very complicated mass spectra, in which the peaks of different
chemical compositions with the same nominal mass overlap. In this study, a detailed structural characterization of EVA wasattempted using a high-resolution TOFMS instrument with a spiral ion trajectory（Spiral-TOFMS）.
The EVA samples with different VA composition（12-40 wt%）were used. Because each EVA sample has wide molecular weight
distribution, fractionation by size-exclusion chromatography（SEC）was performed. The fractions of the oligomer region（> ca. 3000
Da）were subjected to MALDI measurements. DCTB matrix and NaI cationization salt were used to generate［M + Na］+ ions.
MALDI mass spectra were observed using a Spiral-TOFMS（JEOL）.
Complicated mass spectra were observed due to their random comonomer distribution. The monoisotope peaks of EVA chains
having ethylene（E）with m units and VA with n units（EnVAm）were overlapped with the isotopes of（En+3VAm-1）, whose massdifferences were only 0.06 Da. The peak resolution of 40,000 - 60,000（FWHM）could be achieved by MALDI Spiral-TOFMS, enabling
the separation of a number of isobaric peaks with different copolymer compositions and the assignment of each peak to a uniquecomponent.
The mass spectra of the degraded EVA samples were much more complicated, because the isobaric peaks of deacetylated and/or oxidized products with different comonomer compositions overlapped with those of intact components with ca. 0.07 Da
diffrence. Even for the complicated mass spectra, detailed chemical structures（comonomer compositions and structural changes）
of each polymer chain could be characterized. The compositional distribution of EVA before and after degradation were evaluatedusing a Kendrick mass defect（KMD）plot, which can depict the distribution of mass deficiency vs mass. The degradation of EVA
chains will form unsaturated bonds and/or ketone groups, resulting that mass deficiency from the mass of saturated hydrocarbon
chains increase. Therefore, the KMD plot could reveal the degradation profiles of EVA. In conclusion, MALDI Spiral-TOFMS has
potential as a powerful tool for the structural and compositional characterization of EVA copolymers.

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Polymer sciences

PTu-189

11:10 － 12:20

Determination of Volatile Organic Compounds in Polypropylene Raw
Materials by Thermal Desorption-Gas Chromatography/Mass
Spectrometry and Head Space-Gas Chromatography-Mass/Spectrometry

Shuqi Sun, Ying Zhang, Song Chen, Yang SongSINOPEC Beijing Research Institute of Chemical Industry, Beijing, China

Keywords:
VOCs, HS-GC/MS, TD-GC/MS
Novel aspects:
The TD-GC/MS methods was used in the determination of volatile compounds emitted from polypropylene（PP）raw materials,
and campaired with HS-GC/MS method.
Abstract:
Thermal desorption-gas chromatography/mass spectrometry （TD-GC/MS） and head space-gas chromatography-mass/
spectrometry（HS-GC/MS）had been applied to the determination of volatile compounds emitted from polypropylene（PP）raw
materials.
During the TD analysis, 0.30g of sample was heated in a stainless steel tube at 90&#8451； for 3 min, the emitted volatile
substances was cooled with the help of a helium gas stream in the cold trap. After completion of the baking phase, thoseabsorbed in the cold trap are rapidly heated from -10&#8451； to 280&#8451；. The target compounds were evaporated and thenisolated in the gas chromatographic separating column and detected by the mass spectrometer. Chromatographic separation was
carried out on an HP- 5 MS column（30m×0.25μm×0.25mm）with a temperature program, and the mass spectrometric detection
was operated by a quadrupole mass spectrometer with an electron impact ion source. The mass spectrums of eachchromatograph peak obtained from the TD-GC/MS method were searched in the NIST Mass Spectral Library to obtain thequalitative results.
And the HS technique had been carried out at 120&#8451； for 5 hours, 4.50g PP raw materials were sealed in a 20mL HS glass
bottle which was heated in the head space sampler. The head space gas which composed of air and the volatile matter were
injected by the headspace sampler into the inlet of the gas chromatography. The GC/MS experimental conditions were the sameas described above.
A series of PP raw materials were detected by both of HS-GC/MS and TD-GC/MS analysis methods. The research results showedthat the volatile organic compounds（VOCs）released from the PP raw materials were composed of alkanes, alkenes and oxygen
compounds with low molecular mass. Compared with HS method, the results of TD technology attained a large number ofchromatographic peaks from which indicated that more substances would be determinate and showing a wide application
prospect in the determination of the VOCs of the PP raw materials.

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Polymer sciences

PTu-190

13:30 － 14:40

Characterization of a multi-component liquid crystalline copolyester byhigh-resolution MALDI-spiral-TOF-MS

Banri Hashimoto, Takanori Sasaki, Hajime Ohtani
Nagoya Institute of Technology,Aichi,Japan

Keywords:
charactrization, MALDI-spiral-TOF-MS, LCP, copolymer
Novel aspects:
high resolution MS study to characterize copolymer systems
Abstract:[INTRODUCTION]
In recent years matrix-assisted laser desorption/ionization time of flight mass spectrometry（MALDI-TOF-MS）has been widely
utilized in the field of characterization of synthetic polymers. In the case of copolymer samples, however, their structuralcharacterization is generally difficult by ordinary MALDI-TOF-MS mainly because of its insufficient resolution for complex
composition and chemical structures of the copolymers. In this work, by using recently developed high-resolution MALDI-spiralTOF-MS, a liquid crystalline terpolyester（LCP）composed of p-hydroxybenzonic acid（HBA）, isophthalic acid（IA）, andp-aminophenol（PAP）was characterized in detail.
[EXPERIMENTAL]
The LCP sample was dissolved in hexafluoro- 2 -propanol（ 5 mg/mL）to prepare the sample solution. An aliquot（ 1 mL）of thesample solution was mixed with 10 mL of the matrix solution（1,8,9-trihydroxyanthracene 20 mg/mL in THF）. 1 mL solution of
cationizing reagent（NaI, 10 mg/mL in THF）, and then 1 mL of the sample/matrix mixture solutions were stepwise spotted on thesample plate. High-resolution MALDI mass spectra of the LCP sample were acquired in spiral mode using a JEOL/JMS-S3000 timeof flight mass spectrometer.
[RESULTS AND DISCUSSION]
In the MALDI mass spectrum of the LCP sample, several series of peaks were observed with the interval of m/z 120 whichcorresponds to the molar mass of a HBA unit. Moreover, in an expanded mass spectrum, isotopic peaks of a component in the
LCP sample were clearly separated. For example, a group of isotopic peaks was observed from m/z 1040 to 1044. Since measured
accurate mass of m/z 1040 peak is 1040.2220, this component was identified to be a monoisotopic ion of HOOC-C 6 H 4 O-（IA）3 （PAP）3-（HBA）-COCH3（calculated exact mass, m/z 1040.2273）. Meanwhile, the relative intensities of the isotopic peaks（m/z1041-1044）to the monoisotopic ion peak（m/z 1040）observed in the MALDI mss spectrum was significantly larger than thecalculated intensities based on the theoretical isotopic pattern. This fact suggests that the peaks larger than m/z 1041 were not
only composed of the isotopic ions for m/z 1040 monoisotopic ion but also overlapped with other monoisotopic ions. From
accurate molar mass of each peak, corresponding monoisotopic ions were then identified to be a HOOC-C6H4O-（IA）2-（PAP）2（HBA）3-COCH3（m/z 1041）, and HOOC-C6H4O-（IA）-（PAP）-（HBA）5-COCH3（m/z 1042）. Moreover, by comparison between
measured and theoretical intensities for the corresponding isotope clusters, relative abundance of each monoisotopic ion in theobserved peak was estimated to be 92.9% in m/z 1040, 17.0% in m/z 1041, and 19.7% in m/z 1042 respectively. Basically in a similarmanner, various peaks observed in the MALDI mass spectrum were successfully interpreted in terms of peak identification andrelative abundances of the LCP components.

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Polymer sciences

PTu-191

11:10 － 12:20

Mass-analysis of enzymatic hydrolysis of nylon by argon cluster SIMS

Kensuke Iuchi, Kosuke Moritani, Kozo Mochiji, Keisuke Nagai, Kazuki Iida, Seiji Negoro
University of Hyogo, Hyogo, Japan

Keywords:
Ar cluster SIMS, cluster ion, enzymatic hydrolysis, nylon
Novel aspects:
This is the first attempt to apply SIMS to evaluation of hydrolysis of nylon.
Abstract:
In the secondary ion mass spectrometry（SIMS）of organic substances, the molecular weight of the intact ions currently detectable
is at best only as high as 1000, which for all practical purposes prevents the technique from being applied to organic materials ofhigher mass. We have developed SIMS equipment in which the primary ions were argon（Ar）cluster ions. Ar cluster ions consistof hundreds to thousands of Ar atoms aggregated by Van der Waals forces. The average kinetic energy per atom in the cluster
ion is equal to the energy used to accelerate the cluster ion divided by the number of atoms in the cluster ion. For example, whena cluster of 1000 atoms is accelerated to 10 keV, the average kinetic energy per atom of the cluster ion is only 10 eV. Thus a large
cluster ion can generate many low-energy constituent atoms in a collision with the surface. The intensity of fragment ions ofseveral organic molecules was decreased by a factor of 102 when the kinetic energy per atom was decreased below 5 eV.
Moreover, intact ions of insulin（molecular weight： 5,808）and cytochrome C（molecular weight： 12,327）were successfully
detected without using any matrix［ 1 ］. These results indicate that fragmentation can be substantially suppressed without
sacrificing the sputter yield of intact ions when the kinetic energy per atom is decreased to the level of the target’s dissociationenergy.
We applied the Ar cluster SIMS to mass-analysis of the enzymatic hydrolysis of nylon. Nylon are synthetic polymers that containrecurring amide groups（R-CO-NH-R’）as integral parts of their main polymer chains. The high strength, elasticity, abrasion
resistance, chemical resistance, and shape-holding characteristics of nylons over wide temperature ranges make these polymers
suitable for the production of fibers and plastics. On the other hand, methods to recycle nylons or decrease the waste of them at
low cost are required. Biodegradation of nylon is a strong candidate from environmental view point. We have investigated the
enzymatic hydrolysis of nylon［ 2 ］. The enzyme studied here is 6 -aminohexanoate oligomer hydrolase（NylC）which is extracted
from bacteria. This enzyme is a member of the N-terminal nucleophile hydrolase superfamily that is responsible for the
degradation of the nylon- 6 industry byproduct. We prepared thin film of nylon oligomer, ACO（ 6 -aminohexanoate-cyclicoligomer）on silicon substrate. Enzyme reactions were performed by dipping the nylon samples in the enzyme solution at
60&#8451； for 2 h. Secondary ion mass spectra of the sample before and after enzyme reaction were measured. As a result, theintensity of several components of the nylon oligomer was significantly decreased by the enzyme reaction. The experiments using
more highly polymerized nylon are in progress. We propose that NylC should be designated as a nylon hydrolase and Ar cluster
SIMS is promising tool to evaluate the enzymatic degradation of nylon.
［ 1 ］K. Mochiji, et al., Rapid Commun. Mass Spectrom. 23, 648（2009）.
［ 2 ］S. Negoro, et al., J. Biol. Chem., 287, 5079（2012）

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Polymer sciences

PTu-192

13:30 － 14:40

Characterization of poly(ethylene oxide-propylene oxide) alkyl ether(R-EO-PO) copolymer by LC-ESI-MS

Haruka Yamada1, Hirotaka Hisatomi1, Mendra Ritonga2, Sana Ito2, Masaki Morita2, Hideya Kawasaki1,
Ryuichi Arakawa1
1

Kansai University, Suita, Japan, 2Niitaka Co., Ltd., Osaka, Japan

Keywords:
poly（EO-PO）alkyl ether（R-EO-PO）, EO/PO copolymer, nonionic surfactant, structural analysis, LC-ESI-MS
Novel aspects:
The numbers of carbons in the alkyl chain R, EO and PO units for R-EO-PO copolymers were unambiguously determined by LC-ESIMS.
Abstract:
Introduction
Mass spectrometry（MS）has become increasingly important in the characterization of synthetic polymers. For the MS analysis of
the polymers, different ionization methods have been used, especially matrix-assisted laser desorption/ionization（MALDI）and
electrospray ionization（ESI）. The both methods are well suited for the determination of the number of repeat units, end groups,
linear or cyclic structure, and molecular weight distribution of homopolymers. However, for the MS analysis of copolymers, it ismuch more difficult to obtain the structural information, because their mass spectra are extremely complicated. Recently, a few
LC/MS applications with ESI or APCI ionization have been reported concerning the characterization of copolymers. It is expectedthat mass spectra of copolymers are simplified by LC separation. In this study, poly（ethylene oxide-propylene oxide）alkyl ether（R-EO-PO）was characterized in detail using poly（ethylene oxide）alkyl ether（R-EO）as a reference of LC-MS data by LC-ESI-MS.
Methods
The LC-MS data were obtained with an Exactive orbitrap mass spectrometer and Accela LC system（Thermo Fisher Scientific Inc.）
with a column of Inertsil ODS- 3 3 um 2.1×150mm（GL Science）. The best LC separation for the R-EO and R-EO-PO polymers wasachieved using a gradient method with 5 mM ammonium acetate aqueous solution and acetonitrile/THF solution（ 1 /1, v/v）.
Preliminary data
The LC-MS data plot was shown with the column retention times as x-axis, the m/z values as y-axis and the ion intensities
adjusted with the color density as z-axis. The data plot for the R-EO-PO copolymer exhibited clear distributions of the peaks withm/z differences of 44 and 58（singly charged）along the y-axis attributed to the masses of EO（44 Da）and PO（58 Da）. The
distributions along the retention time（x-axis）originate from alkyl chains（R）and PO chains with an ODS column； the longer alkyl
chains（R）and the PO chains, the more hydrophobic the copolymer and the longer the retention times become. The hydrophobic
property increases in the order of EO<PO<R. In other words, the R-EO-PO copolymer was separated based on the number ofcarbons in the alkyl chain, EO and PO units. However, the mass difference 14 Da between PO and EO is equal to CH2 repeating
unit in an alkyl chain R, so that the same m/z isomers with different combinations of CH2（R）, EO and PO units are considered.
These isomers cannot be distinguished from the m/z values. To solve the problem, the R-EO polymer without PO was used as areference for the LC-MS data plot. The numbers of carbons in R and EO units for the R-EO polymer were unambiguously
determined from the m/z values. Therefore, comparing the LC data plot of the reference R-EO with that of R-EO-PO, we could
successfully determine the numbers of carbons, EO and PO for the R-EO-PO copolymers using LC-ESI-MS.

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Polymer sciences

PTu-193

11:10 － 12:20

Novel Method for Verification of Trace Amounts of Polymer Contaminantsin Photoresist Solution

Min-Soo Suh, Da-Hee Lee, Taehyo Choi, Minjung Kim, Dongju Oh, Jung Dae Park, Pilkwon Jun,
Jong Soo Kim
Samsung Electronics, Hwasung, Korea

Keywords:
Polymer impurity analysis, photoresist, Headspace gas chromatography mass spectrometry（HS-GC/MS）, Matrix-assisted laser
desorption/ionization time-of-flight mass spectrometry（MALDI-TOF MS）
Novel aspects:
Rapid and reliable strategy to identify and quantify the polymer impurity in photoresist solution by using two types of mass
spectrometry
Abstract:
The levels of organic and inorganic impurities are controlled to minimize the defect and maximize the manufacturing yield insemiconductor business, because even a few ppb-level impurities generate various defects. Hence, an adulteration of polymer
solution occurred by human or instrumental errors must be a severe problem. The verification of the origin of a trouble is the key
for preventing a recurrence of the mixing problem.
In this study, headspace gas-chromatography mass spectrometry（HS-GC/MS）and matrix-assisted laser desorption/ionization
time-of-flight（MALDI-TOF）MS were used to identify the source of polymer impurities. Although GC/MS is not compatible withhigh-mass organics such as polymers, HS-GC/MS was employed to identify the solvents of polymer without damages on GC
column and narrow down the candidates of impurity. Considering the ratio of contents and the molecular weights of polymer andsolvents, the number of moles of solvent molecule was much larger than that of polymer, which helped the qualitative andquantitative analysis of trace amounts of contaminants. The solvent contaminant was identified with the scan mode of HS-GC/MS
and quantified by monitoring the selected ions（m/z 59）. To convince the identification results at a polymer level, thecontaminated polymer solution was measured by MALDI-TOF MS using the optimal analysis condition for the impurity candidate
determined by HS-GC/MS. Based on the HS-GC/MS and MALDI-TOF MS results, the route of contamination were determined andblocked, which resulted in disappearance of the bridge-type defects.
The combination of solvent analyzer（HS-GC/MS）and polymer analyzer（MALDI-TOF MS）gives a rapid and reliable solution for
the identification and quantification of the ppm-level polymer contaminants.

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Polymer sciences

PTu-194

13:30 － 14:40

Differential Analysis in sulfenamide-based vulcanizing accelerators forrubber products by High mass Accuracy MS and Multivariate Statistical
Technique

Takahiro Goda, Hiroki Nakajima, Satoshi Yamaki, Tsutomu Nishine, Masaru Furuta, Naoki Hamada
Shimadzu Co., Kyoto, Japan

Keywords:
vulcanizing accelerator, Differential Analysis, high mass accuracy MS, multivariate statistical technique, searching out structural
analogues
Novel aspects:
The structures of impurities which were structural analogues of main compounds in vulcanizing accelerator reagent were identified
by high mass accuracy MS and multivariate statistical technique.
Abstract:
Vulcanization is a cross-linking reaction for forming bridges between individual polymer chains via addition of sulfur. The purposeof vulcanization is to convert rubber into more durable materials. In general, a reaction rate of vulcanization is increased by adding
vulcanizing accelerator to mixture of rubber and sulfur. Each tire manufacturer employs particular one from among a lot ofcommercially available reagents by their own evaluation criteria. Therefore, analyzing them is a crucial in the development of tires.
However, it is difficult to detect differences in the reagents which have the same main compounds produced by different
manufacturers. Here, we show the differential analysis of sulfenamide-based vulcanizing accelerators which have the same main
compounds produced by different manufacturers using high mass accuracy MSn and multivariate statistical technique.
Five N-（tert-butyl）- 2 -benzothiazole sulfenamide（NS； NS-1, NS-2, NS-3, NS-4, NS- 5 ）and five N-cyclohexyl- 2 -benzothiazole
sulfenamide（CZ； CZ-1, CZ-2, CZ-3, CZ-4, CZ- 5 ）were used in this study. Each NS and each CZ were produced by different
manufacturers. Sample solutions were prepared at 100 mg/L with tetrahydrofuran and acetonitrile. Equal amount of NS solutions
were mixed and it was used as a quality control（QC）sample for NS analysis to identify the robustness and reproducibility of theanalytical data. The QC sample for CZ analysis also was prepared by the same method. LCMS measurement was conducted using
LCMS-IT-TOF（high performance liquid chromatograph / ion trap time-of-flight mass spectrometer, Shimadzu Corporation）.
SIMCA-P+ software（Umetrics）was used for multivariate statistical analysis. MetID Solution software（Shimadzu Corporation）was
used for searching out structural analogues using MSn data acquired by LCMS-IT-TOF measurement. Formula Predictor software（Shimadzu Corporation）was used for predicting the molecular formulae of the statistical analogues.
As a result of principal component analysis（PCA）for NS, the groups of each sample were located at the different sites on thescore plot. And, the unique peaks of each sample were observed on the loading plot. Then, the candidates of structural analoguesof NS were searched out from among those unique peaks based on the similarities of fragment ions and neutral losses calculated
by partial least square（PLS）. The extracted ion chromatograms（EICs）of the structural analogues suggested that they werecharacteristic compounds of each sample. The molecular formulae of them were predicted using the accurate mass and isotopic
ratio. We also determined the validity of the structures of these compounds using MSn measurement.
For example, a unique peak of m/z 253 in NS- 2 was observed on the loading plot. It was cleared to be a structural analogue of NS
by the similarities of fragment ions and neutral losses. The molecular formula was predicted as C12 H 16 N 2 S 2 . The same neutral
losses as NS were detected, and product ion showing the addition of -CH2 to the phenyl group was observed in MS4 data of thiscompound. These results suggested that this compound had the structure of the addition of -CH2 to the phenyl group of NS. The
structures of other compounds also were identified by the same procedure. These compounds are considered as the impurity or
by-product in the process of synthesis. In the analysis of CZ, we also could determine the structures of impurities or by-productsin the process of synthesis by the same procedure.
It was confirmed that the kind of impurities in each sulfonamide-based vulcanizing accelerator differed from manufacture to
manufacture.

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Polymer sciences

PTu-195

11:10 － 12:20

Diagnostics of degradation levels for thermoplastics using pyrolysisGCxGC-TOFMS

Fumie Kabashima, Fumihiko Tsuchiya
LECO Japan Corporation

Keywords:
GCxGC, TOF-MS, Pyrolysis-GC/MS, Highpolymers, Insulating materials
Novel aspects:
TSUGE Shin, Nagoya University OHTANI Hajime, Nagoya Institute of Technology WATANABE Chuichi, Frontier Laboratories Ltd.
,Pyrolysis-GC/MS Data Book of Synthetic Polymers
Abstract:
Abstract
At present, cable ties are used to bundle wires and cables, such as those used in electric devices. When these cable ties are used
outdoors, the cable ties gradually become degraded due to exposure to ultraviolet rays, for example. In order to evaluate thedegradation level of insulating materials, there are physical tests, such as the loop tensile test. However, there are several
problems with this method； significant variance of the test results, the need to use a lot of samples, and the test itself is acumbersome and time-consuming task. Therefore we employed pyrolysis-GCxGC-TOFMS as a new method for easily andaccurately assessing degradation levels with a small sample. This system separates and identifies the components in the sample
according to the separation characteristics of the two columns enables the separation of the components, which could not be
separated by using a normal GC system, on the 2 nd dimensional chromatograms.
Method and experimental
Three types of cable ties made of polyamide resin were used as test samples； 2 kind of PA66 and PA11. The artificially degradedsamples were made by irradiating ultraviolet rays onto each sample. For the weather resistance test, the Eye Super UV Tester
manufactured by Iwasaki Electric Co., Ltd. was used, and the ultraviolet radiation exposure time was set to 0, 50, and 100 hours
respectively. The irradiation time of 100 hours corresponds to the outdoor exposure time in one year or more.
Using 2 nd dimensional chromatograms of the polyamide resin cable ties, it was confirmed that there were peaks for which theintensities increased as the ultraviolet radiation exposure time increased and peaks for which the intensity was unchanged,
without being affected by the degradation. The peak-area ratio was calculated by dividing peak area of the degraded peaks by
the standard peaks. A master curve was obtained by plotting the peak-area ratios and ultraviolet radiation exposure time.
Separately, the service life was determined by performing a loop tensile strength test on the samples artificially degraded byirradiating ultraviolet radiation. The end of the service life was defined as the point at which the tensile strength has been reduced
to the one half of the initial value. By creating a master curve, which shows the relationship between the results of pyrolysisGCxGC-TOFMS and the loop tensile strength test, it became possible to assess the degradation level of the cable ties made of
the polyamide resin samples evaluated in this study. Therefore, using a master curve and calculating the peak-area ratio of theinsulating material actually being used, it is expected that the degradation level and the residual life can be accurately determined.
Conclusion
Using this method which enables the correct peak identification, it is possible to quickly evaluate the degradation level withultralow volume sample. It is expected that this method can also be applied to quality control and the investigation of causes fordefective products released to the market. The application of this method for the assessment of the degradation level of insulating
materials described in this report is not limited to polyamide resins, but can also be applied to various insulating materials.
Moreover, we can be replaced the loop tensile strength test to the flexural strength test or mass-decrease rate.

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Others

PTu-196

13:30 － 14:40

Yellow Sand and Seawater Interaction from viewpoint of Silica Speciation

Miho Tanaka1, Kazuya Takahashi2
1Tokyo University of Marine Science and Technology, Tokyo, Japan, 2RIKEN, Wako, Japan

Keywords:
Yellow sand, FAB-MS, silica speciation
Novel aspects:
From our measurement by FAB-MS, it becomes clear that the dimer and linear tetramer are produced at the surface of seawater,when yellow sand falls into seawater, .
Abstract:
1. Yellow sand is a kind of loess and distributed in N. W. China. It is carried eastward by prevailing winds and passes over China,
Korea, and Japan. Sulfur, carbon monoxide, and other toxic pollutants including heavy metals（such as mercury, cadmium,
chromium, arsenic）often accompany the dust storm, as well as viruses, bacteria, and combustion products. The dust is known tocause a variety of health problems. In Japan, large amounts of yellow sand is mainly transferred in spring. If yellow sand does notcontain toxins, it could be a good supplier of silicic acid（silica）to the Sea of Japan, especially in spring. Silica species are
nutrients for diatoms in seawater. During spring, silica tends to be lacking due to“blooming“ in some regions. The authors havestudied dissolution states in aquatic phases, particularly seawater. In this study, the behavior of silica species in yellow sand withmodel solution of seawater（sodium chloride（NaCl）solution）was examined under several conditions, and the role of yellow
sand is considered.
2. Sample of yellow sand was obtained at Hotan in N. W. China by 200 mesh sieve. A portion of yellow sand（0.2g）was shaken
with 20 mL of several kinds of sodium chloride at 25oC for several days. The silica concentration was determined using molybdic
yellow methods of spectrophotometrically. The chemical species of silica was identified by FAB-MS spectrometry（JEOL JMS 700）
（negative ion detection mode）. The conditions for mass spectrometry were as follows： for FAB（Xe, 1 mA emission）, theresolving power was m/m=1000 and mass range was 0 -1000.
3. pH dependenceThe pH of solution was changed from 0.33 to 14.1 and shaken for 7 days. A change in pH from 0.52 to 14.2 ofsolution containing yellow sand with a 0.5 mol L-1 sodium chloride solution was attained. However, the pH before shaking was
between 3.2 and 9.0 and remained within 8.1-8.2 after shaking for 7 days. The silica concentration in NaCl was almost a constant
30 μmol L-1. When the pH of the solution was 0.5, 12.4, and 14.2, silica concentration was 1320, 158, and 81μmol L-1, respectively.
When the yellow sand was poured into a NaCl solution, pH equilibrium was attained and NaCl solutions with various pH, were
almost constant. In this pH range, the silica species in solution were observed as［monomer-Na + complex］- ,［monomer-Ca +complex］- , dimer, cyclic tetramer and linear tetramer. When pH of the solution was 0.5, peak intensity of silica species was
extremely low and the same species were observed. When pH solutions were 12.4, and 14.2, the silica species were clearlyobserved. The silica concentration in solution with pH 0.5 was high, but silica species not clearly observed. It is considered thatsilica could be dissolved as particles in the low pH solution but it could be dissolved as ions in high pH.
Time dependence When yellow sand was dissolved for 0, 1, 3 7, and 14 days, the silica concentration increased. For 1 day, dimer
and linear tetramer were observed compared to cyclic tetramer. But for 3 days and more, dimer and cyclic tetramer increased
compared to linear tetramer. When the solution did not attain equilibrium for 1 day, yellow sand dissolved as linear tetramer first,
then changed to cyclic tetramer. According to our previous studies, it has become clear that“food“ of diatoms was dimer andlinear tetramer. When yellow sand falls into seawater, the dimer and linear tetramer would be produced immediately, at thesurface of seawater.

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Others

PTu-197

11:10 － 12:20

High Plasmalogen Content in the Gut of Silkworm (Bombyx mori)Takako Aboshi, Ritsuo Nishida, Naoki Mori
Kyoto University

Keywords:
plasmalogen, LCMS-IT-TOF, phospholipid
Novel aspects:
We found the high plasmalogen content in the gut of silkworm.
Abstract:
Phospholipids are a major component of membrane and exhibit great diversity in the structure of the apolar and the polar
moieties of the lipid molecules. They are distributed unevenly in the tissues and display characteristic phospholipid compositions.
Phospholipids perform various functions in the cells and tissues of the body such as natural surfactants, signaling molecules, andantioxidants. The different pattern can be regarded as an adaptation to environment. In this study, we analyzed phospholipid
composition in tissues of silkworm, Bombyx mori by TLC and LMS-IT-TOF. As a result, we found the ratio of plasmalogen
phosphatidylethanolamines to phospholipids was higher in the gut compared to that in other tissues. Plasmalogens are uniquesubclass of glycerophospholipids characterized by the presence of a vinyl ether at the sn- 1 position of the glycerol backbone,whereas usual glycerophospholipids have an ester bond at the same position. Plasmalogens are reported to inhibit the metal ion
induced oxidation. Midgut tissues suffer from oxidative stress associated with phenolic oxidation in midgut lumen whencaterpillars ingest phenolics, known as defense chemicals in plants. Plasmalogens may protect gut tissues against the oxidative
stress.

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Others

PTu-198

13:30 － 14:40

Characterization on Oxidation and Hydrolysis of Titanium Ion Using ESIMSIo Ryumae, Miho Tanaka

Tokyo University of Marine Science and Technology

Keywords:
ESI-MS, titanium identification, dissolution species
Novel aspects:
As identification of dissolution species, oxidation and hydrolysis process of intermediate products of Ti was succeeded by ESI-MS.
Abstract:
Introduction
ESI-MS（Electrospray ionization mass spectrometry）exhibits a soft ionization technique and can deliver ions in solution to gasphase. Therefore ESI-MS gives information on the dissolution state of metals at molecular level. It is possible to observe chemicalchange, even if change occurs instantaneously. On the other hand, titanium（Ti）ions in solution exists as Ti（III）and Ti（IV）. Ti（III）
is stable in chloride ion（Cl-）solutions of pH 1 - 2 and Ti（IV）causes precipitation gradually when it is exposed to oxygen in air.
For this reason, ESI-MS is an effective technique to measure elements which show similar reactions as Ti ion. Previous studies with
ESR and potentiometric titration, the electronic state and dissolution states based on theoretical calculations of Ti ion wereobserved.
In this study, identification of dissolution species, oxidation and hydrolysis process of Ti with ESI-MS is carried out.
Experimental
ESI-MS（Shimadzu LCMS-2010A）was employed for measurements. The spray voltage was set at +4.5kV or -3.5kV. Ultrapure water（18.2MΩ）was used as the mobile phase. Titanium tri-chloride（TiCl3）, titanium sulfate（Ti（SO4）2）used were of chemical gradefrom Wako pure chemical industries, Ltd. Hydrogen peroxide（H2O2）used was ultrapure grade from Kanto chemical co Ltd.
To aliquots of 0.1, 1, 3, 5, 10 and 15 mL of TiCl 3 , were added 50 mL of H 2 O 2 and Ti（SO 4 ）2 solutions. Oxidation and hydrolysis
process of Ti（III）using 5 mL TiCl3 by ESI-MS was observed.
Results and Discussion［ 1 ］Speciation of titanium（III）and titanium（IV）
In chloride（Cl-）and sulfate ion（SO4 2-）solutions（higher than 1 mM）, the observed mass spectra were examined for the singly
charged hydrolyzed species of Ti（III）and Ti（IV）in positive ion mode.
For Ti（III）solution, the peaks at m/z 154 and 172 proved the existence of［TiCl（OH）（H2O）n］+. As there have been no reports oftitanium species with Cl- ligand in positive ion mode, it is necessary to discuss it. Moreover, the existence of the dimer identified
as a mixed-valancy species of Ti（III）and Ti（IV）is already reported by ESR. ESI-MS gives information on the intermediate
products during the oxidation of Ti（III）to Ti（IV）.
For Ti（IV）solution, previous studies using potentiometric titration had carried out for doubly charged species like TiO2+ and Ti（OH）2 2+. However, they were not observed by ESI-MS. This indicates that electrochemical reduction occurred due to high voltage
in ESI nebulization, which may have changed the dissolution state of titanium species.
［ 2 ］Behavior of oxidation and hydrolysis in Ti（III）
For TiCl3 solution, peaks of Ti（III）and Ti（IV）were observed and their peak intensities decreased with increasing time. The peakintensity of Ti（III）decreased more rapidly than that of Ti（IV）. Furthermore, it was observed that the time-dependent peakintensities of Ti（III）/ Ti（IV）dropped. Therefore, it was considered that Ti（III）was oxidized to Ti（IV）in solution and Ti（IV）
which was hydrolyzed and not present in its ionic form, was rapidly excluded from the solution.
The behavior of oxidation and hydrolysis of Ti（III）could be examined by ESI-MS. On the other hand, there is a possibility of the
dissolution state changing during measurements when using ESI-MS. In this presentation, we expect to examine the complete
conversion of Ti（III）to Ti（IV）in solution.

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Others

PTu-199

11:10 － 12:20

The Development of Multi Elemental Analysis of Ferromanganese Nodule
by LA-ICP-MS

Junichi Hirata, Miho Tanaka

Tokyo University of Marine Science and Technology, Tokyo, Japan

Keywords:
LA-ICP-MS, Ferromanganese nodule, Standards, Multi elemental analysis
Novel aspects:
Multi elements in ferromanganese nodules were determined by LA-ICP-MS and the data were compared with those of ICP-MS inorder to obtain accurate determination values by LA-ICP-MS.
Abstract:
Introduction
Laser ablation inductively coupled plasma mass spectrometry（LA-ICP-MS）is a useful method for determination of trace elements
distributions. It shows higher sensitivity than EPMA or SEM-EDX which are also employed for similar purposes. Furthermore, it ispossible to observe samples under atmospheric pressure by LA, thereby protecting samples against structural changes. However,the drawbacks of LA-ICP-MS are obtaining. Matched standards as established standards do not exist. Secondly, it is difficult toobtain stable signals and accurate determination values by LA-ICP-MS compared with ICP-MS. The data obtained is more accurate
when subjected to internal standard correction and selection of internal standard for the element of interest is to be carefully
considered paying attention to the features of the sample. Therefore, the purpose of this study is to obtain accurate determination
values by LA-ICP-MS.
Multi elements in ferromanganese nodules, which are one of the notable and important geological samples, were determined by
LA-ICP-MS and the data were compared with those of ICP-MS in this work. Certified reference material（CRM）of ferromanganese
nodule and manganese dioxide（MnO2）were used to prepare matrix matched standards. Magnesium which is one of the major
elements in ferromanganese nodules was selected for internal standard correction.
Sample preparation
JMn 1 which is a CRM of ferromanganese nodule collected in the Pacific Ocean（purchased from AIST）and high purity MnO 2
powder（99.5 % , Wako Chemical Co.）were used for preparation of standards for LA-ICP-MS. JMn 1 and MnO 2 with the weightratios of 0, 25, 50, 75, 100% were mixed. These samples were pressed into φ 5 mm pellets for IR analysis. Ferromanganese nodule
collected in Pacific Ocean near Hawaii（10°10’N, 147°00’W, JOGMEC）was cut across the center, and the obtained powder wascollected and made into pellets using the same procedure as standards. These pellets and powder are mentioned as Hawaii
samples in this paper.
Instrumentation and parameters
UP213（NEW WAVE）, LA instrument and ELEMENT XR（Thermo Fisher）ICP-MS instruments were used. The conditions of LA are
as follows： 213 nm wavelength, 100 µm diameter, 10 Hz repetition rate, 15 µm/sec scan speed, 0.023 mJ pulse energy, and rastersample scan mode. The conditions for ICP-MS were 1.23 L/min sample gas and middle resolution. The elements measured were 11
B, 24Mg, 52Cr, 56Fe, 60Ni, 63Cu, 95Mo, 111Cd, 140Ce, 153Eu, 175Lu, 208Pb, and 238U.
Results and discussion
Calibration curves for LA-ICP-MS measurements were successfully obtained. The square of correlation coefficient values（R2）of
the calibration curves were 0.932~0.984. R2 values of the calibration curves obtained by internal standard correction using 24Mg,
which contained 4 points except for blank, were 0.924~0.999. Good linear calibration curves were obtained with internal standard
corrections. For example, R2 value of 56Fe without correction was 0.978, and correction with 24Mg was 0.991. Calibration curvesof 52Cr and 111Cd were not obtained due to its low ablation efficiency due to their chemical property or low isotopic abundances.
Accuracy is defined as［CLA-ICP-MS］/［CICP-MS］×100 in this study.［CLA-ICP-MS］and［CICP-MS］are the determination values of the Hawaii
sample obtained by LA-ICP-MS and ICP-MS respectively. The accuracy obtained without internal standard correction were in therange 97.4~173%. That with internal standard correction using 24Mg were 80.4~110%. Internal standard correction showed accurate
determination values. For example, the accuracy of 56 Fe without correction was 135 % and with internal standard correction
using 24Mg was 110%. Thus, accurate determination values were obtained by this method.

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Others

PTu-200

13:30 － 14:40

Study on Reaction Process of the Complexation of Silicomolybdic andPhosphomolybdic Acids by ESI-MS

Mariko Takahashi, Miho Tanaka

Tokyo University of Marine Science and Technology, Tokyo, Japan

Keywords:
ESI-MS, Silicomolybdic and phosphomolybdic acids
Novel aspects:
ESI-MS as a useful tool to observe complexation reaction processes of silicomolybdic and phosphomolybdic acids.
Abstract:
Introduction
In order to get information on the reaction process of silicomolybdic and phosphomolybdic acids, ESI-MS exhibits a powerful tool.
X-ray analysis, NMR and other instruments portray information on these acids, but ESI-MS is able to track each reaction step on
addition of reagent. ESI-MS has a soft ionization system and produces multiple-charged ions. Furthermore, any compound may beobserved at the same time, when compounds are ionized in ion source. ESI-MS enables us to elucidate reaction process ofcomplexation of silicomolybdic and phosphomolybdic acids in solution.
Silicomolybdic and phosphomolybdic acids are heteropolymolybdic acids, which are used for the determination of silicic acid andphosphoric acid by absorption spectrophotometry known as molybdate yellow and blue methods. These methods were generally
used for. These acids are composed of one molecule of silicic or phosphoric acid which is surrounded with 12 molecules ofmolybdic acids at low pH values. The complexation reaction process of silicomolybdic and phosphomolybdic acids is complicatedand has not been elucidated. From previous studies, it has been known that the reaction is affected by the reagent concentration,
solution pH, and elapsed time for formation. Therefore, it is necessary to measure not only metal ions but sulfuric acid ion or otherions that affect the complexation in order to examine the complexation reaction process. In this study, we report ESI-MS as a
useful tool to observe complexation reaction processes of silicomolybdic and phosphomolybdic acids.
Experiment
ESI-MS（LCMS-2010A： Shimadzu Ltd.）was used in negative ion mode. The mobile phase was ultra pure water.
Generally, the standard solution for silicic acid is prepared using sodium chloride or sodium hydroxide solution with silicon
dioxide. In measurements with ESI-MS, the standard solution for silicic acid causes a serious problem. When sample solution
contains high concentration of volatile ions with objective ions, the ion suppression of the objective ions occur. To resolve thisproblem, a new method to dissolve silicic acid in pure water was developed. We use reagent for silicic acid which were obtainedby rice nutrient.
Results and Discussion
Silicic or phosphoric acid solution form colored complexes by addition of sulfuric acid and ammonium molybdate solutions. These
complexes were measured by ESI-MS. In the reacted solution derived from silicic acid, the peaks assigned as silicomolybdic acid
were observed, in addition to molybdic acid. Furthermore, peaks of silicic acid were not observed. Therefore, it indicates that allof silicic acid in solution is converted to silicomolybdic acid. In the reacted solution derived from phosphoric acid, the peaks
assigned as phosphomolybdic acid were observed, while main peaks were original molybdic acid. Furthermore, deficientheteropolymolybic ions （PMo 9 species） were also observed. Therefore, an incomplete reaction of phosphoric acid tophosphomolybdic acid is considered. And, PMo9 is regarded as the intermediate of phosphomolybdic acid.
In the molybdate blue method for phosphate, the complexes are prepared by adding antimonyl potassium tartarate in addition
to ammonium molybdate. The solution prepared by this method was measured using ESI-MS. From previous studies,
phosphomolybdic acid was the reduced form of［PSb2Mo10O40］by antimonyl potassium tartarate. But the peaks assigned as thiscomplex were not observed. Nevertheless, the peaks of phosphomolybdic acid were higher than those without antimonyl
potassium tartarate. Therefore, the role of antimony was considered to improve the stability of phosphomolybdic acid.
By ESI-MS and solution improvement, new information on complexation reaction process of silicomolybdic and phosphomolybdic
acids could be obtained in this study.

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Others

PTu-201

11:10 － 12:20

The Development of Analytical Method for Speciation of Transition Metal
Ions in Seawater by CE-ESI-MS

Daisuke Nakamoto, Masashi Wakasugi, Miho Tanaka

Tokyo University of Marine Science and Technology, Tokyo, Japan

Keywords:
CE-MS, transition metals, speciation, seawater
Novel aspects:
For seawater analysis, the separation of trace metal ions from high concentration of NaCl in keeping with the dissolution states ofthe original metal ions by CE-MS was accomplished.
Abstract:
Introduction
It is difficult to determine concentration of transition metals dissolved in seawater. Acquisition of their dissolution states is moredifficult than their determination. In order to characterize transition metal ions in seawater, a new analytical system should be
required. ESI-MS（Electrospray Ionization Mass Spectrometry）is known to be one of the soft ionization methods, and many
reports on the dissolution states have been presented. However, detection of these dissolution states in seawater is difficult aswell as avoiding the“ion-suppression effect“. A high concentration matrix（sodium ion）suppresses detection of trace metal ions.
To resolve this problem, pretreatment is required to separate trace metal ions from high concentrations of matrices while keeping
dissolution states of original metal ions. In this study, capillary electrophoresis（CE）was used as a separation technique, andonline hyphenated CE（-ESI）-MS was developed. The separation method with CE is simple, while it reasonably maintains thedissolution states of objective metals, compared with other separation methods. For CE-MS analysis, volatile backgroundelectrolytes（BGEs）are required to obtain high sensitivity. In this study, non-volatile hydrochloric acid（HCl）was used as the BGE
because volatile BGEs, such as formic or acetic acids, easily coordinate with transition metal ions. Manganese（II）and Cobalt（II）,
which have analogous properties, were measured in high concentrations of NaCl solutions by CE-MS.
Application
CE-MS is composed of online hyphenation of CAPI-3300（Photal）and LC-MS 2010A（Shimadzu）with self-designed tri-axial ESI
interface. Separation voltage（+30kV）was applied by CAPI-3300, and ionization voltage as +4.5kV by LC-MS 2010A. Ultrapure water（18.2MΩ）was used as sheath liquid with 0.01 mL/min flow rate. The uncoated separation capillary（I.D. 70μm, length 110cm）wasused.
Experimantal
All sample reagents（Kanto Chemical Co）used are of special grade. 1 ）Solutions containing 0.1mM of manganese chloride（MnCl2）or cobalt chloride（CoCl2）were measured by ESI-MS. 2 ）Solution containing 5 mM of MnCl2, CoCl2 and 500mM of NaCl
was measured by CE-MS with 1 mM of HCl as BGE. The concentration of 500mM NaCl is almost the same as that of seawater.
Conditions of sample injection to separation capillary were examined（ 5 kPa for 5 ~120 sec）.
Results and Discussion
1) Measuring solutions of MnCl2 and CoCl2 with ESI-MS
When an aliquot of MnCl2 solution was measured by ESI-MS,［Mn（OH）（H2O）0~5］+,［MnCl（H2O）3~4］+, and［Mn（H2O）3~5］2+ wereobserved. It is previously reported that［M（OH）（H2O）］+（M： Metal）is derived from hydrolysis of［M（H2O）2］2+ at liquid-phaseand gas-phase in ESI-MS. When CoCl2 solution was measured,［Co（OH）（H2O）0~5］+,［CoCl（OH）3~4］+ were also observed but［Co（H2O）n］2+ was not observed. Cobalt ion is more easily hydrolyzed than manganese ion. Thus, accurate information on sample
solutions could be obtained by ESI-MS.
2) Measuring solutions of MnCl2 and CoCl2 in high concentration of NaCl with CE-MS
First, amounts of sample solution for the capillary were examined. When injection times of sample solution were 30~60 sec, sharp
and high peaks of objective metal species were observed on the electroferrogram. When samples contained extremely highconcentration of components, stacking of minor component occurred in the CE system（Self-stacking）. When injection time ofsample solution was over 80 sec, cluster ions of NaCl（m/z 81+58n）were also stacked and led to ion-suppression effect. Therefore,
the optimal condition of sample injection was 5 kPa for 30~60 sec. Observed metal species with CE-MS were good accordancewith ESI-MS. Thus, the separation of trace metal ions from high concentration of NaCl in keeping with the dissolution states of theoriginal metal ions by CE-MS was accomplished.

Poster Session
Tuesday, 18th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Others

PTu-202

13:30 － 14:40

Identification of Silk Proteins Excavated from the Ruin of the Makimuku
Site in 3-4 AD Japan by MALDI Mass Spectrometry

Kazuki Kawahara1, Miho Muguruma1, Teruhiko Hashimoto2, Kaoru Terasawa2, Atsuko Miyaji1,Takashi Nakazawa1
1

Nara Women’s University, Nara, Japan, 2Research Center for Makimuku-Gaku, Nara, Japan

Keywords:
MALDI mass spectrometry, Silk fiber, Sericin, Fibroin, Archaeology
Novel aspects:
Mass spectrometry has proved effective to identify silk proteins in archaeological textile materials even if they are heavily
contaminated and degradated.
Abstract:
INTRODUCTION： Very few proteins remain intact even in several days of storage unless some care is taken for preservation. It istherefore extremely difficult to detect a protein supposedly contained in materials of archaeological interest. A few exceptional
proteins include fibroin and sericin constituting silk textile, which represents one of the key materials associated with ancient
culture and civilization, although these proteins usually suffer from severe biological degradation. The species of a silk mothproducing the silk proteins have been identified by optical or electron microscopic observation of characteristic appearance ofthe threads as far as the morphology is preserved in good condition whatever old the specimen might be. Infrared（IR）
spectroscopy can also be used to distinguish between silk proteins and other fibrous materials of plant origin if their molecular
structures are not seriously damaged and the level of contamination is reasonably low. However, it is desirable to identify thesilkworm even when the specimen is available only a very small amount and severely contaminated with other proteins. In thepresent study, we suggest a new methodology based on mass spectrometry and proteomics to identify silk fiber. We applied thismethod on a drawstring pouch recently excavated from the Makimuku site situated in Nara Prefecture, which covers almost theentire capital area of ancient Japan during the last stage of the Yayoi period（about 300 BC to 300 AD）to the early Tumulus period（about 300 AD to 400 AD）.
METHODS： A small fragment（about 1 mg）of drawstring pouch was washed with pure water and triturated manually with asaturated solution of LiSCN to dissolve sericin and fibroin inclusively. The supernatants obtained by centrifugation at 14,000g for 15
min were dialysed five times against 25 mM NH 4 HCO 3 solution for 48 h. As a reference, solutions of sericin and fibroin wereindependently prepared from cocoon shells of silkworm Bombyx mori（B. mori）. These samples were then digested with trypsin
at 37 o C for 24 h and the digests were analyzed by matrix-assisted laser desorption/ionization time-of-flight（MALDI-TOF）mass
spectrometry using CHCA as a matrix.
RESULTS： The MALDI-TOF MS spectra of tryptic digests prepared from the small fragment of drawstring pouch indicate that thematerial is highly contaminated with an overwhelmingly large number of contaminant peaks originated from human keratins,
microbial proteins, and synthetic polymers such as（CH 2 -CH 2 -O）n - characterized by a series of peaks evenly spaced by 44 Da.
Nevertheless, we could identify several peaks with the m/z values identical to those of the corresponding peptides of silk proteins（sericin 1, sericin 3, and fibroin）prepared from cocoon shells of silkworm B. mori. In particular the detection of the peak at m/z
1497（assigned to QSNTNYADQNSVR）excludes the possibility that the peptide originates from the wild-type species B. mandarina.
Note that fibroin was detected, but with some difficulty, along with sericin- 1 and -3, while this protein was missing altogether insilk fibres embedded in the debris of lacquer-coated coffin of 7 th century Japan. This implies that fibroin is much more
susceptible to degradation, probably by bacteria, than sericin for more than 1000 years’ burial in the soil. Such bacteria include
Variovorax paradoxus, which can selectively use fibroin as a sole nutrient for growth（Forlani, G., et al., 2000）. Although extending
the research to the contemporaneous silk textiles together with reference silk fiber other than B. mori and B. mandarina would benecessary, we demonstrated that our methodology shown here will provides a clue to investigating the relationship between thesociological value of silk and sericulture in ancient culture and civilization worldwide.

Oral Session
Wedensday, 19th September

Main Hall

09:00 － 11:00

Session 21: Platform Technology for Metabolomics
Chair:

Yoshiya Oda（Eisai Inc, USA）

S21-0900
09:00 － 09:40[Keynote Lecture] New generation high-field Orbitrap instrumentation in
untargeted metabolic profiling

Annie M Evans, Qiang Liu, Brandi Bridgewater, Matthew Mitchell, Hongping Dai, Corey DeHaven,
Sandy Stewart
Metabolon, Durham, NC, USA

Keywords:
Metabolomics, UPLC/MS, Orbitrap Elite, Q-Exactive
Novel aspects:
The application of the new generation Orbitrap instrumentation, OrbiElite, and Q-Exactive, to untargeted metabolic profiling.
Abstract:
Untargeted metabolic profiling requires identification and quantification of as many metabolites in a biological system as possible,
by which the cellular outcomes of some test perturbation, examples, individual genotype, proteomic variation, and toxin exposure,can be revealed. Due to the diversity of metabolites and dynamic range, untargeted metabolic profiling in a high-throughput
manner remains a challenge. The recent advances in high-resolution MS instrumentation incorporate desirable features, including
fast spectral acquisition rate and high mass accuracy. In this study, the recently introduced Orbitrap Elite and Q-Exactive both
equipped with UPLC are evaluated for high-throughput untargeted metabolic profiling. Extracted and vacuum-dried human plasma
and/or urine samples were reconstituted with a solution of mixed internal standards in metabolic profiling experiments, orreconstituted with serial-diluted standard solutions for examining detection sensitivity and dynamic range. The Orbitrap Elite and
Q-Exactive were equipped with Waters Acquity UPLC. These instruments alternated between MS and data dependent MS/MS
scans. Samples were eluted with a 7 -min gradient from BEH C18 columns（2.1×100mm, 1.7μm）and ionized using electrospray/heated electrospray. Data were processed with Xcalibur and in-house software. The key features of the Orbitrap Elite and
Q-Exactive and the resulting advantages utilized in untargeted metabolic profiling have been characterized and reported below.
The spectral acquisition rate of both the Orbitrap Elite and the Q-Exactive was significantly increased in comparison to its
predecessor, the LTQ Orbitrap. This ensured improved data points across a typical chromatographic peak（ 3 - 8 s at baseline）forthe UPLC system as well as an increase in the number of MS/MS fragmentation spectra obtained. Meanwhile, similar massaccuracy（< 5 ppm）and precision were achieved with Orbitrap Elite, Q-Exactive and LTQ Orbitrap based on the measurement ofinternal standards and endogenous metabolites in human urine samples. The high-resolution and accurate mass capabilities of the
Orbitrap instruments reduced background thus leading to more than a magnitude improvement in lower limit of detection whencompared to a low-resolution mass spectral analysis. A linear response range over 3 - 4 orders of magnitude was demonstrated
for the Orbitrap Elite and the Q-Exactive in our study. The detection reproducibility of the Orbitrap Elite and the Q-Exactive wasrevealed with relative standard deviation（RSD）of peak area for internal standards（<10% in positive mode and <15% in negative）
in pooled plasma and urine, which demonstrated an improvement in RSD when compared to similar low-resolution mass spectral
analysis. The average number of qualified extracted-ion-chromatographic（XIC）peaks detected per urine sample injection
increased nearly 4 fold using the Orbitrap Elite and Q-Exactive. This suggests a potential increase in the number of compounds
detected in metabolic profiling. As demonstrated by the peak integration of N-acetylhistidine in individual urine samples,
quantification of metabolites（thus the statistic output）was also improved i.e. made more accurate, by minimizing the massinterference in XIC.

Oral Session
Wedensday, 19th September

Main Hall

09:00 － 11:00

Session 21: Platform Technology for Metabolomics
Chair:

Yoshiya Oda（Eisai Inc, USA）

S21-0940
09:40 － 10:00

Improving comprehensive analysis for myxobacterial secondary
metabolome mining

Thomas Hoffmann1,2, Daniel Krug1,2, Rolf Mueller1,2Helmholtz Institute for Pharmaceutical Research Saarland (HIPS), Saarbruecken/Germany, 2Pharmaceutical
Biotechnology, Saarland University, Saarbruecken/Germany

1

Keywords:
secondary metabolites, screening, statistics, LC-MS/MS
Novel aspects:
Identification of pharmaceutically relevant compounds using in-house database queries and statistical evaluation of complex
bacterial extracts. MS² precursor selection based on statistics improve finding of new compounds in screening projects.
Abstract:
As stated by many researchers, extracting relevant information from complex data sets is an important bottleneck in（microbial）
metabolomics research. Compared to a focused targeted approach this is even more important in untargeted metabolomics,
aiming for the identification of all compounds produced by a particular bacterial strain. Many of these compounds are part ofprimary metabolism and therefore out of scope when research concentrates on pharmaceutically active secondary metabolites.
Other metabolites may be very common for a certain genus of bacteria and already well known but the large subset ofcompounds that is“really new“ is hard to identify.
By combining targeted and untargeted metabolomics approaches it is possible to narrow down/identify numerous featuresderived from HPLC-high resolution MS/MS measurements of highly complex bacterial extracts. An automated feature finding
algorithm extracted around 3000 5000 features within one HPLC-MS chromatogram. In a first step these features were subjected toa search against an in-house database using accurate mass, isotope pattern fit, and retention time in order to identify alreadyknown compounds. Untargeted metabolite profiling, using statistical methods such as t-test and PCA analysis could identify
features related to the growth of the bacteria by comparing myxobacterial extracts to blank samples（growth medium）. Thesefeatures were added to a scheduled precursor list（SPL）to focus fragmentation experiments to this relevant subset of compounds
within the complex mixture. This enabled a fragmentation of even low abundant features which might have been missed during an
automatic precursor selection without predefined compounds of interest. High resolution full scan MS and MS/MS spectra wereused to identify target metabolites by queries in an in-house database to identify putative derivatives of known compounds basedon a similar fragmentation pattern. The combination of this methodology with genome based metabolome mining approaches
further improves the classification of new non-ribosomally synthesized peptide compounds in an early state of research. The
whole process covers the very first identification of new secondary metabolites up to the elucidation of the structure and thebiosynthetic pathway to end up with final bioactivity studies.

Oral Session
Wedensday, 19th September

Main Hall

09:00 － 11:00

Session 21: Platform Technology for Metabolomics
Chair:

Yoshiya Oda（Eisai Inc, USA）

S21-1000
10:00 － 10:20

Understanding alterations of platelets metabolism during storage by using
UPLC-Q-TOF-MS strategy.

Giuseppe Paglia1,2, Olafur E Sigurjonsson3, Manuela Magnusdottir2, Steinun Thorlacius2,
Sveinn Gudmundsson3, Bernhard O Palsson2, Ines Thiele2

University of Iceland, Reykjavik, Iceland, 2Center for Systems Biology, University of Iceland, Reykjavik, Iceland, 3The
Blood Bank, Landspitali-University Hospital, Reykjavik, Iceland

1

Keywords:
Metabolomics, Platelets, HILIC, UPLC-Q-TOF
Novel aspects:
To date this is the first metabolomics study on platelets, and it provides understanding of metabolic changes occurring during
platelets concentrates storage in transfusion medicine.
Abstract:
Platelets are small anucleate cells having an important role in thrombosis, haemostasis, inflammation, and wound healing. For
many years, it was thought that the majority of platelets proteins were synthesized by megakaryocyte and acquired from themduring their maturation. Recent evidence has shown that de novo protein synthesis occurs in mature platelets. Consequently,
platelets appear to be metabolically more active than previously thought.
Platelets are routinely stored and used in transfusion medicine for clinical purpose. However the storage time is limited（ 5 to 7days）, because of biochemical and structural changes occurring in platelets during the storage, named platelet storage lesions.
Metabolic profiling provides a global picture of the metabolome by maximizing the number of detected metabolites without apriori knowledge of the metabolites present in a sample. The complete set of all metabolites formed by a cell is defined as themetabolome and it comprises the endo-metabolome（all intracellular metabolites）and the exo-metabolome（all the metabolites
secreted into the extracellular fluid）. During cell culture, continuous exchange of metabolites occurs between the extracellularand intracellular environment, as cells take nutrients up and secrete metabolites. This exchange of metabolites between
intracellular and extracellular environment together with the intracellular metabolite profiling provide a complete picture of themetabolism of cells in culture.
In this study we have used an UPLC method（HILIC）coupled with a Q-TOF mass spectrometer（Synapt G2, waters）to study
metabolomic changes occurring in the intracellular and extracellular environment of platelets concentrates during their storage.
Three platelet units, each one derived from 5 blood donors（15 different individuals）, were stored for 10 days at 22 °C undergentle agitation. Samples were collected at day 0, 2, 4, 6, 8 and 10, cells and medium were separated by centrifugation, andextracted with 0.5 mL of methanol：water（ 7 ： 3 ）and 0.5 mL of acetonitrile, respectively.
During storage, platelets concentrates are maintained in a saline solution together with carry over plasma（between 20 to 30%）at
22°C. Carry over plasma provides nutrients to sustain platelets metabolism.
Significant changes of the exo-metbolome composition over the time were found during the analysis. For instance, platelets use as
main fuel glucose and glutamine and secrete large amount of lactate. Moreover, during the storage, platelets take citrate up,
which is present in the platelets storage solution at high concentration as chelating agent. Aconitic acid instead resulted increasedin the supernatant, suggesting secretion of this metabolite.
Accumulation in the supernatant of intermediates of purine metabolism, xanthine and hypoxanthine, was also found in samples.
On the other hand inside the cells, it was found accumulation of citrate, pyruvate and lactate, while level of aconitic acid resulted
depleted over the time. Intermediates of glycolysis seem to be nearly constant over the time.
The changes in the exo- and endo-metabolome composition suggest that storage affects the TCA cycle. The cells continuously
metabolize glucose, and pyruvate is mainly converted in lactate, instead of feeding the TCA cycle, which shows a reduced
metabolic activity.

Oral Session
Wedensday, 19th September

Main Hall

09:00 － 11:00

Session 21: Platform Technology for Metabolomics
Chair:

Yoshiya Oda（Eisai Inc, USA）

S21-1020
10:20 － 10:40

Neuroactive Steroids in Plasma Measured by a Novel Mass Spectrometry
PlatformAndre Kopoyan, Karin M Green, Kristina M Deligiannidis, Scott A Shaffer
University of Massachusetts Medical School, Worcester, MA, USA

Keywords:
steroids, quantitation, electrospray, LC-MS, metabolites
Novel aspects:
A nanoflow LC-MS/MS assay for neuroactive steroids capable of detection limits similar to those found by GC-MS methods.
Abstract:
Neuroactive steroids（NAS）have been implicated in a variety of disorders including epilepsy, multiple sclerosis, neurodegenerative
diseases, traumatic brain injury, depression, schizophrenia, alcohol dependence, pain and anxiety disorders. Several neuroactive
steroids are potent modulators of the GABAA receptor and consequently can alter the excitability of the central nervous system.
Measurements of these analytes are most commonly approached by gas-chromatography mass spectrometry（GC-MS）methods
requiring chemical derivatization to decrease polarity and increase sample volatility. Our motivation was to develop an LC-MS/MS
method to quantify neuroactive steroids that precluded the need for chemical derivatization and minimized the steps involved
with sample preparation. Here we present a novel non-derivatizing LC-MS/MS assay to screen a panel of eight neuroactive
steroids in plasma from women classified either as low risk（LR）or high risk（HR）to postpartum depression（PPD）during theperinatal period. The feasibility study measured plasma concentrations from six cohorts from each group and were measured atthree gestational age time points and 3 - 9 weeks post-partum.
The assay set out to measure plasma concentrations of progesterone, deoxycorticosterone, 5 a-dihydroprogesterone,
5 b-dihydroprogesterone, allopregnanolone, allotetrahydroxycorticosterone, pregnanolone, and pregnenolone, in addition to
GABA（via additional LC-MS/MS assay）. The LOQ for progesterone was determined to be ~200 pg/mL, comparable to the ~100 pg/
mL value commonly reported for GC-MS. Preliminary data suggest that allopregnanolone, a potent modulator of the GABA A
receptor, is elevated for the HR over the LR group： 61.2 versus 13.7 ng/mL for the <36 hours cohort, respectively. For GABA,
increasing GABA levels were positively correlated to gestational age and into the postpartum（p=0.0029）. Further, HR subjects
had an average GABA level 0.34 μg/mL lower than LR subjects（p=0.78）. Results for the NAS pilot study will be presented alongwith a discussion of measuring neuroactive steroids by LC-MS/MS approaches.
Neuroactive steroids were extracted from human plasma and analyzed against a calibration curve prepared from human charcoalstripped plasma over the 200 pg to 0.1 microgram per mL range. Samples and calibrants（300 uL）were both spiked with deuterium
labeled analogs and processed through a two-step procedure using C18 followed by aminopropyl SPE cartridges. Final eluateswere resuspended and injected onto a Waters NanoAcquity UPLC fitted with a 2 cm C18 trap column configured to a 20 cm long（180 um ID）C18 fused silica capillary column with gravity-pulled tip. Neuroactive steroids were eluted using a fast gradientfollowed by an isocratic elution at 95% methanol. Multiple reaction monitoring was performed using a Waters Quattro Premiere
XE operating in the positive ion electrospray mode； data was processed further by QuanLynx.

Oral Session
Wedensday, 19th September

Main Hall

09:00 － 11:00

Session 21: Platform Technology for Metabolomics
Chair:

Yoshiya Oda（Eisai Inc, USA）

S21-1040
10:40 － 11:00

Metabolomics Studies Using a Versatile Microfluidic Platform

Steven A Cohen, Giuseppe Astrarita, Giorgis Isaac, Angela Doneanu, Jay Johnson, Jim Murphy
Waters Corporation, Milford, USA

Keywords:
Metabolomics, microfluidics, lipidomics
Novel aspects:
A microfluidic platform suitable for analyzing small molecules was developed for the analysis of metabolomics and lipidomics
samples. This versatility is unprecedented in a microfluidic LC-MS system.
Abstract:
One of the fastest growing“’omics“ applications today is the broad based screening of small molecules in biological systems
known as metabolomics. Because metabolite studies encompass a diversity of analyte types and a wide dynamic range ofconcentrations, comprehensive analysis challenges both the chromatographic separation and the mass spectrometry.
This presentation will describe novel microfluidic devices optimized for LC-MS analysis of small molecules and applied to theanalysis complex sample mixtures such as those analyzed in metabolomics and lipidomics studies. These devices are fabricatedfrom ceramic materials that permit operation at high pressure with sub 2 micron particles and thus are capable of producing
highly efficient UHPLC operation. By optimizing the channel design, and incorporating an integral mass spectrometer emitter, onboard column heating and flexible column formats, the devices are suitable for analyzing a variety of small molecule samples withperformance comparable to that achieved with analytical scale LC-MS analysis.
Results： Devices with an internal channel diameter of 150 microns permit no-loss direct injection of microliter sample volumes,
with a sensitivity enhancement compared to 2.1 mm ID columns of 20 - 50x. Column efficiency is equivalent to analytical scalechromatography, with peak capacities exceeding 200 within a 10 minute analysis, and greater peak capacities achieved with longer
gradients. Typical samples were plasma, urine and brain, heart or liver tissue extracts, and were screened for metabolites by
reversed phase and HILIC chromatography. Lipids were analyzed from solvent extracted samples, and amines（including aminoacids and biogenic amines）in deproteinized plasma or urine were analyzed after a rapid, simple one step derivatization protocol.
Q-Tof and triple quadrupole mass spectrometers were used for untargeted and targeted analyses, respectively. Comparisons withanalytical scale columns demonstrated equivalent results, with solvent consumption reduced by > 50 x.

Oral Session
Wedensday, 19th SeptemberRoom A

09:00 － 11:00

Session 22: Instrumentation Developments in Mass Spectrometric Imaging
Chair:

Anastassios Giannakopulos（ThermoFisher Scientific, Germany）

S22-0900
09:00 － 09:40[Keynote Lecture] High Resolution in Mass and Space: New Developments
and Trends in MALDI Mass Spectrometry Imaging

Bernhard Spengler, Andreas Römpp, Sabine Guenther, Oliver Schulz, Yvonne Schober, Zoltan Takats,
Klaus-Peter Hinz, Christian Lotze, Joerg-Ulrich Poetzl, Christian Schinz
Justus Liebig University, Giessen, Germany

Keywords:
Mass Spectrometry Imaging, atmospheric pressure, accurate mass, high spatial resolution, biomolecules
Novel aspects:
Mass spectrometry imaging at a high resolution in mass and space for a high validity of topological analyses of animal and planttissue.
Abstract:
MALDI Mass Spectrometry Imaging（MSI）, 18 years after its first announcement［ 1 ］, has turned into a routine method of highest
performance for the molecular histology of biological tissue［ 2 ］. The method, providing high resolution in mass and space, hasbeen developed for a reliable identification and localization of individual tissue components, and has been applied to a number of
research areas in pathology, cancer diagnostics, metabolic pathway analysis and plant research. Targeted compound classes
include phospholipids, peptides, proteins, drug compounds and metabolites.
An atmospheric pressure ion source was developed in our laboratory, based on a dedicated microoptical setup which was
designed for highest optical resolution under mass analytical sampling conditions, providing a spatial resolution on tissue of 3 to
5 micrometers. The ion source was coupled to orbital trapping and ion cyclotron resonance analyzers, at a mass accuracy of
better than 1 ppm RMS. Ion images were acquired with an acquisition speed of more than 2 pixels per second in asynchronousand about 1 pixel per second in synchronous sampling mode.
The presentation will describe technical principles of the new instrumentation in comparison to alternative approaches describedin the literature, including optical focusing, ion formation and transfer at atmospheric pressure, image acquisition and substanceidentification. It will also include examples for biomolecular imaging of phospholipids［ 2 ］, peptides［ 3 ］and drug compounds［ 4 ］
in mammalian, insect and plant tissue at high spatial resolution, confirmed by MS/MS imaging experiments and histologicalstaining of measured samples. Tumor tissues were characterized by their phospholipid patterns, providing structural features
invisible in histological staining experiments. Phospholipids are less specific than proteins, but their pattern can be used to
distinguish different tumor types and also to distinguish primary tumors from their metastases.
Direct detection and identification of proteins on tissue is limited by a number of factors including limited mass range andfragmentation efficiency as well as incompatibility with formalin-fixed samples. On-tissue digestion of proteins and detection of theresulting peptides can overcome some of these limitations. In a current study we used a newly developed spraying device toapply trypsin on tissue sections［ 5 ］. With this setup we were able to obtain a spatial resolution of 50 µm for tryptic peptides.
Combining high spatial resolution down to 3 µm with high mass resolution, high mass accuracy, high imaging selectivity, MS 2
capability and an improved sample preparation technology resulted in a significantly improved quality of MS imaging data in our
experiments. One of the main conclusions from the results is that imaging mass spectrometry of biological tissue in general
requires highly accurate raw data from FT instruments, since low accuracy data were found to contain a high risk of creating
ambiguous or invalid information in imaging experiments of complex biological tissues.
Acknowledgements
Financial support： State of Hesse（LOEWE Research Focus ’AmbiProbe’）, European Union（STREP project COMPUTIS）,
European Reseach Council（ERC Starting Grant Zoltan Takats）, and Deutsche Forschungsgemeinschaft（DFG, Sp314/10-1, 12-1, 131 ）.
［ 1 ］Spengler B, Hubert M, Kaufmann R, MALDI Ion Imaging and Biological Ion Imaging with a new Scanning UV-Laser Microprobe,
Proceedings of the 42nd Annual Conference on Mass Spectrometry and Allied Topics, Chicago, IL, May 29 - June 3, 1994, pp 1041.
［ 2 ］Römpp, A., S. Guenther, Y. Schober, O. Schulz, Z. Takats, W. Kummer, and B. Spengler（2010）Angewandte ChemieInternational Edition. 49：3834-3838.
［ 3 ］Guenther, S., Römpp, A., Kummer, W., Spengler B.（2011）Int. J. Mass Spectrom. 305：228-237.

［ 4 ］Römpp, A., Guenther, S., Takats, Z., Spengler, B.（2011）Analytical and Bioanalytical Chemistry 401：65-73.
［ 5 ］Schober, Y., Guenther, S., Spengler, B., Römpp, A.（2012）Rapid Communications in Mass Spectrometry 26：1141-1146.

Oral Session
Wedensday, 19th SeptemberRoom A

09:00 － 11:00

Session 22: Instrumentation Developments in Mass Spectrometric Imaging
Chair:

Anastassios Giannakopulos（ThermoFisher Scientific, Germany）

S22-0940
09:40 － 10:00

Development and Applications of New Stigmatic Mass Microscope withHigh Mass and Spatial Resolving Power using Multi-Turn Time-of-Flight
Mass Spectrometer

Jun Aoki, Hisanao Hazama, Kunio Awazu, Michisato Toyoda
Osaka University, Osaka, Japan

Keywords:
Imaging mass spectrometry
Novel aspects:
We developed new stigmatic imaging mass spectrometer and achieved mass resolving power of 10000 and spatial resolution of 1
micron. Preliminary data of some applications were also obtained.
Abstract:
Measurement methods of spatial distribution of molecules such as proteins and drugs at cellular-scale are required in many fields
including pathology, pharmacology, etc. Recently, sanning type imaging mass spectrometry（IMS）with matrix-assisted laser
desorption/ionization（MALDI）is intensively used for biomolecular analysis. However, the spatial resolution of sanning MALDI-IMS
is limited by the laser focus diameter to about 10 - 100 μm and inadequate for cellular-scale observation. Therefore, we aredeveloping a stigmatic MALDI imaging mass spectrometer, in which spatial resolution of sub-micron can be achieved irrespectively
to the laser focus diameter. We developed a new imaging mass spectrometor to improve measurement performance.
The experimental apparatus consists of a matrix-assisted laser desorption/ionization（MALDI）ion source, a multi-turn time-offlight mass spectrometer（MULTUM-IMG）and a time and position sensitive delay line detector. The components of the ion opticalsystem were designed and assembled to achieve high accuracy alignment. Ion distributions at the sample plate are magnified andprojected with the ion optical lens system onto the detector. MULTUM-IMG which has four toroidal sector electric fields
constitute a figure-eight trajectory is inserted into the ion flight path. The ion optical system of MULTUM-IMG satisfies the perfectspatial and temporal focusing condition, so that the spatial distributions of ions before entering the multi-turn circuit are conserved
after circulation.
A result of experiment carried out with our prototype apparatus indicate that an alignment accuracy of the ion optical system iscritically important to obtain high quality ion image. Thus we designed new optical system as high rigidity block component to beassembled with high accuracy. Main components of the optical system are fabricated with titanium for high rigidity. We observed
a dried droplet of crystal violet dye solution covered by a metal foil with fine slits of 1 micro-meter width for evaluating
performance of our new apparatus. The slits were formed by fast ion beam etching. As a result, ion image of the sample can beobserved after 4 cycles in MULUM-IMG with appropriate voltage applied to electrodes consistent with the theoretical value. The
flight path length of ions reached 6.2 m. We also evaluated a new ion extraction method optimum for a stigmatic IMS. Ion
distribution is not conserved with ordinary delayed extraction method. By using post extraction differential acceleration（PEDA）
method, time focusing and image conservation can be simultaneously satisfied. Our evaluation experiment demonstrated thatboth the stigmatic observation of ion images with spatial resolution of 1 micro-meter and the mass resolving power of 10000 was
simultaneously achieved.
We applied this new apparatus to several practical applications； i.e. observation of sustance distribution in biological tissue andfine structure of highly-functional organic material. Preliminary data of these experiments are reported in presentation.

Oral Session
Wedensday, 19th SeptemberRoom A

09:00 － 11:00

Session 22: Instrumentation Developments in Mass Spectrometric Imaging
Chair:

Anastassios Giannakopulos（ThermoFisher Scientific, Germany）

S22-1000
10:00 － 10:20

An Evolution of TOF-SIMS for Biological Samples: From 2D Imaging to 3D
FIB-TOF Tomography

John S Hammond, Gregory L Fisher, Scott R Bryan
Physical Electronics, Chanhassen, Minnesota, USA

Keywords:
Imaging Mass Spectrometry, FIB-TOF-SIMS
Novel aspects:
3 D TOF-SIMS Tomography of organic and inorganic materials with chemical and molecular sensitivity, < 0.5 micron spatial
resolution and depth of analysis > 10 microns
Abstract:
TOF-SIMS has emerged as an important tool for 2 D and 3 D imaging mass spectrometry of biological and complex material
specimens due to it’s unique capability to detect molecular and elemental ions at a spatial resolution of < 0.3 μm with a highmass resolution of ~ 15,000 m/Δm without the sample treatments required by e.g. MALDI or fluorescence microscopy. For many
biological and materials specimens, TOF-SIMS also has the capability to image samples having a large degree of surface
topography. The resulting elemental and molecular images provide important information regarding the composition of
biointerfaces, tissues and cells, as well as many complex structures such as solid oxide fuel cells and OLEDs.
An example of the high spatial resolution 2 D chemical imaging capability of TOF-SIMS will be discussed to provide thedifferentiation of epicuticular components present at the surfaces of Arabidopsis thaliana organs. The mass spectra were acquiredwith an analytical sampling depth of ~ 2 nm. Mass spectra acquired from specialized cells forming the stomates and trichomes
reveal that, even for single cells on the surface of an organ, the wax composition may vary.
To extend the TOF-SIMS chemical imaging and molecular characterization deeper than the outer 2 nm of the sample, cluster ion
beams have been developed to remove multiple layers of material between analysis（chemical imaging）cycles. Examples of thisapproach to provide 3 D reconstructions of tissue samples and cells to a depth of 1 µm will be discussed. Nevertheless, there arepractical limitations of the use of ion beam sputtering for probing both organic and inorganic specimens beyond the surface
region. Accumulated ion beam-induced damage may occur and give rise to incorrect chemical distributions. Certain matrix
components may sputter at a different rate than others, called preferential or differential sputtering, which results in a distortion
or complete loss of the true 3 D chemical distribution as a function of sputtered depth. Finally, many specimens contain void
spaces that are impossible to preserve in 3 D images obtained by sputter depth profiling.
An alternative approach to achieve 3 D chemical imaging of chemically complex and porous specimens is to utilize in situ FIB
milling and sectioning in conjunction with TOF-SIMS chemical imaging what we have called FIB-TOF tomography. With FIB milling,
the interior of a specimen is revealed to depths of more than 100 μm. 3 D chemical imaging with a 3 D spatial resolution < 0.5 µm
and with a z-dimension of greater than 10 μm may be achieved within a reasonable analysis time. The advantage of the FIB-TOF
approach is that artifacts caused by sputter depth profiling, such as differential sputtering and accumulated ion beam damage tomatrix molecules, are avoided. Examples of 3 D FIB-TOF tomography will be illustrated and discussed for drug distributions inbiological applications as well as for complex inorganic samples.

Oral Session
Wedensday, 19th SeptemberRoom A

09:00 － 11:00

Session 22: Instrumentation Developments in Mass Spectrometric Imaging
Chair:

Anastassios Giannakopulos（ThermoFisher Scientific, Germany）

S22-1020
10:20 － 10:40

Study of Toxin Distribution in Sprouted Potatoes by Electrospray Laser
Desorption Ionization Imaging Mass Spectrometry (ELDI/IMS)

Yichia Huang1, Min-Zong Huang1, Yi-Tzu Cho2, Jentaie Shiea1

1National Sun Yat-sen University, Kaohsiung, Taiwan, 2Yuh-Ing Junior College of Health Care & Management, Kaohsiung,TaiwanKeywords:
Electrospray laser desorption Ionization, Imaging mass spectrometry, glycoalkaloids
Novel aspects:
Using electrospray laser desorption ionization imaging mass spectrometry to study of toxin distribution in sprouted potatoes.
Abstract:
As an ambient ionization source, electrospray laser desorption ionization mass spectrometry（ELDI/MS）has been demonstrated
to be useful in detecting proteins and small organic compounds on the solid without sample pretreatment. The analyte molecules
were desorbed using a pulse laser then post-ionized in an ESI plume. With the assistance of the precise movement of a stepper
motor and laser desorption for sampling at high spatial resolution, it is possible to obtain the information of predominant chemicalcompounds from a particular sample surface via ELDI/MS, and makes it become a promising analytical tool to perform ambient
imaging analysis on tissues.
When exposing potato tubers under sunlight, it has known that sunlight triggers the production of specific toxin glycoalkaloids.
This is a natural defense mechanism for preventing the uncovered tuber from being consumed by animals. The distribution of
glycoalkaloid toxins on the surface of a sprouted potato was examined in this study via ELDI/MS analysis, and the best
glycoalkaloid ion signals were obtained. The ELDI imaging of potato slices indicated that the majority of toxins was detected in
the cortex and the amount of the toxins decreases with the distance from the potato skin. In addition, more glycoalkaloids weredetected as the potato was exposed to sunlight for a longer time. It was also found that different types of the toxins were
produced at the cortex part of potatoes near the skin and sprouting buds.

Oral Session
Wedensday, 19th SeptemberRoom A

09:00 － 11:00

Session 22: Instrumentation Developments in Mass Spectrometric Imaging
Chair:

Anastassios Giannakopulos（ThermoFisher Scientific, Germany）

S22-1040
10:40 － 11:00

Conventional and stigmatic laser desorption/ionization-imaging mass
spectrometry applied to analyses of organic layers in electronic devices

Yuko Tachibana1, Yoji Nakajima1, Tsuguhide Isemura1, Kiyoshi Yamamoto1, Takaya Satoh2, Jun Aoki3,
Michisato Toyoda3
1Asahi Glass Co., Ltd., Yokohama, Japan, 2JEOL Ltd. , Akishima Tokyo, Japan, 3Osaka University, Osaka, Japan

Keywords:
Imaging mass spectrometry, Laser desorption/ionization, High-resolution time-of-flight mass spectrometry, Stigmatic imaging mass
spectrometry, Electronic device
Novel aspects:
Imaging LDI-TOFMS with high mass resolution was shown to have potintial to be a powerful analytical technique for application toelectronic devices such as OLED.
Abstract:
In recent years, the electronic devices composed of organic and inorganic materials such as organic light-emitting diodes（OLED）
and organic semiconductor devices have been widely used. These organic materials are often easily degradable as comparedwith inorganic materials. Thus, it is important to analyze the degradation area of organic materials in devices and to understandthe degradation mechanism.
There are various techniques for elemental analysis such as electron probe microanalysis（EPMA）, x-ray photoelectron
spectroscopy（XPS）and so on. However, the analytical techniques to obtain the molecular information on organic layers indevices are limited and insufficient, typically, time-of-flight secondary ion mass spectrometry（TOF-SIMS）. In TOF-SIMS, molecularions do not appear because of strong fragmentation in many cases, and the unknown material is not often determined because
the accurate mass is difficult to be acquired. Besides, extremely high surface sensitivity sometimes prevent from getting theinformation of the objective material under surface contamination, because the information of surface contamination constitutethe majority and the desired information of organic layer is obscured.
For analyses of organic layers in electrical devices, we investigated to apply laser desorption/ionization high-resolution time-offlight mass spectrometry（LDI-HRTOFMS）utilizing SpiralTOF-TOF tandem mass spectrometer（SpiralTOF）with the spiral trajectoryion optical system. It might be expected that most of the organic layer materials used in OLED or organic semiconductor devices
are ionized by LDI without or less fragmentation, because they have optical absorption in UV region. In addition, SpiralTOF makes
possible to obtain the accurate mass of materials, and high-energy collision-induced dissociation（CID）provides characteristic
structural information. The information obtained by SpiralTOF and CID can provide us not only molecular weight but also
elemental composition information and characteristic structural information. These information could be clarified the phenomenathat had occurred in organic layers in electrical devices and the degradation mechanism that suggest to solve various problems.
Besides, the ionization depth in LDI is around several tens nanometers, so the information of organic layer is obtained without
disturbing that of surface contamination. For these, it could be expected that LDI-HRTOFMS utilizing SpiralTOF has potential to bea powerful technique to analyze organic layers in electronic devices.
We observed the organic layer materials of OLED on glass are desorped and ionized by laser radiation without matrix materials
and cation agent. The measured OLED panel was prepared by removing the device sealing and aluminum cathode, andsucceeding exposure in air for one year. The organic layer material, N,N’-di- 1 -naphthaleyl-N,N’-diphenyl-1,1’-biphenyl-4,4’-diamine（NPD）, was measured with accurate mass and several tens of thousands of mass resolutions by direct measurement of glass
sample with LDI-HRTOFMS utilizing SpiralTOF.
We also examined imaging mass spectrometry（IMS）on this glass sample with SpiralTOF and obtained a clear image of thedegraded area of NPD layer. The spacial resolution was around 20 micron, similar to that of XPS which is widely used in analysis ofelectrical devices. Furthermore, We also challenged to examine the stigmatic type imaging mass spectrometer using a multi-turn
TOF mass spectrometer ’MULTUM-IMG’ developed by Osaka University group. The spacial resolution was a few micron and therequired time to measure was remarkably reduced.
For these investigations, it can be expected that imaging LDI-HRTOFMS has potential to be a powerful technique to analyze theorganic layers in electronic devices, for clarifying the phenomena that occurred in devices and obtaining solution to resolve
various problems.

Oral Session
Wedensday, 19th SeptemberRoom B-1

09:00 － 11:00

Session 23: Gas Phase Fragmentation Mechanisms of Biomolecular Radicals
Chair:

Shigeo Hayakawa（Osaka Prefecture Uiniversity, Japan）

S23-0900
09:00 － 09:40[Keynote Lecture] Gas Phase Chemistry of Biomolecular Radicals - An
Overview

Michael Leeming1,2, William A Donald1,2, Richard A J O’Hair1

1
The University of Melbourne Victoria 3010 AUSTRALIA, 2ARC Centre of Excellence for Free Radical Chemistry andBiotechnology

Keywords:
radical； amino acids； peptides； nucleic acids； fragmentation
Novel aspects:
Keynote lecture that provides an overview of the gas phase chemistry of biomolecular radicals
Abstract:
The presence of a radical site opens up new types of gas phase chemistries that are not available for even-electron biomolecularions. In this keynote lecture, a brief overview is given on the： ways of forming biomolecule radical ions； types of radical ions thatcan be formed； types of radical directed reactions that can occur； rearrangement of radical sites via intramolecular hydrogen
atom transfer or tautomerization reactions. Amino acids, peptides and nucleic acids will be used to illustrate key concepts using
examples from our and other laboratories.

Oral Session
Wedensday, 19th SeptemberRoom B-1

09:00 － 11:00

Session 23: Gas Phase Fragmentation Mechanisms of Biomolecular Radicals
Chair:

Shigeo Hayakawa（Osaka Prefecture Uiniversity, Japan）

S23-0940
09:40 － 10:00

Mechanism of“Oxygen rearrangement“ in mass spectrometry

Nino G Todua, Karl K Irikura, Stephen E Stein, Anzor I Mikaia
National Institute of Standards and Technology

Keywords:
Oxygen rearrangement, EI mass spectrometry.
Novel aspects:
Mechanism of“oxygen rearrangement“ is presented.
Abstract:
Introduction. Hydrogen migration under electron ionization（EI）was discovered in the late 1940s. Later this process was studied
and explained by Happ [1] and Nicholson [2] and subsequently utilized by McLafferty [3]. However the migration of the oxygen atom
connected to carbon atom under mass spectrometry conditions was not observed or even thought to be possible. Recently [4]carbonyl oxygen migration has been suggested in the EI mass of N-alkyl-N-arylsulfonylbenzamides. The results of study of thisphenomenon will be presented.
Materials and Methods. N-Benzoyl-arylsulfonamides and their 18O-benzoyl-analogs were prepared by reaction of arylsulfonamides
with benzoic acid or 18 O-benzoic acid in pyridine. The EI mass spectra are recorded on GC-MS systems with quadrupole andmagnetic sector analyzers. Exact mass determinations and linked-scan are performed on a double-focusing mass spectrometer.
Theoretical calculations employed the B 3 LYP hybrid density functional, together with 6 -31G（d）basis sets. Vibrational zero-point
energies were scaled by 0.9757. Stationary points were characterized by Hessian analysis, and the reaction mediated by eachtransition structure was confirmed by IRC calculations. The standard uncertainty of the calculated energetics is estimated to be 24
kJ/mol.
Results. Mass spectra of mono-, di- and trimethyl-sulfabenzamides（I a-c）revealed an unexpected base peak at m/z 118（ion a）；
this peak was shifted by 3 Da from 118 to 121 for N-trideuteromethyl-, N,N’-di（trideuteromethyl）- and N,N’,N’-tri（trideuteromethyl）
analogs. The exact mass measurements（118.0657）confirmed the molecular formula for the ion a, and MS-MS spectra indicated theformation of the ion a from M +. and from the radical cation ［M-SO 2 ］+. All the above supported a formation of themethylbenzonitrilium cation ［C 6 H 5 CN（CH 3 ）］+ , which involved a migration of the amidic O-atom. The unsubstituted
sulfabenzamide（II）demonstrates a base peak of para-aminophenol cation radical under electron ionization.
Theoretical calculations investigated possible mechanisms of this phenomenon. An ipso attack of the amide oxygen upon the aryl
sulfonyl moiety had an especially small energy barrier（about 40 kJ/mol for I and 57 kJ/mol for II）. SO2 is expelled, an entropicallyfavorable process, leading to a carboximidate intermediate. For II, the intermediate dissociates to the thermodynamically favored
products： a phenol radical cation（the observed base peak）and benzonitrile. There is a barrier for I, leading instead to kinetic
products of simple bond homolysis： a phenoxyl radical and methylbenzonitrilium ion（the observed base peak）.
The theoretical mechanism was supported by subsequent isotopic experiments. Thus, substituted phenol radical cations in the
mass-specta of N-（arylsulfonyl）benzamides were shifted by 2 Da in the spectra of 18O-benzamide-analogs. It was concluded
that the mechanism involves an ipso attack of the amide oxygen.
Literature［ 1 ］G.P.Happ, D.W.Stewart. J. Amer. Chem. Soc., 1952, 74, 4404-4408［ 2 ］A.J.C.Nicholson. Transactions Faraday Soc., 1954, 50, 1067-1073［ 3 ］G.P.Happ, D.W.Stewart. J. Amer. Chem. Soc., 1952, 74, 4404-4408［ 4 ］N.G.Todua, et.al. Rapid Commun. in Mass Spectrometry, 2011, 25, 750-754.

Oral Session
Wedensday, 19th SeptemberRoom B-1

09:00 － 11:00

Session 23: Gas Phase Fragmentation Mechanisms of Biomolecular Radicals
Chair:

Shigeo Hayakawa（Osaka Prefecture Uiniversity, Japan）

S23-1000
10:00 － 10:20

The Cn Ions Formed by the Dissociation of an N-terminal Deuterohemin
Containing Hexapeptide

Xinhua Guo, Bing Wang, Jiayi Yu
Jilin University, Changchun, China

Keywords:
deuterohemin； peptide dissociation； cn ions； tandem mass spectrometry
Novel aspects:
The Cn ions are detected in CID mass spectra of a deuterohemin containing hexapeptiede which involve the McLafferty
rearrangement reaction
Abstract:
Gas phase dissociations of an N-terminal deuterohemin modified hexapeptide are studied with an ion trap mass spectrometer.
Due to a fixed charge in the N-terminal of the peptide characteristic precursor ions that run short of an added proton in single,
double and treble charge states are observed in the mass spectra. Upon collision induced dissociation（CID）the singly charged
peptide ion generates bn+17（cn）and bn+18 ions in addition to bn and bn derived product ions while the doubly charged peptide
ion forms normal sequencing b and y ions. The formation pathway of the c n ions is proposed that involves a McLafferty
rearrangement reaction in which companying a γH transfer from carbon to carbonyl oxygen a cleavage at N-Cα bond instead ofan amide bond occurs to the peptide. In contrast, the formation of bn+18 ions is attributed to the nucleophilic attack of hydroxyl
or carboxyl oxygen in the side chain of an amino acid C-terminal to the cleaved amide bond, which competitively happens prior to
the McLafferty rearrangement reaction. Since the cn ions are normally detected in electron capture dissociation（ECD）or electrontransfer dissociation（ETD）fragmentation mode and are seldom observed in CID mass spectra, the factors that lead to theformation of the cn ions are investigated through replacing the crucial amino acid residues, removing or changing the N-terminal
deuterohemin group in the original peptide and analyzing the fragments of resulted peptides. The results provide significant
insights into the formation and mechanism of the cn ions in the specific pepetide upon CID process, alternatively, suggest an easy
way to generate c n ions which will be useful for providing much sequence information to improve peptide and protein
identifications.

Oral Session
Wedensday, 19th SeptemberRoom B-1

09:00 － 11:00

Session 23: Gas Phase Fragmentation Mechanisms of Biomolecular Radicals
Chair:

Shigeo Hayakawa（Osaka Prefecture Uiniversity, Japan）

S23-1020
10:20 － 10:40

Isomerization of Radical Peptide Ions and Subsequent Radical-Induced
Dissociations

Ivan Keung Chu

Department of Chemistry, The University of Hong Kong, Hong Kong, China

Keywords:
Dissociation, Isomerization, Peptide Radical
Novel aspects:
Effect of isomerization on radical-induced bond cleavage of peptide ions
Abstract:
Dissociation of peptide radical ions involves competition between charge-induced and radical-induced reactions that can bepreceded by isomerization. A better understanding of the interconversions and fragmentations of odd-electron radical peptideions enrichs the scientific basis underlying peptide dissociation in the gas phase--the underpinning technology in proteomics. The
dissociations of peptide radical cations（M·+）differ from and complement those of the corresponding even-electron protonatedpeptide ions of the type［M + nH］n+； the latter fragment through amide bond cleavages to give predominantly y and b ions, while
the peptide radical cations undergo peptide backbone cleavages of their N-Cα and Cα-C bonds to give c/z pairs and a/x productions, respectively（a, b, and c ions are N-terminal containing ions, while y and z ions are C-terminal containing ions）. Despitesome recent advances from investigations of the reactivities of radical cationic peptides, their fragmentation mechanisms remain
poorly understood. In this talk, we will monitor the effect of a basic amino acid residue on various radicalinduced bond cleavages
such as C α-C and N-C α   backbone bonds as well as C α-C β and C β-C γ bonds of side chain cleavage. A combination of mass
spectrometric experiments and theoretical calculations were used to examine the isomerization of radical peptide cations andtheir radical-site-specific fragmentations. The laser-induced dissociation（LID）and low-energy collision-induced dissociation（CID）
spectra of the radical cationic peptides with well-defined initial locations of the radical centers are significantly different fromthose of their basic analogs. DFT calculations revealed that the basic radical cations have their charge densities largely
sequestered on the guanidine groups of the side-chain arginine residues； the lower charge densities on the backbones result ingreater flexibility, decreasing the barriers for interconversion between these α-carbon-centered radicals； thus the energy barriers
for the isomerization of radical cationic isomers were comparable with those of its competitive fragmentation reactions. It isapparent that the highly basic arginine residue in the peptide radical cations could mediate the facile migration of the various
radical centers prior to promoting their subsequent side chain cleavages.

Oral Session
Wedensday, 19th SeptemberRoom B-1

09:00 － 11:00

Session 23: Gas Phase Fragmentation Mechanisms of Biomolecular Radicals
Chair:

Shigeo Hayakawa（Osaka Prefecture Uiniversity, Japan）

S23-1040
10:40 － 11:00

Cascade Dissociations of Peptide Cation Radicals

Frantisek Turecek

University of Washington, Seattle, WA, USA

Keywords:
cascade dissociations, peptide cation radicals
Novel aspects:
Mechanisms have been elucidated for the consecutive dissociations of peptide cation-radicals using experimental andcomputational methods.
Abstract:
Amino acid residue-specific backbone and side-chain dissociations of peptide z ions in MS 3 spectra were elucidated for over 40
pentapeptides with arginine C-terminated sequences of the AAXAR and AAHXR type, nonapeptides of the AAHAAXX“AR and
AAHAXAX“AR type, and AAHAAXX“AAR decapeptides. Peptide z（n）ions containing amino acid residues with readilytransferrable benzylic or tertiary beta-hydrogen atoms（Phe, Tyr, His, Trp, Val）underwent facile backbone cleavages to form
dominant z（n- 2 ）or z（n- 3 ）ions. These backbone cleavages are thought to be triggered by a side-chain beta-hydrogen atomtransfer to the z ion C-alpha radical site followed by homolytic dissociation of the adjacent C（alpha）-CO bond, forming x（n- 2 ）cation-radicals that spontaneously dissociate by loss of HNCO. Amino acid residues that do not have readily transferrable betahydrogen atoms（Gly, Ala）do not undergo the z（n）  to z（n- 2 ）dissociations. The backbone cleavages compete with side-chain
dissociations in z ions containing Asp and Asn residues. Side-chain dissociations are thought to be triggered by beta-hydrogen
atom transfers that activate the C（beta）-C（gamma）or C（beta）-heteroatom bonds for dissociations that dominate the MS 3
spectra of z ions from peptides containing Leu, Cys, Lys, Met, Ser, Arg, Glu and Gln residues. The Lys, Arg, Gln, and Glu residues
also participate in beta-hydrogen atom transfers that trigger other side-chain dissociations.
Dissociations of z（ 4 ）ions from pentapeptides AAXAR where X = H, Y, F, W, and V produce dominant z（ 2 ）ions that accountfor >50% of the fragment ion intensity. The dissociation has been studied in detail by experiment and theory and found to involve
several isomerization and bond-breaking steps. Isomerizations in z（ 4 ）ions proceed by amide trans-cis rotations followed byradical-induced transfer of a beta-hydrogen atom from the side chain, forming stable C（beta）radical intermediates. These
undergo rate-determining cleavage of the C（alpha）-CO bond at the X residue followed by loss of the neutral AX fragment,
forming x（ 2 ）intermediates. The latter were detected by energy-resolved resonant excitation collision-induced dissociation（CID）
and infrared multiphoton dissociation（IRMPD）experiments. The x（ 2 ）intermediates undergo facile loss of HNCO to form z（ 2 ）fragment ions, as also confirmed by energy-resolved CID and IRMPD MS 4 experiments. The loss of HNCO from thex（ 2 ）ion fromAAHWR is kinetically hampered by the Trp residue that traps the OCNH radical group in a cyclic intermediate.

Oral Session
Wedensday, 19th SeptemberRoom D

09:00 － 11:00

Session 24: Regulated Bioanalysis
Chair:

Shinobu Kudoh（Shimadzu Techno-Research, Inc , Japan）

S24-0900
09:00 － 09:40[Keynote Lecture] Drug regulation and bio-analysis

Tatsuo Kurokawa

Keio University, Tokyo, Japan

Keywords:
JBF, drug development, regulatory science, international harmonization, guideline
Novel aspects:
Activities of recently formed Japan Bio-analysis Forum will be intrduced. Progress of draft bio-analysis guideline in Japan in JBF
will be touched upon.
Abstract:
Approach and pathway of drug development as well as scenes of drug therapy have been dramatically changed since introduction
and application of molecular biology and human genomic sciences. At the same time, many countries and regions now realize theabsolute and urgent needs of optimizing the use of medical resources in front of economic difficulty and rapid increase of healthcare demands. Bio-analysis technology is now considered as one of key factors for our societies to find the very best solution
upon this almost inconsistent issues. The bio-analysis technology can be a strong tool for providing people with the best
recommendable therapy in accordance with his/her particular condition. Appropriate utilization of bio-analysis technology in the
drug development stage may allow us to reduce the total time and process for a drug development and it will obviously improve
the availability and affordability of such new therapeutic options and proposals for patients. This is one of the important subjectsof regulatory science.
A group named Japan Japan Bio-analysis Forum, which consists of regulators, industries and academia, has been formed in 2010
with consideration of progressing international harmonization in this area and it started works for establishment of necessary
guidelines in Japan.
The above subjects will be introduced and discussed in this session.

Oral Session
Wedensday, 19th SeptemberRoom D

09:00 － 11:00

Session 24: Regulated Bioanalysis
Chair:

Shinobu Kudoh（Shimadzu Techno-Research, Inc , Japan）

S24-0940
09:40 － 10:00

Bioanalytical method validation: Process of preparation and notable
points of the draft Japanese guideline

Noriko Katori

National Institute of Health Sciences, Tokyo, Japan

Keywords:
Bioanalysis, Bioanalytical Method Validation, Japanese Guideline
Novel aspects:
This presentation is the first announcement of the Japanese BMV guideline draft in English.
Abstract:
The FDA released the Guidance for Industry on Bioanalytical Method Validation（BMV）in May 2001. A draft of the revised
guidance is expected later this year. Also EMA released the Guideline on BMV in July2011.
A Draft Japanese Guideline on BMV will be released this year. The history of discussion around regulated BMV and the
preparation process of the Japanese BMV guidelines will be presented.
The first symposium for regulated BMV held in Japan was the workshop titled“Discussion on AAPS/FDA White Paper forbioanalytical method validation“ planned in The 56th Annual Conference on Mass Spectrometry（Tsukuba, Japan）in 2008, where
Dr. Viswanathan（FDA）was invited. After the symposium, the interest to regulated BMV was getting higher in Japan year by year.
The organization of Global Bioanalysis Consortium（GBC）and the First Asia Pacific Conference on Recent Issues in Regulated
Bioanalysis（Shanghai, China）triggered a formation of the first and only association for scientific discussion of regulated
bioanalysis and BMV in Japan. The group became the Japan Bioanalysis Forum（JBF）which has helped to assign Japanese
representatives for all Harmonization Team（HT）in GBC. In spite of the Great East Japan Earthquake in March 2011, a lot ofJapanese have applied to the Harmonization Teams of GBC with willingness. At the same time, JBF has created a discussion
group to support all Japanese HT members in the GBC.
The first Symposium of JBF was held in August 2011 in Tokyo, and full-fledged discussions of regulated bioanalysis have been
started in Japan. The JBF symposia have been held about every six months until now.
Responding to the enthusiastic discussion around BMV, the Japanese regulatory authority prepared a study group in which the
BMV has been discussed among regulation sides and companies. The study group is preparing the draft Japanese guidelines on
BMV based on the tremendous contributions by JBF. The policy of the preparation of guideline is as follows；* Applied to bioanalytical methods generating quantitative data in toxicokinetics and clinical studies.
* Sections for Small molecule are prepared firstly.
* Sections for Large molecule will be following.
* Analysis of metabolite can be considered.
* Analytical methods： Chromatographic methods, e.g. LC-MS, LC-MS/MS.
* Terminology； conform to ICHQ 2 a, b.
* Aligned with the FDA Guidance /EMA guideline.
I will show you the summary of the Draft Japanese Guideline on BMV.

Oral Session
Wedensday, 19th SeptemberRoom D

09:00 － 11:00

Session 24: Regulated Bioanalysis
Chair:

Shinobu Kudoh（Shimadzu Techno-Research, Inc , Japan）

S24-1000
10:00 － 10:20

Using of Multistage Mass Spectrometry Techniques in Combinations with
Electrophoretic and Chromatographic Separation Techniques for Analysis
of Biological Samples

Jozef Marak, Andrea Stanova, Monika Kondekova, Monika Radicova, Nikoleta Biherczova,
Katarina Krcova
Comenius University, Bratislava, Slovak Republic

Keywords:
multistage MS, separation techniques, complex biological samples, therapeutic drugs
Novel aspects:
Multistage IT-TOF MS technique was used for the identification of the analytes present in complex samples and for the proposalof their structures and possible changes under in vitro conditions.
Abstract:
Bioanalytically important compounds, such as low- and high-molecular weight compounds naturally occurring in the living beings,
or different xenobiotics and/or their metabolites, are often needed to be determined in various biological samples at（ultra）trace
concentration levels. Apparently, the complex characters of all biological samples are representing the top challenges for the
analytical chemistry technologies used nowadays. For example, consider several hundreds of different constituents as these stillspanning various chemical/biological properties and, in addition, concentrations of analytes as differing several orders ofmagnitude. Due to these facts, a combination of a powerful separation technique is required, especially, to reflect for anadequately sensitive and/or selective detection method. There are two main analytical separation techniques used for solving
these problems, liquid chromatography（LC）and capillary electrophoresis（CE）. LC techniques, mainly in nanoLC variation, served
as“standard“ analytical separation technique due to its“universality“ and flexibility. However, its mostly not sufficient separation
efficiency and the production of toxic waste are limiting factors for its acceptance as the“universal“ analytical separation tool.
On the other hand, while CE techniques offer some favorable properties, like very high separation efficiencies along with very low
consumption of both the samples and the operating solutions, they suffer from the detection sensitivity. MS detection itself or in
the combination with LC or CE techniques provides an excellent selectivity while keeping sufficient detection sensitivity. However,
because of the complex character of biological samples and different concentration levels of the analytes of interest and thematrix components even LC-MS and/or CE-MS combinations are not always able to solve successfully the above mentioned
analytical problems and some sample pretreatment technique is required to be implemented into the analytical scheme.
The experiments with micropreparative capillary isotachophoresis（pITP）, used in our approach as the sample pretreatment
technique, were performed in a hydrodynamically closed CE separation system. Different biological samples（urine, saliva and tearsamples served as the model matrices in our case）were used alone and/or spiked with several low- and high molecular weightcompounds（model analytes）and pITP served as a tool for isolating analytes while accompanied only by minimum interferents
from the biological matrices. Such an isolation procedure was performed with the help of a fractionation valve. pITP was included
for discrete spacer constituents as added to the sample solutions. The isolated fractions were lyophilized and then reconstituted
for final mass spectrometric analyses by：（i）a direct injection mode,（ii）HPLC-MS（UPLC-MS）and（iii）CE-MS combinations.
Our work deals with different approaches and analytical aspects as regarding low- and high molecular weight compounds
containing samples. Multistage MS based on IT-TOF technique was used for the identification of the analytes of interest present incomplex samples and for the proposal of their structures and possible changes under in vitro conditions. A very low concentrationlevel of the analyte by using pITP and, subsequently, as loading to chromatography and electrophoresis techniques as coupled toMS. MS and MS n spectra were obtained from the reconstructed fractions as included both the pITP clean-up effect and its ITP
concentration power as regarding the analyte as present in the complex sample at very low concentration levels. This study is
showing high potentialities and compatibility of pITP as the sample pretreatment technique before several modes of MS analysisof the complex samples of biological origin.

Acknowledgement:This work was supported by： the Slovak Research and Development Agency（the project
No. VVCE-0070-07）and the Slovak Grant Agency（the project No. 1 /1305/12）.

Oral Session
Wedensday, 19th SeptemberRoom D

09:00 － 11:00

Session 24: Regulated Bioanalysis
Chair:

Shinobu Kudoh（Shimadzu Techno-Research, Inc , Japan）

S24-1020
10:20 － 10:40

Quantitative Analysis of Beta casomorphin-7 by SRM using stable isotope
labeled peptide as internal standard

Xiaomin Song1, Thiri Zaw1, Ardeshir Amirkhani1, Andrew Clarke2, Mark Molloy1
1

APAF Macquarie University, Sydney, Australia, 2A2 Co Ltd, Australia

Keywords:
SRM, Beta casomorphin, BCM-7, peptide quantitation
Novel aspects:
Our MS based BCM- 7 quantitation method is of better specificity and similar level of sensitivity comparing to ELISA for detecting
BCM- 7 in body fluids.
Abstract:
Beta casomorphin- 7 （BCM- 7 ）is a peptide released from beta-casein when bovine milk is digested. The health effects of BCM- 7
in the body have been investigated and shown to play a role in the aetiology of some human diseases, including Type 1 diabetes.
In 2009 the European Food Safety Commission（EFSA）released a review relating to BCM- 7 ； it reported that BCM- 7 was an
opioid receptor agonist, with the potential to impart activity on a range of tissues including immune and digestive cells. However it
concluded that a cause and effect relationship could not be established between the dietary intake of BCM 7 and noncommunicable diseases； owing in part to lack of data on exposure to BCM- 7 following the consumption of milk products.
Historically, the quantitation method for BMC- 7 lacked either sensitivity or selectivity and the analysis of BCM- 7 in body fluid has
been difficult. To address this, we developed a SRM based BCM- 7 analysis method using stable isotope labeled BCM- 7 *（H-TyrPro-*Phe-Pro-Gly-Pro-Ile-OH, where *Phe = 13C9, 15N phenylananine, mass increase 10Da）as internal standard. BCM- 7 * was spiked
into analyte before BCM- 7 enrichments. Because they have identical chemical character, simultaneously acquiring SRM data fornative BCM- 7 （MW=790.4Da）and stable isotope labeled BCM- 7 *（MW=800.4Da）effectively compensated the sample matrix
effect, sample preparation effect and instrument drifting effect. This allows highly sensitive and specific absolute quantitation of
the peptide without the need to run standard calibration curve. Our tests showed that BCM- 7 in plasma can be accurately
quantitated in low ng/ml range.
Human plasma was used as matrix in the method development and validation. However, this method can be applied to otherbody fluids or milk products after some sample specific sample preparation tests.

Oral Session
Wedensday, 19th SeptemberRoom D

09:00 － 11:00

Session 24: Regulated Bioanalysis
Chair:

Shinobu Kudoh（Shimadzu Techno-Research, Inc , Japan）

S24-1040
10:40 － 11:00

Accurate Mass MS/MS an Attractive Option For the Quantification ofBiotherapeutics in DMPK Studies

Robert S Plumb1,2, Christopher Evans3, Paul D Rainville1, Joanne Mather1
1

Waters Corporation, 2Imperial College, London, UK, 3GlaxoSmithKline, DMPK, PA, USA

Keywords:
Bioanalysis, Protein, accurate mass, LC/MS/MS
Novel aspects:
The use of accurate mass LC/MS with narrow mass extraction range to detect and quantify protein therapeutics in plasma extract
Abstract:
In this paper we discuss the application of LC-HRMS for the quantification of a the rapeutics protein. Biotherapeutics now
represent approximately20% of a typical pharmaceutical companies drug pipeline, and must be evaluated for safety and efficacy ina similar manner to small molecules. Tandem quadrupole LC/MS/MS is the technology of choice for the quantification of small
molecules in DMPK studies. The application of accurate mass MS for quantification in DMPK has attracted considerable interest in
recent years, allowing simultaneous quantitative and qualitative analysis. By employing a narrow mass window assay the
specificity is significantly improved, increasing the S/N and assay accuracy. The results generated in this study were compared tothose previously acquired on a tandem quadrupole MS system.
The plasma samples（50 µL）was digested with an equal volume of Lys-C solution overnight, the sample centrifuged and theresulting solution isolated by means of cation exchange SPE. The resulting sample was evaporated to dryness and thenreconstituted in mobile phase starting conditions for analysis by LC/MS. The LC/MS analysis was performed on a hybrid
quadrupole TOF mass spectrometer operated in positive ion mode. A the separation was performed on a 2.1 x 5 cm 1.7um C18
column operated in reversed phase gradient mode over 2 minutes. The quadrupole was set to 503.5 the TOF analyser range was
50 1000 m/z. Quantification was performed using the mass fragment m/z = 616.3821, with 510.5 => 623.399 employed for the internal
standard.
The two most common approaches for the quantification of biotherapeutic drugs are either the analysis of the intact protein or
analysis of a signature peptide following protein enzymatic digest. The use of a proteolytic digestion followed by measurement ofa signature peptide is the preferred approach frequently for the quantification of proteins due to the poor the sensitivity of intact
proteins LCMS/MS.
In this study a Q-TOF type instrument was employed in V mode with a mass resolution of 20,000 FWHM. The data was processed
to investigate the effect of reducing the mass analysis window from 1 Da, to 100, 40 and 20mDa. The analysis of a 50ng/mL
standard extracted protein digest from plasma showed that peak of interest elutes with a retention time of 1.20 minutes； m/z=616.3821. The use of a 100mDa extraction window significantly improved the specificity of the assay compared with the 1 Daanalysis, removing multiple endogenous peaks from the analysis. The 40mDa extraction window the assay showed further
improvements in specificity. The blank exhibited no peak with a S/N greater than 3 ： 1 eluting at the retention time of 1.2
minutes.
The acquired data was reprocessed using a 20mDa mass window, the results showed that with a 10ng/mL sample an intensity
increased by a factor of 1.8 compared to the 40mDa mass window analysis. The 20mDa mass window analysis also showed an
improved signal/noise by a factor of 4 over the 40mDa analysis. A typical calibration curve obtained from the 20mDa window
analysis had a coefficient of determination（r 2 ）of 0.994545 with an intercept of 0.00025.
A comparison of the sensitivity of the accurate mass method with a tandem quadrupole methodology showed that the accuratemass method was within one order of magnitude in terms of sensitivity.

Oral Session
Wedensday, 19th SeptemberRoom E

09:00 － 11:00

Session 25: New Approaches to Defining the Diversity of Glycans
Chair:Catherine E Costello（Boston University School of Medicine, USA）

S25-0900
09:00 － 09:40[Keynote Lecture] Development and Application of a Rapid and Sensitive
Method for Screening Cellular Models of Congenital Disorders of
Glycosylation

Jane Thomas-Oates1,2,3, Salina Abdul Rahman1,2,3, Jennifer E Dodd1,3, Ed Bergstrom1,2,3,
David A Ashford1,2,4, Jerry Thomas1,2,4, Daniel Ungar1,4

University of York, York, UK, 2Centre of Excellence in Mass Spectrometry, 3Department of Chemistry, 4Department of
Biology

1

Keywords:
congenital disorders of glycosylation（CDG）； glycan analysis； protein glycosylation；permethylation；filter-aided sample
preparation
Novel aspects:
development of simple, fast, sample handling approaches for release and analysis of whole cell glycans； reducing cell numbers tosingle culture dishes for medium-throughput glycan screening
Abstract:
Congenital Disorders of Glycosylation（CDG）are a group of rare inherited metabolic diseases caused by defects in protein
glycosylation. Most CDG subtypes arise due to a defect in one of the enzymes or transporters in the protein glycosylation
pathway. However, there are several subtypes of CDG that arise not by mutation of the enzymes or transporters themselves, butrather from mutations in the components responsible for organising the glycosylation machinery in the Golgi（Wu et al）； these
defects lead to much less predictable patterns of glycosylation and are thus harder to diagnose, to understand, and to treat.
In order to study this class of CDGs, we are developing cellular models of the different patient disorders using appropriate chinese
hamster ovary cell mutants（Krieger et al, Ungar et al）. These cellular models allow us to study the effects of such mutations byscreening the cellular glycosylation patterns in the laboratory, a particularly important approach given the rarity of diagnosed
patients. When potential patients do present, fibroblast samples can be cultured and studied using the same approaches.
We have consequently been developing generic approaches to screening whole cell glycans, with an emphasis on working withrealistic cell numbers for medium-throughput screening, and convenient, sensitive, robust, MS-compatible sample handling
protocols. We have based our approach on the filter-aided sample preparation procedures described for proteomic analyses（Manza et al, Winiewski et al）and developed these for glycan release, followed by permethylation and MS analysis. The
approach relies on solubilising（glyco）proteins from whole cell lysates by heating in SDS, followed by exchange of the SDS for
more MS-friendly buffers in a spin filter device. The glycans are then released from the protein mixture above the membrane andthe released glycans retrieved from the reaction mixture by centrifugation of the spin filter. Permethylation of the retrieved
released glycans makes the samples ready for MS analysis.
The talk will describe our sample handling approaches, illustrated with some of our data from cultured cells.

Acknowledgements
SAR very gratefully acknowledges her PhD Scholarship and travel support from the Ministry of Health Malaysia. The York Centre of
Excellence in Mass Spectrometry was created thanks to a major capital investment through Science City York, supported byYorkshire Forward with funds from the Northern Way Initiative.
References
Krieger, M., Brown, M.S. & Goldstein, J.L. J Mol Biol 150, 167-184（1981）.
Manza, L.L., Stamer, S.L., Ham, A.J., Codreanu, S.G. & Liebler, D.C. Proteomics 5, 17421745（2005）.
Ungar, D., Oka, T., Brittle, E.E., Vasile, E., Lupashin, V.V., Chatterton, J.E., Heuser, J.E., Krieger, M. & Waters, M.G. J Cell Biol 157, 405415（2002）.
Winiewski, J.R., Zougman, A., Nagaraj, N. & Mann, M. Nat. Methods 6, 359362（2009）.
Wu, X., Steet, R.A., Bohorov, O., Bakker, J., Newell, J., Krieger, M., Spaapen, L., Kornfeld, S. & Freeze, H.H. Nat Med 10, 518-523（2004）.

Oral Session
Wedensday, 19th SeptemberRoom E

09:00 － 11:00

Session 25: New Approaches to Defining the Diversity of Glycans
Chair:Catherine E Costello（Boston University School of Medicine, USA）

S25-0940
09:40 － 10:00

Studies of Oligosaccharides and Monosaccharides by LEISD MALDI and
2D-CID-ESI Mass Spectrometry Techniques

shuying Liu1,2, hongmei Yang1, Debin Wan1

Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, changchun, China, 22Changchun Universityof Chinese Medicine, 130117, China

1

Keywords:
oligosaccharide, monosaccharide, LEISD MALDI, 2 D-CID-ESI mass spectrometry, isomer
Novel aspects:
This is the first report that carbohydrate isomers were successfully differentiated by LEISD MALDI and 2 D-CID-ESI mass
spectrometry.
Abstract:
Studies of Oligosaccharides and Monosaccharides by LEISD MALDI and 2D-CID-ESI Mass Spectrometry Techniques
Hongmei Yang1, Debin Wan1 and Shuying Liu1,2*
1
Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, 130022, China
2
Changchun University of Chinese Medicine, 130117, China
Carbohydrates play crucial roles in multiple biological processes including human diseases1-3. The complete structural elucidationof carbohydrates is very important to understand their biological functions. Despite the recognized importance, structural analysis
of carbohydrates is still challenging due to their enormous structural diversity and micro-heterogeneity.
Here we describe two efficient and practical methods of Mass spectrometry to study the isomer structures of oligosaccharides
and monosaccharides.
First, laser enhanced in source decay (LEISD) technique of the MALDI MS allows highly reliable and abundant fragmentation of
the neutral oligosaccharides.
MALDI MS technique described to date has proven to be a convenient and rapid method for sequencing purified peptides andproteins. However, for oligosaccharides even the application of the conventional ISD can not produce adequate fragments for thedetailed structural elucidation, due to the low internal energy for precursor ions. In this study, we applied an ultrahigh irradiation
laser of mJ level to obtain better quality ISD spectra of fragment ions. The LEISD of oligosaccharides demonstrated significantly
better signal-to-noise ratio spectra and more structural information obtained. The results showed that the intensities of A-typeions derived from cross-ring cleavage of oligosaccharides allowed the distinction among α（ 1 → 4 ）-, α（ 1 → 6 ）-, β（ 1 → 4 ）-,and β（ 1 → 3 ）-linked isobaric structures according to fragment types and intensities.
We also discussed several parameters（doped metal ions, gas-phase sodium affinity, and proton affinity of the matrices）affecting
LEISD of oligosaccharides in detail.
Second, 2D-CID-ESI mass spectrometry was employed to discriminate the isomers of monosaccharides.
Electrospray ionization tandem mass spectrometry（ESI-MS/MS）is another common and well-studied approach for carbohydrate
discrimination, according to differences in MSn fragmentation patterns. Unfortunately, the mass spectra may be too similar toassign isomers based on the product ion ratios.
The collision energy as an additional dimension is added to display the related product-ion information of the isomers in the
2 D-CID mass spectra, which proposed by Han 4 . The CID spectra associate with the internal energy of precursor ions, whichmainly depend on collision energy and related reactions. We employed 2 D-CID-ESI-MS in the negative ion mode, and thediscrimination of isomers of monosaccharides was achieved completely. With the change of collision energy, the ratios of productions are detected obviously different among the four isomers, glucose, fructose, mannose, and galactose.
References
1. Hakomori, S. The Glycosynapse. Proc. Natl. Acad. Sci. U.S.A.2002,99, 225-232.
2. Dwek, R. A. Glycobiology： Toward Understanding the Function of Sugars. Chem. Rev.1996,96, 683-720.
3. Zhang, X. L. Roles of Glycans and Glycopeptides in Immune System and Immune-Related Diseases. Curr. Med. Chem.2006, 13,1141-1147.
4. Han, X.； Gross, R. W. Quantitative Analysis and Molecular Species Fingerprinting of Triacylglyceride Molecular Species Directlyfrom Lipid Extracts of Biological Samples by Electrospray Ionization Tandem Mass Spectrometry. Anal. Biochem.2001,295, 88-100.

Oral Session
Wedensday, 19th SeptemberRoom E

09:00 － 11:00

Session 25: New Approaches to Defining the Diversity of Glycans
Chair:Catherine E Costello（Boston University School of Medicine, USA）

S25-1000
10:00 － 10:20

A new approach to the rational synthesis of MALDI matrix. Keeping
functional groups and its location but changing the stereochemistry

Maria L Salum2, Lucia M Itovich2, Hiroshi Nonami1, Rosa Erra-Balsells2
1

Ehime University, Matsuyama, Japan, 2University of Buenos Aires, Argentina

Keywords:
isomers, geometric-isomers, trans-cinnamics, cis-cinnamics, carbohydrates.
Novel aspects:
New strategy to prepare MALDI matrix. Improving MALDI MS carbohydrate analysis.
Abstract:
The main idea of the approach followed in the present project for the synthesis of several potential MALDI matrix was based on
the fact that each cinnamic acid presents two different isomeric forms： trans-cinnamic and cis-cinnamic. The pair of compounds,
are called geometric isomers, in which the nature and position of the functional groups in the benzenic moiety is the same but the
spatial relation between de substituent present in the alkenic exocyclic group is different（1,2-anti, trans or E； 1,2-gauche, cis or Z）.
As consequence of this different geometric distribution the bulky substituents present at de vicinal 1,2 positions push the molecule
to adopt quite different tridimensional structure. Classically for the trans isomer the thermodynamically more stable conformationis almost flat（bencenic and alkene moieties in the same plane）and for the cis isomer bulky substituents at de 1,2 positions brakethe molecular planarity and the total volume and tri dimensional structure of the cis isomer is completely different than those of
the trans form. As a reality both isomers are different chemical species whose physical, chemical and photochemical properties
can be quite different too.
As commercial cinnamic acids（sinapinic, coumaric, ferulic, caffeic, 4 -mthoxy- 3 -hydroxy cinnamic, etc.）are 97-99 % thecorresponding trans isomer, we decided to find a proper a technique to prepare the corresponding pure cis forms. Thus, after a
one-pot preparation method was developed in our laboratory we started the MALDI experiments. Each pair of trans/cis isomers
was inspected in comparative way with regard to its mass spectrum and its performance in protein and carbohydrate MALDI MS
analysis.
For our delaight, cis-cinanmic acids showed in general better performance than the trans isomers. In the field of carbohydrates thedifferent behavior is amazing. Particularly cis-sinapinic acid showed to be an excellent matrix for carbohydrate analysis. Thus,
neutral sugars（maltoses, fructanes, cyclodextrins, trehalose）showed in positive ion mode, signals with absolute intensity andsignal/noise ratio similar or better than those obtained with gentisic acid. Furthermore, in the field of sulfated sugars, cis-cinnamic
acids, particularly cis-sinapinic showed better spectra with a drastic diminishing of number and intensity of signals produced bydesulfated fragments； this experiments were conducted comparing the spectra with those got using gentisic acid and norharmane as matrix.
For better understanding of the relative performance of the new matrices and its trans isomer computational chemistry（molecularmodeling）has been used, after optimization of the geometry, to calculate formation energy, proton affinity, volume, surface area,
hydratation energy, log P and dipolar moment.

Oral Session
Wedensday, 19th SeptemberRoom E

09:00 － 11:00

Session 25: New Approaches to Defining the Diversity of Glycans
Chair:Catherine E Costello（Boston University School of Medicine, USA）

S25-1020
10:20 － 10:40

Automated Identification of Intact Glycopeptides from Complex Samples

Christopher Becker1, Marshall Bern1, Yong Kil1, Doron Kletter2, Mudita Singhal2, Julian Saba3,David Horn3, Sergei Snovida4, Rosa Viner3
1
4

Protein Metrics Inc., 2Palo Alto Research Center, Palo Alto, CA, USA, 3Thermo Fisher Scientific, San Jose, CA, USA,
Institute of Biological Chemistry, Academia Sinica, Taipei, TAIWAN

Keywords:
glycopeptide identification, glycoproteins, glycosylation
Novel aspects:
Large-scale intact glycopeptide identification by integrated HCD and ETD analysis and database searching
Abstract:
Introduction
About half of all human proteins are believed to be glycosylated, and glycosylation plays a major role in both normal cell signaling
and disease processes. Understanding glycosylation on an“omics“ scale, as well as discovery of glycoprotein disease biomarkers,
however, has been limited by the analytical challenge of identifying glycopeptides in complex samples such as blood plasma or
cellular extract. Here we present a new database search engine named Byonic that allows large-scale glycoprotein identification,
including glycans. Byonic was applied to product-dependent Orbitrap data that uses HCD-generated MS/MS fragments to target
glycosylated peptides for ETD analysis in the following scan.
Methods
Human blood plasma and cellular protein samples were digested by trypsin and enriched for glycopeptides by strong anion
exchange, and then labeled with Tandem Mass Tags（TMT 0 ）to improve ionization efficiency and increase precursor charge state
distribution, yielding superior ETD fragmentation. Product-dependent ETD acquisition uses characteristic glycoside（138.055 Da）
and oxonium ion peaks（204.087, 366.139 Da）in high-accuracy HCD Orbitrap scans to trigger ETD scans of glycopeptides. Byonicdata analysis uses expert-systems, for both fragmentation modeling and O- and N-linked glycan table generation, to makeidentifications from search spaces that can involve ~ 1000 glycan forms and > 4000 proteins, with > 10,000 potential N-glycosylationsites, and roughly a million potential O-glycosylation sites.
Preliminary Results
Glycopeptide identification is limited by relatively poor fragmentation and very large search spaces, even with the advances of
TMT labeling and product-dependent ETD data acquisition. The size of the search space is dependent upon the product of thenumber of peptides with potential glycosylation sites, the number of glycan masses, and the number of combinations of othermodifications（sodiation, potassiation, methionine oxidation, etc.）. With Modification Fine Control capability, Byonic allows theuser to customize the search with separately settable limits on each modification type, capped by a limit on the total number ofmodifications. This avoids the all-to-familiar combinatorial explosion of other search engines while providing great flexibility and awide range of types of modifications allowed. We used a table of the ~600 most plausible mammalian N-glycan masses, as
determined by Cartoonist, expert-system software for glycan profiling. From plasma proteins, of 2540 ETD scans almost sure tocontain glycopeptides, Byonic found identifications for 700 scans with accuracy > 90 % , as judged by manual curation and priorknowledge（Uniprot KB）. Computation time was less than a few minutes on a desktop PC. Investigation of cellular samples will
also be reported. We compare independent HCD / ETD analysis with integrated HCD / ETD analysis, in which peaks in the HCD
spectrum focus attention on glycopeptides containing, for example, fucosylated or sialylated glycans. We also show the effect oflimiting the search space along the three dimensions of protein, glycan, and other-modification complexity.

Oral Session
Wedensday, 19th SeptemberRoom E

09:00 － 11:00

Session 25: New Approaches to Defining the Diversity of Glycans
Chair:Catherine E Costello（Boston University School of Medicine, USA）

S25-1040
10:40 － 11:00

Ion mobility mass spectrometry of IgG Fc glycopeptides from different
subclasses

Michiko Tajiri1, Yayoi Hongo2, Takemichi Nakamura2, Kenji Hirose3, Yoshinao Wada1
1
3Osaka Medical Center and Research Institute for Maternal and Child Health, Izumi, Japan, 2RIKEN, Wako, Japan,
Nihon Waters K.K., Osaka, JAPAN

Keywords:
ion mobility MS, glycoproteomics, glycopeptide, glycan-peptide interaction, collision cross-section
Novel aspects:
Ion mobility MS of glycopeptides suggests an interaction between N-glycan and peptide backbone sequence to specify different
IgG subclass functions.
Abstract:
Introduction
IgG molecules are glycosylated with a complex biantennary-type oligosaccharide at Asn-297 of the CH 2 domain within the Fc
region. These glycans are essential for Fc effector functions of IgG such as complement activation and Fcγ receptor-mediatedactivities. These functions have been studied mainly for IgG 1 among four subclasses whose structures are different each other inthe residues Y（Tyr）or F（Phe）at 296 and 300. Considering the differential affinities of IgG subclasses to Fcγ receptors, wehypothesized an interaction between glycan and amino acid residues near the glycosylation site, and analyzed glycopeptides
containing the region by ion mobility MS to address the issue.
Methods
The tryptic glycopeptide from IgG Fc region were enriched by a hydrophilic affinity method using Sepharose CL- 4 B（Wada et al.,Anal Chem 76：6560, 2004）. Truncation of oligosaccharides was carried out by specific glycosidic enzymes, and the glycopeptides
from different IgG subclasses were separated each other by reversed phase chromatography. The glycopeptides were dissolvedin 20 % methanol containing 0.1 % formic acid, and the sample solution was loaded in a nanospray tip for ion mobility MS using a
Waters SYNAPT G 2 system. The drift time of doubly protonated molecules of glycopeptides was measured. For CID, the ionswere activated in the Transfer T-wave collision cell. For calibration of collision cross section, tryptic peptides of BSA, bovine apotransferrin, human transferrin and horse myoglobin were employed according to a report by Valentine et al.（JASMS 10： 1188,
1999）.
Results
The amino acid sequences of tryptic glycopeptides from the IgG 1 and IgG 2 Fc regions are EEQYNSTYR（exact mass 1188.5；
296Y/300Y）and EEQFNSTFR（1156.5； 296F/300F）, respectively, and the N-glycans are attached to Asn297 in the middle of thesequence. The drift time of doubly protonated molecules of the synthetic peptides without glycans was normally dependent on
the molecular mass of these peptides. The protonated molecular ions of IgG 1 glycopeptide bearing mature glycans displayed asmaller collision cross section relative to IgG2, despite the former is larger in the molecular mass. This feature of conformationalchange was found in the glycopeptide bearing a single GlcNAc attached to Asn297, suggesting an interaction between the
innermost GlcNAc and peptide backbone sequence. Analysis of IgG 3 （296Y/300F）and IgG 4 （296F/300Y）glycopeptides suggested
that both 296 and 300 were involved in the interaction. More specifically, regarding the glycopeptide bearing core trisaccharide（GlcNAc 2 Man）, IgG 1 or IgG 4 displayed smaller collision cross section than IgG 2 or IgG 3 glycopeptides, suggesting acontribution of Tyr300. Finally, an energy-resolved CID experiment suggested the compact conformation of IgG 1 glycopeptideions compared with IgG 2 ions, apparently consistent with the results described above.
Conclusion
The contribution of innermost GlcNAc of N-glycans to the entire acceleration of folding and native state stabilization of
glycoproteins has been reported by Hanson et al.（PNAS 106： 3131, 2009）. Our findings suggested that the aromatic residues at
296 and 300 of IgG Fc region are involved in the GlcNAc-peptide interaction, thereby making differences in the biological propertiesof four IgG subclasses.

Oral Session
Wedensday, 19th September

Main Hall

15:00 － 17:00

Session 26: Lipidomics : Recent New Techniques and Applications
Chair:

Stephen J Blanksby（University of Wollongong, Australia）

S26-1500
15:00 － 15:40[Keynote Lecture] High-Resolution ’Shotgun’ Mass Spectrometry andChemical Labeling for Comprehensive and Quantitative Lipidome Profilingof Disease

Cassie J Fhaner1, Hong Ji2, Richard J Simpson2, Gavin E Reid1
1

Michigan State University, USA, 2Latrobe University, Australia

Keywords:’fixed-charge derivatization； colon adenocarcinoma； lipidomics
Novel aspects:
Chemical derivatization enables enhanced lipidome using ultra-high resolution ’shotgun’ mass spectrometry
Abstract:
A large number of studies have demonstrated that disruption of lipid metabolism or signaling can play a key role in the onset andprogression of human diseases, including cancer and diabetes. Thus, comparative analysis of changes in individual lipids or lipid
profiles（i.e., the lipidome）between normal and diseased cells, tissues or accessible bodily fluids, may enable the identificationand characterization of lipids that can serve as effective biomarkers of the disease. In this presentation, we will describe thedevelopment and application of a straightforward and high throughout analysis strategy consisting of ultra high-resolution ’shotgun’ mass spectrometry（MS）coupled with functional group specific chemical modification and ’targeted’ MS/MS for theidentification, characterization and quantification of >600 individual glycerolipid, glycerophospholipid, sphingolipid and sterol lipid
species from whole cell or tissue samples, across more than 4 orders of magnitude dynamic range of abundance, and withminimal need for sample handling or lipid extraction prior to analysis.

Oral Session
Wedensday, 19th September

Main Hall

15:00 － 17:00

Session 26: Lipidomics : Recent New Techniques and Applications
Chair:

Stephen J Blanksby（University of Wollongong, Australia）

S26-1540
15:40 － 16:00

Tandem MS Methods For Assignment Of Double Bonds And Chain
Branching In Fatty Acid Methyl Esters

Tom Brenna, Rinat Ran-Ressler, Peter Lawrence
Cornell University, Ithaca, New York, USA

Keywords:
lipidomics, FAME, BCFAME, chemical ionization
Novel aspects:
Lipidomic methods have traditionally ignored isomeric chains； this paper highlights emerging methods for determining structure
via MS.
Abstract:
The position of double bonds and chain branching in fatty radyl chains determines their chemical and biological properties.
Conventional lipidomics methods provide carbon number and double bond number of fatty radyl chains in their native lipid class,but do not provide information on isomeric structure. The most common analytical form of fatty radyl groups in a wide range of
biomedical and biochemical studies is as fatty acid methylesters（FAME）because of the very high resolving power of capillary gas
chromatography. Neither electron ionization nor conventional chemical ionization provide ions diagnostic of double bond positionor chain branching in FAME. The usual means to evaluate these isomers is by specialized derivatization with charge localizing
groups（e.g. DMOX, picolinate）. Apart from the need for special chemistry, these techniques alter retention times, can induce
double bond migration, and reduce sensitivity, all of which present difficulties. For more than a decade we have developed
covalent-adduct chemical ionization tandem mass spectrometry for assignment of double bond position in monoene, honoallylic
polyene, conjugated diene, and several other classes of conjugated polyene FAME. Under CI conditions, acetonitrile（CH 3 CN,
MW=41）self reacts to form a reagent ion［CH 2 =C=N=CH 2 ］+（m/z 54）which undergoes an ion-molecule reaction with double
bonds to form cyclic structures. Collisional activation yields a small number of diagnostic ions that reveal double bond position.
Fragmentation is systematic and depends on the double bond configuration, either vinylic or allylic to the site of the original
double bond, and occurring with transfer of a H either to or from the neutral. The method is particular amenable to quantitative
analysis since a small number of strong ions are formed. We extended these results to atmospheric pressure ionization of
triacylglyerols with mono and diene fatty acyl groups. More recent work has focused on branching in saturated monomethyl
branched chain FAME（BCFAME）. BCFAME are prominent in cow and other ruminant milks, beef and other ruminent meat,bacteria, and in certain human substances generally related to skin secretions. EI spectra provide fragments that enableassignment of branching position in many but not all cases. Zirroli and Murphy（JASMS, 1993）showed that the EI-derived
molecular ions of a few BCFAME are rearrangement products that, upon further collisional dissociation, produce highly specificand strong fragment ions. We recently tested this method for a broad range of BCFAME from C12 to C31. Bond breaking was on
the carboxyl side of the branch point for iso BCFAME yielding an isopropyl neutral loss. For most other BCFAME, bonds on bothsides of the branch point break yielding two high intensity fragments. In all cases the expected diagnostic ions were observed,
enabling unambiguous assignment of branch structure. The method was applied to vernix caseosa, the white, waxy substancefound on the skin of human newborns, and for BCFAME found in lanolin. These very convenient and rapid methods improve theconfidence with which the very diverse set of isomers found in FAME can be assigned.

Oral Session
Wedensday, 19th September

Main Hall

15:00 － 17:00

Session 26: Lipidomics : Recent New Techniques and Applications
Chair:

Stephen J Blanksby（University of Wollongong, Australia）

S26-1600
16:00 － 16:20

The Missing Link between Neurodegeneration and Sphingolipid
Metabolism: Integration of Lipidomics and Proteomics

Sarita Hebbar, Mayura Nakade, Dominik Schwudke

National Centre for Biological Sciences, Tata Institute of Fundamental Research, Bangalore, India

Keywords:
Lipidomics, Label-Free Quantitative Proteomics, Sample Preparation, Drosophila melanogaster, Sphingolipids
Novel aspects:
We present an analytical strategy enabling us to perform simultaneously quantitative lipidomics and proteomics experiments fromsmall tissue samples.
Abstract:
In recent years the importance of lipid metabolism in maintaining the integrity of the nervous system was established in a numberof studies［reviewed in（ 1 ）］. Perturbations of lipid biosynthetic pathways due to stress and pathological processes are ofcomplex and quantitative in nature. D.melanogaster is the ideal model for this study because of the wealth of genetic tools
available to modulate lipid metabolism and the simplicity of its cultivation. The aim of this study was to develop efficient sample
preparation strategies for integrating proteomics and lipidomics approaches for studying Drosophila lipid biology.
We developed a workflow which enabled us to routinely perform lipidomics and proteomics measurements from only 5
dissected adult brains. Dissected adult brains were processed with the MTBE lipid extraction（ 2 ）. The upper organic layer was
utilized for shotgun lipidomics and LC-MS/MS（3,4）. Lipidomics analysis was performed on a hybrid LTQ Orbitrap XL mass
spectrometer（Thermo Fisher Scientific, Bremen, Germany）equipped with a robotic nanoflow ion source TriVersa（Advion
BioSciences Ltd, Ithaca NY）. Liquid chromatography was performed on a 1200 micro-LC-system（Agilent Technologies, Waldbronn,
Germany）with a split flow setup enabling nano-ESI-MS. Mass spectrometric data interpretation was performed with proprietary
software LipidXplorer（ 5 ）and Xcalibur（Thermo Fisher Scientific, Bremen, Germany）. The lower aqueous layer was processed
for total protein determination and proteomics starting with 1 D SDS-PAGE analysis. Total protein amounts were determined byimage analysis of coomassie stained gels using Image J. Gel lanes were sliced into 11 pieces which were processed for In-gel
digestion. The extracted peptides were spiked with BSA peptides and analyzed by Agilent 1200 NanoLC system and a LTQOrbitrap Discovery（Thermo Fisher Scientific, Bremen, Germany）coupled with an Advion Nanomate Triversa. MASCOT databasesearches and label-free quantitation were performed using Proteome Discoverer 1.3（Thermo Fisher Scientific, Bremen, Germany）.
In the course of such experiment we can access quantitative information of approximately 800 proteins, 200 lipids, and determine
the total protein content of a sample. For the analysis of such small samples amounts we apply the abundance of housekeeping
and structural protein as reference for lipid quantities. Furthermore, we can evaluate the efficiency of genetic manipulation of
biosynthetic genes on the protein level as exemplified by Ceramidase（Dmel ￥CDase）over-expression.
Currently we apply this methodology to analyze the influence of lipid metabolic perturbation in spinster（Dmel ￥spin）mutants（ 6 ）, an established model for Lysosomal Storage Diseases（LSD）. We could show that the abundance levels of ceramides arepreceding the manifestation of neurodegenerative pathology as characterized by electron microscopy and behavioral assays.
Finally, we show that membrane lipid classes like PE, PC and CerPE start to accumulate in the brain in conjunction with theappearance of abnormal structures like membrane whorls and lipofuscin. We will now manipulate sphingolipid metabolic
pathways in the brain using the UAS-Gal 4 system in the background of the Spinster mutation.
References
1. Haughey, N. J.（2010）Neuromolecular Med12, 301-305
2. Matyash,V., Liebisch, G., Kurzchalia, T. V., Shevchenko, A., and Schwudke, D.（2008）The Journal of Lipid Research49, 1137-1146
3. Schwudke, D., Schuhmann, K., Herzog, R., Bornstein, S. R., and Shevchenko, A.（2011）Cold Spring Harb Perspect Biol3
4. Schwudke, D., Liebisch, G., Herzog, R., Schmitz, G., and Shevchenko, A.（2007）Methods Enzymol433, 175-191
5. Herzog, R., Schwudke, D., Schuhmann, K., Sampaio, J. L., Bornstein, S. R., Schroeder, M., and Shevchenko, A.（2011）Genome
Biol12, R 8
6. Sweeney, S. T., and Davis, G. W.（2002）Neuron36, 403-416

Oral Session
Wedensday, 19th September

Main Hall

15:00 － 17:00

Session 26: Lipidomics : Recent New Techniques and Applications
Chair:

Stephen J Blanksby（University of Wollongong, Australia）

S26-1620
16:20 － 16:40

What is a complete lipidome? A snipers approach to shotgun analysis of
the human lens.

Jane M Deeley, Jessica R Hughes, Shane R Ellis, Jo Ann Seng, Roger J Truscott, Stephen J Blanksby,
Todd W Mitchell
University of Wollongong, Wollongong, Australia

Keywords:
Lipidomics, ozone-induced dissociation, TLC-DESI, ambient ozonolysis, MS imaging
Novel aspects:
We have developed new mass spectrometry based techniques for lipid characterization and used them to identify novel lipids inthe human lens.
Abstract:
The lens is an essential component of vision, allowing light impinging upon the eye to be focused onto the retina. Presbyopia andcataract are age-related disorders of the lens and are the two most common causes of visual impairment and blindness
worldwide. Considerable knowledge has been attained regarding the biochemistry of lens proteins and their possible role in thedevelopment of these diseases, yet our knowledge of lens lipids is limited in comparison. To address this disparity we have
utilized numerous modern mass spectrometry techniques, e.g. shotgun lipidomics, MALDI imaging, DESI imaging, liquid extraction
surface analysis（LESA）and TLC-DESI and novel techniques, e.g. ozone-induced dissociation（OzID）and TLC-ambient ozonolysis
to investigate the lipid composition of the human lens and how it is altered as we age. Through the use of OzID we have beenable to identify that most phospholipid classes in the lens are dominated by alkyl ethers and that numerous double bond isomers
are found in both the ether and ester-linked acyl chains. For example, the most abundant glycerophospholipid ion observed
during MS analysis of human lens lipids extracts was found to be a mixture of up to four double bond isomers, i.e. PE（11Z-18：
1 e/ 9 Z-18： 1 ）, PE（ 9 Z-18： 1 e/ 9 Z-18： 1 ）, PE（ 9 Z-18： 1 e/11Z-18： 1 ）and PE（11Z-18： 1 e/11Z-18： 1 ）. OzID and TLCambient ozonolysis-DESI have also uncovered the presence of several molecular lipids that had not previously been identified, e.g.
PS（13Z-18： 1 e/ 9 Z-18： 1 and SM（d18： 0 /19Z-24： 1 ）. Through the use of TLC-DESI we have also obtained the first
identification of molecular sulfatides, dihydrosulfatides, lactosylceramide sulfates and dihydrolactosylceramide sulfates within thehuman lens. Based on this information we have developed specific precursor ion and neutral scans for the detection andquantification of these lipids using a shotgun lipidomics approach or directly from lens tissue using LESA. Finally, we have also
been able to observe large changes in both the concentration and distribution of molecular lipids in the lens with age utilizing
shotgun lipidomics in combination with both MALDI and DESI imaging.
From the example of the human lens it is clear that there is no single mass spectrometry platform capable of comprehensively
analyzing a complete lipidome. Through the use of various techniques however, we are edging towards such an understanding in
the human lens. In this talk I will discuss some of the techniques we have developed and how we have applied them to thecharacterization and measurement of molecular lipids in the human lens.

Oral Session
Wedensday, 19th September

Main Hall

15:00 － 17:00

Session 26: Lipidomics : Recent New Techniques and Applications
Chair:

Stephen J Blanksby（University of Wollongong, Australia）

S26-1640
16:40 － 17:00

Toward Total Structural Analysis of Complex Lipids: Electrospray
Ionization Multiple-stage Linear ion-trap with High-resolution Mass
spectrometric Analysis of Intact Ions

Fong-Fu Hsu

Washington University School of Medicine in St. Louis

Keywords:
Complex lipids； Double bond localization； Multiple-stage mass spectrometry； glycerolipids
Novel aspects:
This report presents linear Ion-trap MSn with high-resolution mass spectrometry for complete structural characterization ofcomplex lipids that would be difficult to define using traditional approaches
Abstract:
Recent development in mass spectrometric techniques has advanced lipid research to a new level. The mass spectrometricapproaches facilitate characterization of complex lipid structures, including features such as the identity of fatty acid substituents,
their sn-position on the glycerol backbone, and the positions of carbon-carbon double bonds along the acid chain of theglycerolipids. Among the various approaches including high-energy collision induced dissociation, covalent adduct chemical
ionization, and more recently, ozone-induced dissociation and low-energy collision induced dissociation in line with multiple-stage（MSn）linear ion-trap（LIT）mass spectrometry, the latest two methods are the most comprehensive and allow a near complete
identification of the lipid structure. In this presentation, I will describe LIT MSn with high-resolution mass spectrometry to achieve
structural determination of complex lipid isolated from biological specimen by applying（ 1 ）MSn on the［M - H］- ions for definitionof the lipid class and identification of the fatty acid substituents, as well as determination of their regio-specificities,（ 2 ）MSn on
the corresponding［M + Li］+ or［M - H+ 2 Li］+ ions for confirmation of the structure assignment and more importantly, locating the
double bond（s）, cyclic chain and branch chain positions of the fatty acid substituents. The fragmentation pathways for formationof the fragment ions arising from rearrangement processes and leading to structural assignments will be proposed. Examples
including differentiation among isomers and revelation of isomeric structures in a mixture using high resolution and MS n mass
spectrometry will be given and the prospects of this analytical method in the lipid study in the future, in particular, in the structure
determination of complicate microbial lipids will be discussed.

Oral Session
Wedensday, 19th SeptemberRoom A

15:00 － 17:00

Session 27: Progress in Microbiology
Chair:Catherine Fenselau（University of Maryland, USA）

S27-1500
15:00 － 15:40[Keynote Lecture] Translating metabolic phenotyping and systems
medicine into clinical Practise: Understanding gut microbial-hostInteractions in personal and public healthcare problems

Jeremy K Nicholson

Imperial College London

Keywords:
Microbiome Metabolism Healthcare Stratified Medicine
Novel aspects:
Spectroscopic modelling of complex micro biome-host metabolic interactions to create new healthcare and drug targeting
opportunities
Abstract:
The composition, activities and function of the gut microbiome in health anddisease is of currently of great interest in basic human biology and medical
research. Multiple and diverse discoveries on the close associations between
microbiome variation and the etiopathogenesis of many common non-infectious
diseases has lead to new research initiatives designed to answer key
microbiome questions about“Who is there； what are they doing； how did they
get there； what are their functions； what happens if things go wrong and how
do we manipulate them for human health benefits?“ To answer these questions
on the functional properties of microbiomes requires systems approaches that
go well beyond metagenomic mapping as it is the response of the host to
microbiological variance that is of critical importance in human health andfor the informed use of microbiome data in the development of new
therapeutic approaches. This involves deep metabolic phenotyping via
spectroscopic methods. The extensive cross-talk between the host and the gut
microbiome at the metabolic control and signalling level that is modulated
in exquisitely complex ways by genes and environment and link to diseaserisk factors（1,2）. These symbiotic supraorganismal interactions greatly
increase the degrees of freedom of the metabolic system that poses a
significant challenges to fundamental notions on the nature of the humandiseased state, the etiopathogenesis of common disease and current
analytical modelling requirements that are required for future developmentsin personalised healthcare and“precision medicine“（3,4）. The principal
analytical challenges involve integrative analysis of biofluid metabolic
signatures via mass spectrometric and NMR spectroscopic methods in relation
to to microbiome variation and the extraction of biomarker patterns andmodels that can be used to understand human disease processes and/or theirtreatments. Here we will explore the systems analytical approaches tounderstanding the functional aspects of microbiome activity and describe
possible discovery roadmaps to elucidate the many roles of the microbiome inhuman health and disease.
1. Nicholson J.K. and Lindon, J.C.（2008）Nature 455 1054-1056.
2. Swann, J.R. et al（2011）PNAS 108 4523-4530.
3. Mirnezami, R. Nicholson, J.K. and Darzi, A.（2012）New Eng. J. Med. 366（ 6 ）489-491.
4. Clayton, T.A. et al（2009）PNAS 106 14728-14733.

Oral Session
Wedensday, 19th SeptemberRoom A

15:00 － 17:00

Session 27: Progress in Microbiology
Chair:Catherine Fenselau（University of Maryland, USA）

S27-1540
15:40 － 16:00

Distribution and identification of molecular interactions between tomato
roots and bacterial biofilms

Delphine Debois1, Emmanuel Jourdan2, Nicolas Smargiasso1, Marc Ongena3, Philippe Thonart2,
Edwin De Pauw1

University of Liege, Liege, Belgium, 2Walloon Centre for Industrial Biology, Liege, Belgium, 3Gembloux Agro-Bio Tech,
Gembloux, Belgium

1

Keywords:
MALDI-Imaging, Plant-bacteria interaction, lipopeptides, biofilm
Novel aspects:
MALDI-Imaging allowed mapping tomato roots-biofilm interactions. Different lipopeptides families were shown to be produced
according to the age of the root-biofilm system. Finally, a putative new lipopeptide family was detected.
Abstract:
Some non pathogenic microorganisms evolving in the root micro-environment can trigger a positive effect on plant, increasing hostdefense against disease or/and directly inhibiting growth of pathogen in soil（ 1 ）. To initiate both phenomena leading tobiocontrol activity, microorganisms use plant exudates to grow on roots and to produce in-situ active compounds. In Bacilli, cyclic
lipopeptides of the surfactin, iturin and fengycin families represent important antibiotics involved in biocontrol（ 2 ）. Recent
studies in microbiology allowed a better understanding of plant microorganism interactions but few has been done at themolecular level. In this study, MALDI-MS imaging has been used to study the nature of the secreted lipopeptide molecules, theirrelative quantity and their distribution in the root’s environment.
Disinfected tomato seeds were first germinated at 28°C in sterile conditions for germination. Seedlings were then placed in Petri
dish on ITO glass slide recovered with a thin layer of plant nutritive solution（Hoagland）containing 1,75% of agar and treated withfreshly-grown cells of Bacillus amyloliquefaciens S499. Petri dishes were finally incubated vertically in phytotron at 28°C with a 16h
photoperiod. Different root age / time of incubation were studied： 13 / 3 ； 13 / 7 ； 21 / 14 and 39 / 32. Control tomato root（without bacterial treatment）of the same ages were also analyzed（13 / 0 ；21 / 0 and 42 / 0. For MALDI imaging experiments,
the ITO slide was removed from the agar and dried in a dessiccator under vacuum. The matrix solution（α-cyano-hydroxycinnamic
acid, 5 mg/mL in ACN/0.2 % TFA 70/30）was applied with an ImagePrep automated sprayer（Bruker Daltonics）. An UltraFlex II
TOF/TOF and a Solarix FT-ICR mass spectrometers were used to record molecular cartographies.
The average mass spectra recorded around the tomato root（ 2 - 3 mm on both sides of the root）showed that lipopeptides weremajor compounds detected on the agar. The relative intensity of lipopeptides families varied with respect to the age of the root/
biofilm system. In the 13/ 3 system, 3 homologues of surfactins were essentially detected（C13, C14 and C15）, with very few
iturins and fengycins. Their localizations were identical, whatever the considered homologue. Then the production of iturin andfengycin families increases in older systems（13/ 7 and 21/14）and a novel homologue of surfactin is detected（C12）. Somevariations in localization within families may be observed（around the root or at the close vicinity of it in function of the consideredhomologue or alkali adduct）. Then for the oldest system we studied, iturins and fengycins are not detected anymore and the
localization of surfactins is less precise. In the 39/32 system, we also detected unknown compounds at 986.6, 1000.6, 1014.7 and1028.7 m/z. The mass range of these compounds allied to the mass difference between two consecutive ion peaks let us think thatthese unknown compounds could be a new lipopeptide family. Investigations are in progress to identify these new secondary
metabolites of Bacillus amyloliquefaciens.
References（ 1 ）Lugtenberg B, Kamilova F（2009）Plant-Growth-Promoting Rhizobacteria. Annu Rev Microbiol 63：541-556（ 2 ）Ongena M, Jacques P（2008）Bacillus lipopeptides： versatile weapons for plant disease biocontrol. Trends Microbiol 16： 115125

Oral Session
Wedensday, 19th SeptemberRoom A

15:00 － 17:00

Session 27: Progress in Microbiology
Chair:Catherine Fenselau（University of Maryland, USA）

S27-1600
16:00 － 16:20

Identification of Protein Biomarkers as Diagnostic Targets for Invasive
Aspergillosis using Discovery and Targeted Proteomics

Chengsi Huang1,3, Jason McCarthy2, Marta Feldmesser2, Vicki H Wysocki1,3

University of Arizona, Tucson, AZ, USA, 2Albert Einstein College of Medicine, Yeshiva University, Bronx, NY, USA, 3Ohio
State University, Columbus, OH, USA

1

Keywords:
Aspergillus, Discovery/ Targeted Proteomics, Plasma
Novel aspects:
The ability to detect Aspergillus fungal proteins in host plasma in pulmonary diseases； identification of same Aspergillus protein
in both mouse and human plasma.
Abstract:
Aspergillus fumigatus is a ubiquitous fungus. While an average individual’s healthy immune system will effectively clear out theconidia inhaled, immunocompromised patients may develop invasive aspergillosis（IA）, a potentially fatal disease. Current
diagnostic tests can be time-consuming and/or invasive, and have variable accuracy. Consequently, patients with IA often receive
treatment too late. In addition, A. fumigatus infection can lead to other diseases such as aspergilloma, or allergic
bronchopulmonary aspergilloma. Hence, development of a fast, sensitive, and less invasive diagnostic technique is critical to being
able to correctly and confidently identify IA in patients. The goal of the present study is to identify A. fumigatus proteins that are
specifically present during IA. The identified target proteins will help develop future diagnostic tools.
In this study, mouse is used as the model system. CBA/J mice were divided into three different groups： naïve control, asthmamodel, and IA-model. Lung homogenate, BAL fluid, plasma, and serum were collected from all mice. A bottom-up proteomics
approach was utilized for analysis. Due to the complexity of the sample, several purification treatments were implemented andoptimized in order to provide the greatest number of protein identifications. Plasma samples were depleted via mouse spincolumns（Seppro® Mouse）. The depleted then digested samples were analyzed via LC-MS/MS on the Thermo Scientific LTQ
Velos Orbitrap via either general discovery methods or targeted methods. Results were searched against a database composed ofAspergillus fumigatus, Mus musculus, and common contaminants using various algorithms for protein identification.
The mouse plasma samples yielded identification of A. fumigatus proteins. By comparing the three models, differential expressionof A. fumigatus proteins was found. We found several Aspergillus proteins with confident two peptide hits that were exclusive to
the IA sample. These proteins, however, exhibit shared homology to other organisms, and are hence are not specific enough toserve as diagnostic targets. Based on multiple spectral count single peptide hits, we also found numerous proteins that are
exclusive to either the IA sample or the asthma sample. Out of these proteins, through multiple sequence alignment, we
generated a list of approximately thirty possible candidates that are fumigatus only, pan-aspergillus, and/or pan-fungal. The
presence of these proteins is confirmed via targeted analysis on the Orbitrap in individual mouse samples. Meanwhile, we also
observed similar host protein patterns and the same Aspergillus protein in a patient sample. This gives confidence in using mousemodels for human diagnostic study development.

Oral Session
Wedensday, 19th SeptemberRoom A

15:00 － 17:00

Session 27: Progress in Microbiology
Chair:Catherine Fenselau（University of Maryland, USA）

S27-1620
16:20 － 16:40

Microbial genome mining for novel bioactive natural products with highresolution LC-MS/MS

Lijiang Song1, JuanPa Gomez-Escribano2, Luisa Laureti3, Eshwar Mahenthiralingam4, Bertrand Agile3,
Meryvn Bibb2, Gregory Challis1

University of Warwick, Coventry, UK, 2John Innes Centre, Norwich, UK, 3University of Nancy, Nancy, France, 4Universityof Cardiff, Cardiff, UK

1

Keywords:
genome mining, microbial drug discovery
Novel aspects:
Genome mining as a novel efficient approach for antimicrobial natural product discovery with mordern analytical tools combinedwith molecular microbiology and bioinformatic techniques.
Abstract:
Antimicrobial resistance is a growing health problem worldwide and there is an urgent requirement for new antibiotics to deal
with life-threatening infection, such as those caused by MRSA and VRSA. Meanwhile, the pace of discovery of novel antibiotics has
been slowed significantly by the frequent re-isolation of known compounds with the traditional bioactivity-based screening
approach, which led to the notion that most natural products had already been discovered, and prompted many pharmaceuticaland agrochemical companies to terminate their natural product discovery programs. However, with many microbial genome
sequences becoming available, a successful new approach, genome mining, has been developed in recently years. This new
approach benefits in particular from modern chromatography, mass spectrometry and NMR spectroscopy, combined with modern
molecular biology and bioinformatics tools. Many novel bioactive natural products have been discovered in an efficient manner by
this approach not only from the well-known antibiotic producer, the gram positive actinomycetes, great potential has also beenrealized in gram negative bacterial, such as Burkholderia.
Large number of annotated bacterial genome sequences are completed and deposited in varies database, many of them arefreely available in the public domain, target gene clusters, in particular, PKS and NRPS,（mainly encoding polyketide synthases（PKSs）and non-ribosomal peptide synthetases（NRPSs））were located and mutants with biosynthetic genes deleted or regulator
genes activated or overexpressed were prepared. Structural information of potential products of such gene clusters can often be
predicted by sequence analysis. Comparative metabolite profiling using LC-MS/MS and targeting predicted structural properties ofmetabolic products led to their identification.
This talk will cover the identification, isolation and structure elucidation of coelimycin P, an unusual yellow-pigmented metabolic
product of the cpk cryptic polyketide biosynthetic gene cluster of Streptomyces coelicolor M145, using a genetic engineering
strategy designed to increase metabolic flux through the biosynthetic pathway. The discovery of a family of novel 51-memberedmacrolide antibiotics, the stambomycins, as the metabolic products of the Streptomyces ambofaciences by activation of a silent
type I PKS gene cluster will be discussed. The identification of enacyloxin IIa as the product of the cryptic PKS gene cluster andinsight into enacyloxin biosynthesis provided the basis for potential genetic engineering to generate more analogues. This new
genome mining approach, which relies on modern LC/MS/NMR instrumentation, has been shown to be a highly successful
approach for the discovery of novel bioactive natural products.
Acknowledgement： This project was supported by ActinoGEN an integrated project funded by the European Commission underthe 6 th framework program（FP 6 -5224）.The Bruker MaXis mass spectrometer used in this research was obtained, throughBirmingham Science City: Innovative Uses for Advanced Materials in the Modern World (West Midlands Centre for Advanced
Materials Project 2), with support from Advantage West Midlands (AWM) and part funded by the European Regional
Development Fund (ERDF).

Oral Session
Wedensday, 19th SeptemberRoom A

15:00 － 17:00

Session 27: Progress in Microbiology
Chair:Catherine Fenselau（University of Maryland, USA）

S27-1640
16:40 － 17:00

Novel accurate bacterial identification by MALDI-TOFMS based on
ribosomal protein coding in S10-spc-alpha Operon at Stain level

HIROTO TAMURA2, YUDAI HOTTA2,3, HIROAKI SATO4, KEISUKE SHIMA1, AKIFUMI HOSODA2,NORIYUKI OJIMA1SHIMADZU Corporation, Analytical & Measuring Instruments Division, 2School of Agriculture, Meijo University,
Formulation Technology Institute, Kumiai Chemical Industry Co., 4Research Institute for Environmental Management
Technology, National Institute of Advanced Industrial Science and Technology
1
3

Keywords:
MALDI-TOFMS, bacterial identification, bioinformatics, ribosomal protein, database search
Novel aspects:
Novel Accurate MALDI-TOFMS bacterial identification method at strain level, named S10-GERMS method, using ribosomal proteinscoded in S10-spc-alphaoperon as biomarkers, which are theoretically calculated and validated.
Abstract:
Matrix-assisted laser desorption ionization-time-of flight mass spectrometer（MALDI-TOFMS）is one of the most widely used massbased approaches for bacterial identification and classification because of simple sample preparation and extremely rapid analysis
within a few minutes without any substantial costs for consumables. Two approaches are utilized for MALDI-TOFMS bacterial
identification. First approach is the pattern matching between the experimental MS spectrum of unknown bacteria and MS spectraof known bacteria species in database. This method can realize stable bacterial identification at species level independent on
culture conditions and operator’s skill, because database includes only m/z of the peak, which is stably detected in mass
spectrum of bacteria at any conditions. Some bacteria identification systems on the basis of pattern matching are commercialized
and are rapidly expanding to clinical, food safety sterility testing and so on. However, pattern matching methodology cannot
assure to identify bacteria at strain level, because infrequently detecting strain specific ion peaks are almost missed in database.
In biomarker finding as second approaches, m/z of species or strain specific ion peaks are theoretically calculated andcorresponding peaks to theoretical peaks are found in experimental MS spectra for bacterial identification. This method has highpotential to identify bacteria at strain level, because strain specific peaks are known and validated before measurement. However,the bacterial identification method based on the bioinformatics approach has several caveats；（ 1 ）the being identified or aclosely related strain must have been genome-sequenced；（ 2 ）the accuracy of the genomic information is critical；（ 3 ）becauseof post-translational modification, there can be a significant difference between the theoretical mass based on a genomic
database and the observed mass of the protein by MALDI-TOFMS. To avoid uncertain biomarker peaks, we are proposing thebacterial identification method at the strain level using ribosomal subunit proteins as biomarkers for the rapid identification ofbacteria. Because ribosomal subunit proteins have fewer post-translational modifications except for N-terminal methionine loss, it
is easy to calculate theoretical biomarker masses of the genomic-sequenced strain. Finally, the ribosomal subunit proteins codedin the S10-spc-alphaoperon, which encodes half of the ribosomal subunit protein and is highly conserved in eubacterial genomes,
were selected as reliable biomarkers for bacterial identification at strain level.
We have already reported that our proposal method could successfully distinguish the difference at strain level because of massdifference of 2 ribosomal subunit proteins, despite the difference of only 2 bases in the 16S rRNA gene between two strains ofgenus Bacillus. In this presentation, we reported other application examples of bacterial identification at strain level and propose
newly accurate, robust and easy bacterial identification method by combining pattern matching and biomarker method.

Oral Session
Wedensday, 19th SeptemberRoom B-1

15:00 － 17:00

Session 28: IR Spectroscopy of Gas-phase Ions
Chair:

Dietmar Kuck（Bielefeld University, Germany）

S28-1500
15:00 － 15:40[Keynote Lecture] IR Photodissociation Spectroscopy: A new dimension to
Tandem Mass Spectrometry

Philippe Maitre

Paris Sud University / CNRS - Orsay - FRANCE

Keywords:
Specific Ion Activation； Ion Spectroscopy； IRMPD； Peptide Fragmentation； Gas-Phase Ion Structure
Novel aspects:
The coupling of tunable infrared lasers with commercial tandem mass spectrometer offers a specific activation method for deriving
structural information and thereby provides a new dimension to mass spectrometry.
Abstract:
Recent coupling of tunable and highly intense pulsed IR laser sources with commercial tandem mass spectrometer offers a new
dimension to mass spectrometry. Free-Electron-Laser（FEL）facilities such as FELIX（Nieuwegein, Netherlands）and CLIO（Orsay,
France）have recently been shown to be particularly well suited IR light sources for performing IR spectroscopy of mass-selected
molecular ions. FELs are particularly interesting since they produce tunable mid-IR radiation in a broad wavenumber range
covering the so-called molecular fingerprint region（typically 600-2000cm-1）, and their high intensity makes them very suitable for
IRMPD（IR Multiple Photon Dissociation）spectroscopy of gas phase molecular ions.
At CLIO, experiments are performed using two commercial instruments： a quadrupole ion-trap（QIT）or a 7 teslas Fourier
Transform Ion Cyclotron Resonance（FT-ICR）mass spectrometer coupled with the IR FEL. In addition, IR spectra can be recordedin the NH and OH stretching region using an optical parametric oscillator/amplifier（OPO/OPA）laser system. In this case, inparticular, an auxiliary CO2 laser can be used for improving the sensitivity of the spectroscopy.
Understanding the structures, energetic, and decomposition pathways of the gas phase peptide fragments is central to the widelyused“bottom-up“ approach for proteome sequencing based on mass spectrometry. We will focus our attention on the structureof N-terminal CID fragments（an +or bn +）of peptides. We will see that the 1600-2000 cm-1 spectral range is the most diagnosticallyuseful part of the IR spectrum, especially for distinguishing oxazolone from other isomers of bn + ions. IR spectra of larger bn +, andalso an +, ions will also be presented providing evidence of the formation of a macro-cyclic structure. The isomerisation of peptide
fragments will be discussed in light of very recent results, and we will see that IR signatures of rearranged peptide fragments canalso be found in the NH and OH stretching region.

Oral Session
Wedensday, 19th SeptemberRoom B-1

15:00 － 17:00

Session 28: IR Spectroscopy of Gas-phase Ions
Chair:

Dietmar Kuck（Bielefeld University, Germany）

S28-1540
15:40 － 16:00

Intracavity infrared multiple photon dissociation spectroscopy oftransition metal carbene cations: Agostic interactions or not?

Joost M Bakker1,2, Vivike Lapoutre1, Andrew Sweeney3, Abhigya Mookherjee3, Peter B Armentrout3
1
3

FOM Institute for Plasma Physics Rijnhuizen, The Netherlands, 2Radboud University Nijmegen, The Netherlands,
University of Utah, Salt Lake City, USA

Keywords:
intracavity, transition-metal, agostic-interactions, carbene
Novel aspects:
This study provides the first IR spectroscopic characterization of transition metal carbene cations and reveal details of theirbonding.
Abstract:
Methane, a key fossil fuel, is difficult to transport and difficult to convert to other energy rich chemicals because of the strong CH
bond； however, several third-row metal cations readily activate methane to form MCH 2 + + H2, a metal carbene. Except in one
case, structures of these species have not been experimentally determined, although theory suggests several competing
structures, some of which involve“agostic“ interactions in which the electrons in a CH bond donate to the metal, distorting its
structure. Transition metal carbenes are also interesting because they act as heterogeneous catalysts in the Shell higher olefin
reaction that produces plasticizers and detergents. The low density of states in these four atom species means that efficientdissociation using multiple infrared（IR）photons requires high powers, made accessible recently by the Free Electron Laser for
IntraCavity Experiments（FELICE）at the FOM Institute.
Ta, W, Ir, and Pt rods were ablated with a frequency doubled YAG laser（532 nm）to create gas phase atomic metal ions. These
ions are entrained by a pulse of He gas containing methane gas to form metal carbene cations, MCH 2 +. Ionic species areirradiated by FELICE and the frequency of the light is varied from 300 to 3400 cm-1. Absorption of multiple IR photons can occur at
frequencies corresponding to vibrational modes of the metal carbene cations, leading to dissociation of the molecule either by H
or H 2 loss. Fragments and reactant ions are detected and mass analyzed using time-of-flight mass spectrometry（TOF MS）.
The IR multiple photon dissociation（IRMPD）action spectrum of TaCH 2 +（loss of H）is consistent with the calculated spectra forthe 3 A’ground state of the carbene, TaCH 2 +. Calculations indicate this species has an“agostic“ interaction, such that themolecule no longer has the expected C 2 v symmetry. No evidence for a hydrido-carbyne（HTaCH+）species is observed. The
IRMPD spectrum of WCH 2 +（loss of H）is dominated by contributions from the ground state WCH 2 +（ 4 A“）, which has NO
agostic interaction. Some evidence for contributions from a low-lying HWCH+（ 2 A’）species is possible. The IRMPD spectrum of
IrCH 2 +（loss of H 2 ）is consistent with the calculated spectrum of the ground state species, HIrCH+（ 1 A）with possible
contributions from IrCH 2 +（ 3 A）structures, which have no agostic interactions. The IRMPD spectrum for PtCH 2 +（loss of H）isconsistent with that calculated for the PtCH 2 +（ 2 A 1 ）ground state structure. This spectrum shows clear evidence of an overtone
band at ~2000 cm-1. This late metal carbene cation has NO agostic interaction, consistent with no empty orbitals on the metal.
The high power afforded by the intracavity operation of FELICE allows efficient IR photodissociation of the transition metalcarbene cations, species with a low density of states that would otherwise make them incompatible with IRMPD measurements.
Such high powers can also complicate the spectrum, however, by introducing overtone bands.

Oral Session
Wedensday, 19th SeptemberRoom B-1

15:00 － 17:00

Session 28: IR Spectroscopy of Gas-phase Ions
Chair:

Dietmar Kuck（Bielefeld University, Germany）

S28-1600
16:00 － 16:20

Gas-Phase Structure and Chemistry of the Radical Cation of DNA basepair dGdC

George N khairallah1, Linda Feketeova1, Bun Chan2, Vincent Steinmetz3, Philippe Maitre3, Leo Radom2,
Richard A O’Hair1
University of Melbourne, Melbourne, Australia, 2The University of Sydney, NSW 2006, Australia, 3Universite Paris Sud,
Orsay Cedex, 91405, France

1

Keywords:
Nucleobases, radical cations, CID, IRMPD spectroscopy, Watson-Crick base pair
Novel aspects:
This work is a first combined mass spectrometry/ IRMPD spectroscopy study of a simple model for radical cations of thecomplimentary base pairs found in DNA
Abstract:
Radiation damage of living cells can cause potential biological mutations. It is agreed that the mostradiation sensitive biomoleculein living tissues is DNA, where damage can occur in either the nucleobase or the sugar moiety, leading to strand breaks andcausing mutagenesis and cancer amongst others. Collision-induced dissociation（CID）of a metal-biomolecule complex has beenpreviously used to generate radicals from biomolecules, where loss of the biomolecule from the complex proceeds via electrontransfer to produce the radical cation. Here, we used this method to form the deoxyguanosine（dG）/deoxycytidine（dC）radical
cation（［dGdC］.+）. This latter represents a simple model for radical cation formation of the complimentary base pairs found in
DNA. In order to determine its gas-phase structure, IRMPD spectroscopy was used in combination with theoretical calculations.
IRMPD spectroscopy of mass-selected ions is now an established and powerful technique for the structural characterization of
gaseous ions and has been proven to work well with biomolecules.
We have used a hybrid ion trap/ ICR mass spectrometer（Finnigan LTQ-FT）. This instrument consists of an ESI ion source coupled
to a linear ion trap（LTQ）and a FTICR cell for high resolution accurate mass measurements. Multistage CID experiments wereused to study the unimolecular chemistry of［dGdC］.+. IRMPD spectroscopy was performed at the CLIO facility in Paris using a FEL
in the spectral range 900-1800 cm-1 as well as an OPO/CO2 laser in the spectral range 3100-3700 cm-1. These lasers were coupled toeither a 3 D ion trap or an FTICR mass spectrometer. Ab initio calculations were also undertaken using the Gaussian09 programand geometries were optimized with the M06- 2 X/ 6 -31G（d,p）. Literature scaling factors were employed in the evaluation of the
ZPVE and IR frequencies.
The Unimolecular CID study has revealed a rich gas-phase chemistry including radical migration. Hence, the dissociation of theradical dimer,［dGdC］.+, showed that the radical migrates from the guanine nucleobase to its sugar moiety. From this, it is possible
to conclude that radical damage to the nucleobase can propagate to the sugar backbone, leading to the cleavage of the DNA
strand. The structure of［dGdC］.+ was revealed via a combination of theoretical calculations and experimental gas-phase infrared（IR）spectroscopy. The results obtained provided proofs indicating a hydrogen bonded［dGdC］.+ dimer structure（i.e.； Structureof the Watson-Crick base pair）. The suitability of the theoretical calculation method used, was further proven via the successfulcomparison of the IRMPD spectrum of a simpler molecule, i.e.；［ 9 -methyl-guanine］.+,to its calculated minimum energy structures.
Therefore, the details of the fragmentation mechanisms as well as the IRMPD spectra and calculated structures will be presented.

Oral Session
Wedensday, 19th SeptemberRoom B-1

15:00 － 17:00

Session 28: IR Spectroscopy of Gas-phase Ions
Chair:

Dietmar Kuck（Bielefeld University, Germany）

S28-1620
16:20 － 16:40

Infrared spectroscopic studies on isomerization reactions of ionizedmolecular clusters

Yoshiyuki Matsuda, Naohiko Mikami, Asuka Fujii
Tohoku University, Sendai, Japan

Keywords:
Infrared spectroscopy, cluster, vacuum-ultraviolet photoionization, isomerization
Novel aspects:
Ionization dynamics of molecular clusters has been studied with infrared spectroscopy and theoretical calculation. This
spectroscopic approach has been developed by us and is based on the vacuum-ultraviolet photoionization detection.
Abstract:
Understanding of ionization dynamics of molecular clusters would contribute to provide molecular level insights into ionizationand protonation mechanisms in various ionization methods. We have investigated isomerization reactions upon ionization ofmolecular clusters with infrared（IR）spectroscopy based on the vacuum-ultraviolet（VUV）photoionization detection.［ 1 ］This
spectroscopy enables to observe IR spectra of size-selected neutral and cationic clusters, which correspond to precursors andproducts in the VUV one-photon ionization, respectively. Therefore, this technique is powerful to study ionization dynamics ofclusters.
Ionization-induced isomerization of various protic/aprotic molecules and their hydrated clusters has been investigated by
combining the IR spectroscopic approach and theoretical calculations.［ 1 - 3 ］It has been elucidated that the proton-transfer
reactions occur between the hydrophilic groups without energy barriers in the ionization of the clusters of methanol, ammonia,
and acetic acid.［ 1 ］The proton-transfer reactions from the hydrophobic groups such as an alkyl groups have also been found in
the acetone dimer, hydrated acetone, and hydrated formamide.［2,3］The ionized molecules in these clusters tautomerize to theenol forms through the transfer reactions of an alkyl proton. In the case of the hydrated clusters of acetone and formamide, thecatalytic action of a water molecule for the alkyl proton-transfer reaction has been demonstrated experimentally and theoretically.
［3］
Recently, the cationic structures of diethylether and trimethylamine dimers have been analyzed through the IR spectroscopy.
These results indicate that alkyl groups in the cations have high acidity. The acidity of the cationic alkyl groups as well as the alkyl
proton-transfer reactions will be discussed with the spectroscopic and theoretical results.
［ 1 ］Matsuda et al. Phys. Chem. Chem. Phys. 11, 1279（2009）.
［ 2 ］Matsuda et al. Phys. Chem. Chem. Phys. 14, 712（2012）.
［ 3 ］Matsuda et al. Angew. Chem. Int. Ed. 49, 4898（2010）.

Oral Session
Wedensday, 19th SeptemberRoom B-1

15:00 － 17:00

Session 28: IR Spectroscopy of Gas-phase Ions
Chair:

Dietmar Kuck（Bielefeld University, Germany）

S28-1640
16:40 － 17:00

IR-MPD investigation of steric constraints on hydrogen bonding in
dicarboxylic acid ions

Christine Merkert, Fabian Menges, Yevgeniy Nosenkov, Gereon Niedner-Schatteburg
TU Kaiserslautern, Germany

Keywords:
IR-MPD, hydrogen bonding, Eigen cation, Zundel cation, molecular switch
Novel aspects:
Steric constraints by twisting covalent bonds modulates proton transfer such that a model system for an isolated molecular switch
emerges, elucidated by ab intio calculations and by IR-MPD spectroscopy
Abstract:
When isolated the protonated and deprotonated dicarboxylic acid ions HOOC-（CH 2 ）n -C（OH）2 + and HOOC-（CH 2 ）n -COO stabilize in cyclic form through ionic hydrogen bonding. Other than e.g. in hydrogen bonded clusters the alkyl back bone causes
steric constraints which are expected to vary by the alkyl chain length. Ab initio calculations［ 1 ］predict a possible coexistence ofsymmetric（Zundel- type）and asymmetric（Eigen-type）hydrogen bonds in the case of adipic acid（n= 4 ）. A multitude oftransition states define a stepwise proton transfer reaction path in concert with an equally stepwise twisting of the alkyl chain.
We present Infra-Red-Multi-Photon-Dissociation（IR-MPD）spectra of dicarboxyilic acid cations and anions n=1,.., 6 and discuss
fingerprints of proton localization and / or delocalization within the hydrogen bond. The importance of an appropriate
temperature control will be emphasized. An outlook towards molecular switching will be provided.

［ 1 ］S.K.Min, M. Park, N. J. Singh, H. M. Lee, E. C. Lee, K. S. Kim, A. Lagutschenkov, and G. Niedner-Schatteburg； Chemistry - a
European Journal（2010）, 16（34）, 10373-10379

Oral Session
Wedensday, 19th SeptemberRoom D

15:00 － 17:00

Session 29: The Advances in Biological Mass Spectrometry in Drug Discovery andDevelopment: Current State of the Art and Challenges
Chair:

Ajai K Chaudhary（Merck and Company, USA）

S29-1500
15:00 － 15:40[Keynote Lecture] Advances in the Application of Mass Spectrometry to
Drug Metabolism and Pharmacokinetics Studies

Ragu Ramanathan1,2
1Bristol-Myers Squibb, 2Department of Biotransformation, Princeton, New Jersey 08540, USA

Keywords:
Drug Metabolism, Pharmacokinetics, Metabolites, Quantitation, Qual/Quant, UHPLC-Q-TOF
Novel aspects:
Invited keynote talk providing an overview of current applications of liquid chromatography-mass spectrometry（LC-MS）for
various drug metabolism and pharmacokinetics（DMPK）studies.
Abstract:
The focus of this keynote talk will be to present an overview of current applications of liquid chromatography-mass spectrometry（LC-MS）for various drug metabolism and pharmacokinetics（DMPK）studies. Discovering highly potent and highly target selective
new molecular entities（NMEs）are essential to develop efficacious and safe drugs for patients. Across the biopharmaceuticalindustry, LC-MS based techniques are utilized for many purposes by DMPK scientists due to their sensitivity, selectivity, specificity,and speed. Over the years, several mass analyzer types have been used in DMPK studies, including quadrupole mass filter（QMF）,
quadrupole ion trap（QIT）, linear ion trap（LIT）, time-of-flight（TOF）, and Fourier transform mass spectrometers（FTMS）.
Some of the desired qualities of the individual mass analyzer types and ion manipulation techniques have now been combined
into a single instrument, generally referred to as hybrid mass spectrometers. Hybrid mass spectrometers have become very
selective and specific when either a TOF or FTMS based high resolution mass spectrometer（HRMS）is combined with either ITs
or QMFs. The ability of LITs and QMFs to operate in the vacuum pressure range of 10-5 to 10-6 torr makes these types of mass
spectrometers ideally suited for coupling with LC separation and atmospheric pressure ionization（API）techniques as well as with
TOF and FTMS based mass analyzers, in tandem, for capitalizing on the high resolution capabilities of the later two mass
analyzers. Fast scanning HRMS based mass analyzers provide the option to integrate qualitative and quantitative analysis of drugs
and their metabolites, under ultra high pressure liquid chromatography（UHPLC）conditions, and facilitate an increased throughput
and streamlined workflow. These technological advances may allow a“paradigm shift“ in how MS is used for various DMPK
assays within the pharmaceutical industry.

Oral Session
Wedensday, 19th SeptemberRoom D

15:00 － 17:00

Session 29: The Advances in Biological Mass Spectrometry in Drug Discovery andDevelopment: Current State of the Art and Challenges
Chair:

Ajai K Chaudhary（Merck and Company, USA）

S29-1540
15:40 － 16:00

Development of stable isotope labeling by essential nutrients in cell
culture (SILEC) coupled with LC-MS for drug target discovery

Ian A Blair, Nathaniel W Snyder, Zinan Zhou, Andrew Worth, Sankha S Basu
University of Pennsylvania, Philadelphia, PA 19104, USA

Keywords:
LC-MS, stable isotopes, neurodegenerative disease, CoA, lipids
Novel aspects:
SILEC methodology coupled with LC-MS has revealed potential new targets that can be employed for the development of drugs
for treating neurodegenerative diseases.
Abstract:
Although mechanisms underlying selective cell death in neurodegenerative diseases such as Parkinson’s and Alzheimer’s disease,
Friedreich ataxia, and amyotrophic lateral sclerosis are not well defined, mitochondrial complex 1 dysfunction has long been
implicated. Coenzyme A（CoA）and CoA thioester derivatives are central players in numerous mitochondrial metabolic pathways.
However, the lack of isotopically labeled CoA standards has limited the implementation of rigorous LC-MS-based methods fortheir analysis. We have developed stable isotope labeling by essential nutrients in cell culture（SILEC）methodology to generate
stable isotope labeled CoA and thioester analogs for use as internal standards in LC-MS-based assays. This methodology can also
be used to generate other stable isotope metabolite standards（such as menadione, an inducer of oxidative stress）so that
accurate quantification of CoA species can be conducted. Much of the research on mitochondrial dysfunction has focused on
induction of reactive oxygen species（ROS）and reactive nitrogen species generation. While these reactive species undoubtedly
play a role in mitochondrial dysfunction by inducing macromolecular damage as well as by serving as signaling molecules, thesource of the ROS and their exact role remains controversial. Using stable isotope dilution LC-MS, we have now shown that
inhibition of complex 1 does not initially induce oxidative stress but causes a dramatic decrease in levels of succinyl-CoA andincrease in levels of acetyl-CoA. By using［ 13 C］-labeling and isotopomer analysis, we have characterized the metabolic
derangement in lipid metabolism that occurs. This has revealed potential new targets that can be employed for the developmentof drugs for treating neurodegenerative diseases. Supported by NIH grants T32HL007439 and P30ES013508.

Oral Session
Wedensday, 19th SeptemberRoom D

15:00 － 17:00

Session 29: The Advances in Biological Mass Spectrometry in Drug Discovery andDevelopment: Current State of the Art and Challenges
Chair:

Ajai K Chaudhary（Merck and Company, USA）

S29-1600
16:00 － 16:20

High-resolution metabolic profiling towards G-protein coupled receptors:
rapid and comprehensive screening of histamine H4 receptor ligands

Jeroen Kool1, Anders F Rudebeck1, Frank Fleurbaaij1, Saskia Nijmeijer1, David Falck1, Rogier Smits2,
Henry F Vischer1, Rob Leurs1, Wilfried M Niessen1
1

VU University, Amsterdam, The Netherlands, 2Griffin Discoveries B.V., The Netherlands

Keywords:
Correlation MS data functional activity
Novel aspects:
Identity with bioactivity correlation in metabolic mixtures. Bioactivity assessed as ligand binding selectivity and functional activity.
Most important： correlation to identity of individual metabolites with accurate MS and MS/MS analysis.
Abstract:
In the past years, we developed high-resolution screening platforms involving separation of bioactive mixtures and on-line or atline bioassays for a wide variety of biological targets with parallel mass spectrometric detection and identification. In the current
research, we make a major step forward in the development of at-line bioassays by implementation of（functional）cell-based andmembrane receptor binding assays. We demonstrate a new platform for high resolution metabolic profiling of lead compounds
for functional activity and selectivity toward the human histamine H 4 receptor（hH 4 R）, a GPCR. In this platform, a new postfractionation cell seeding strategy, analytical chemistry, cell biology and pharmacology are merged. The methodology is based on
chromatographic separation of metabolic mixtures by HPLC coupled to high-resolution fractionation onto（multiple）microtiterwell plates for complementary assaying. Bioaffinity and selectivity were measured with radioligand displacement assays, while thefunctional assay was a gene reporter assay with adherent mammalian cells as pharmacological endpoint. The methodology wasused for efficient and rapid metabolic profiling of three histamine hH 4 R selective lead compounds and the drug clozapine.
Metabolites with undesired altered selectivity and/or functional activity profiles can be identified as well as bioactivation. The
parallel structure identification of the metabolites, with accurate mass measurements and MS/MS, allowed identification of liable
metabolic ’hotspots’ for further lead optimization and plays a central role in the workflow and in this study. The methodology canbe easily adapted for use with other GPCRs and GPCR screening formats. The efficient use of combining cell-based and binding
assays with analytical techniques by leveraging high resolution at-line fractionation as a linking technology will allow
implementation of comprehensive metabolic profiling earlier on in the drug discovery process.

Oral Session
Wedensday, 19th SeptemberRoom D

15:00 － 17:00

Session 29: The Advances in Biological Mass Spectrometry in Drug Discovery andDevelopment: Current State of the Art and Challenges
Chair:

Ajai K Chaudhary（Merck and Company, USA）

S29-1620
16:20 － 16:40

THE ROLE OF MASS SPECTROMETRY IN DRUG DISCOVERY AND
DEVELOPMENTANTHONY TSARBOPOULOS

UNIVERSITY OF PATRAS, PATRAS, GREECE

Keywords:
UPLC Tandem Mass Spectrometry, Antibiotics, Protein PTMs, Noncovalent Interactions
Novel aspects:
Novel high-resolution UPLC-tandem MS methodologies for the quantification of cyclic peptide antibiotics. Monitoring of proteinligand noncovalent interactions by ESI MS to shed light into disease processes.
Abstract:
Mass Spectrometry（MS）has played an important role in the design, discovery and development of new pharmaceuticals, fromthe earliest stages of disease determination through the final stages of clinical testing. Today, the whole effort is“target-driven“
rather than the previously followed“disease-driven“ method. Once a target has proven to be related to the disease process,high-throughput screening（HTS）methods may be used to determine initial structural leads among compounds generated fromeither total synthetic process, derivative of natural products, natural product etc. The selected compounds are then used for invivo testing in preclinical species（mouse, rat, dog, monkey, etc.）. LC-MS and LC-MS/MS assays are the methods of choice for thelate-stage discovery studies because they are rapid, sensitive, easy to automate and robust. In general, LC-MS and LC-MS/MS
enters in all phases of drug discovery, i.e., phases I-III clinical trials, and especially providing an assessment of pharmacokinetics
and drug metabolism prior to new drug application（NDA）. The members of the modern（bio）pharmaceutical industry are
engaged in an on-going struggle to balance the needs for finding a new medicine while maintaining the relevant development costin reasonable levels. The advent of electrospray ionization（ESI）and matrix-assisted laser desorption/ionization（MALDI）methods
combined with the rapid proliferation of several types of mass analyzers（especially quadrupole time-of-flight and orbitrap）has
revolutionized the applicability of MS for studying proteins of high molecular weights by themselves or when they are bound toligands. That, in turn, has opened the way to elucidate macromolecular structures and study their interactions with small
molecules or other macromolecules, which are important aspects in understanding their biological function and developing new
therapeutics. One of the main features of the aforementioned MS methods is their unique ability to determine whether the protein
product presents any heterogeneity. This heterogeneity is usually associated with post-translational modifications（PTMs）, suchas glycosylation, phosphorylation, proteolytic processing, deamidation, aggregation and formation of disulfide bonds, with allthese contributing to the diversity of the protein product. Therefore, characterization of these protein molecules with regard topurity and structure is an essential and challenging task for protein therapeutics, and represents an integral part of the overallsubmission records to the regulatory agencies prior to protein’s approval as a drug.
This presentation will outline the development and validation of a rapid, simple and accurate UPLC-ESI MS/MS analytical method
on a hybrid qTOF instrument for the quantification of cyclic peptide antibiotics, which can be used in pharmacokinetic andmetabolic studies of these drugs. In addition, complete structural analysis of recombinant protein pharmaceuticals and MS
identification of PTMs, including identification of the glycosylation sites in a glycoprotein and structural elucidation of the attachedoligosaccharide components will be illustrated. Finally, analysis of certain noncovalent interactions between bioactive moleculesand proteins, which are responsible for certain diseases, will be demonstrated. Monitoring of these protein interactions in realtime by ESI MS will also provide the stoichiometry and the topology of the interacting species. This is nicely shown in thenoncovalent complex detection of amyloid beta（Aβ）peptide with bioactive compounds. The Aβpeptide is currently believed toplay a central role in the pathogenesis of Alzheimer’s disease（AD）, and screening of bioactive antioxidants could lead to novel
aggregation inhibitors for the prevention or treatment of AD. Overall, the elucidation of the structures and formation mechanismof these complexes could offer a better understanding of the disease processes, which in turn may aid in modern drug design.

Oral Session
Wedensday, 19th SeptemberRoom D

15:00 － 17:00

Session 29: The Advances in Biological Mass Spectrometry in Drug Discovery andDevelopment: Current State of the Art and Challenges
Chair:

Ajai K Chaudhary（Merck and Company, USA）

S29-1640
16:40 － 17:00

Evaluation of fully automated DBS extraction system with LC-MS/MS
systems for Sartans and Statins using different DBS card types

Souki Kanda3, YASUHIKO BANDO1,2, Takemoto Kawamukai1, Hiroshi Hike1, Amane Sakurai3
1

AMR,Inc., 2Biosys Technologies,Inc., Tokyo, Japan, 3Toray Research Center, Inc., Kanagawa, Japan

Keywords:
Automated DBS extraction, High sensitive, Robust, Sartan, Statin
Novel aspects:
Fully automated DBS extraction systems overcomes the limitation of off line extraction method for quantitation of LC-MS/MS.
Abstract:
Introduction
The use of Dry Blood Spotting for sample collection gains popularity because of the low sample volume, handling and logistic
benefits. It has been evaluated in a few years for the use in PK and TK studies, however, the reproducibility and sensitivity remains
as an issue with current off-line punching and manual extractions. In addition, the three commercially available DBS cards havedifferent characteristic resulted in the variation of extraction and quantitation of compounds to be analysed. In this study we haveevaluated the fully automated extraction of compounds from DBS card and LC-MS/MS in order to overcome the exisiting
limitations and validated the analytical method including drug stability on the cards for PK and TK application.
Methods
DMPK-A, DMPK-B, DMPK-C cards（GE healthcare）were evaluated with 8 compounds, 6 Sartans and 2 statins in rat whole blood（15ul）. The fully automated DBS extraction system（SCAP systems）integrated with LC-MS/MS（Agilent1100/4000QTrap）were
utilized and evaluated in the aspect of compatibility of compounds to the type of cards. In addition quantitation with the internal
standard method, reproducibility, drug stability on the card, sensitivity, reproducibility of Internal standard and carryover are
examined based on Valsartan on Dry Blood Spot DMPK-A card.
Preliminary Data
Due the pretreatment of DMPK-A and DMPK-B card, the extraction efficiency varied from compound to compound. In general,
DMPK-C card gained better extraction efficiency because of no pretreatment on the paper. No extraction took place on
Simvastatin for all DMPK-A, DMPK-B and DMPK-C cards. The degradation or strong adsorption onto the card can be considered,however, further investigation will be required. Quantitation of valsartan from rat whole blood on the DMPK-A card gave excellent
linearity from 10 - 10,000 ng/mL with 0.9987 correlation coefficient（r）under the internal standard method. The varsartan with 4
different concentration was spotted on the card and analysed in the same day. Accuracy（%）and CV（%）were both within 15%（n= 5 ）and S/N of 10ng/mL（LLOQ）was > 5 although the extraction area is only 1.5mm i.d.. The good reproducibility andsensitivity were achieved under fully automated systems. 15ul spot of whole blood with Varsartan, 20ng/mL（LQC）and 8000ng/mL（HQC）on DMPK-A card were packed with a plastic bag with desiccant and stored at ambient temperature for 14 days to see thestability. As a result of quantitation analysis, accuracy were both within + 15%. Injection of internal standard were examined in thesame analytical batch（n=35）and found CV（%）= 7.14. Ratio of peak area of 2 compounds, Varsartan and Losartan on DMPK-A
card were calculated in order to see reproducibility of 4 extraction（1.5mm i.d.）within 1 blood spot. Variance was less than 15%.
The reproducibility among spot 1 ~ 4 resulted in less than 15 % as well. 2 trap column switching system for the extraction of thecompounds of interest reduced ion suppression. Extra washing mechanism reduced carry over significantly. Fully automatedextraction system integrated with LC-MS/MS systems overcome the limitation of current off line extraction. The result shed thelight on the use of DBS for PK and TK application.

Oral Session
Wedensday, 19th SeptemberRoom E

15:00 － 17:00

Session 30: Data Processing and Informatics for SIMS
Chair:

DaeWon Moon（Center for Nano-Bio Technology, Korea Research Institute of Standards and Science, Korea）

S30-1500
15:00 － 15:40[Keynote Lecture] Image and Spectral Processing for ToF-SIMS Analysis of
Biological Materials

David G Castner

University of Washington, Seattle, USA

Keywords:
ToF-SIMS, multivariate analysis, image processing
Novel aspects:
Latest advances in multivariate analysis processing of ToF-SIMS image and spectral data will be presented. Proper correction anddisplay of 3 D images will also be presented.
Abstract:
Modern time-of-flight secondary ion mass spectrometry（ToF-SIMS）instruments can rapidly produce an enormous amount ofcomplex data. Each spectrum typically contains hundreds to thousands of peaks. Each pixel in a 2 D image contains a complete
spectrum, which means for each 256 x 256 pixel image a total of 65,536 spectra are collected. When extended to 3 D imaging thenumber of spectra increase by the number of 2 D images acquired during the depth profile. Thus, millions of spectra can beacquired in a 3 D image. This presents a significant challenge for the processing of ToF-SIMS image and spectral data. A powerful
method for extracting information from the data sets is to use multivariate analysis（MVA）, either in a supervised or unsupervised
mode. The unsupervised mode uses all peaks in the data set, while the supervised mode uses a subset of the peaks（e.g., aminoacid fragments for analyzing protein samples）.
Principal component analysis（PCA）is one of the most common MVA methods used to process ToF-SIMS data. It determines
which combination of original variables（i.e., peak intensities）account for variation across the data set. For example, it can beused to show that variation of different primary ion beam species（e.g., monoatomic vs. cluster）results in larger changes in theamino acid fragmentation pattern than the variation in amino acid content of different proteins（e.g., albumin vs. fibrinogen）［ 1 ］.
It can also be used to discover the presence of unexpected species such as trace photoresist residue on a polymer film［ 2 ］.
However, PCA results can depend on how the data is normalized and scaled, so it is often beneficial to do further processing withother MVA methods such as discriminant analysis or maximum autocorrelation factors（MAF）. MAF has the advantage that it isscaling independent and can be applied to images from a range of different samples［ 3 ］. Filtering or preprocessing the image
data prior to PCA processing can improve the PCA performance［ 4 ］.
An additional challenge of 3 D imaging is correcting the inaccuracies in the ToF-SIMS data that arise from projecting data from acurved surface onto a 2 D image plane. Software has been developed（ZCorrectorGUI）that allows the entire 3 D data cube to beproperly corrected and displayed［ 5 ］. This software along with other MVA software for processing of ToF-SIMS data is available
for free download at http：//mvsa.nb.uw.edu/.
［1］“ToF-SIMS Analysis of Adsorbed Proteins： Principal Component Analysis of the Primary Ion Species Effect on the Protein
Fragmentation Patterns“ S. Muramoto, D.J. Graham, M.S. Wagner, T.G. Lee, D.W. Moon and D.G. Castner, Journal of Physical
Chemistry C, 115, 24247-24255, 2011.
［2］“Surface Analysis of Photolithographic Patterns using ToF-SIMS and PCA,“ M. Dubey, K. Emoto, F. Cheng, L.J. Gamble, H.
Takahashi, D.W. Grainger and D.G. Castner, Surface and Interface Analysis, 41, 645-652, 2009.
［3］“Multivariate Analysis Strategies for Processing ToF-SIMS Images of Biomaterials,“ B.J. Tyler, G. Rayal and D.G. Castner,
Biomaterials, 28, 2412-2423, 2007.
［4］“Denoising and Multivariate Analysis of ToF-SIMS Images,“ B. Wickes, Y. Kim and D.G. Castner Surface and Interface
Analysis, 35, 640-648, 2003.
［5］“ToF-SIMS Depth Profiling of Cells： Z-correction, 3 D Imaging, and Sputter Rate of Individual NIH/ 3 T 3 Fibroblasts,“ M.A.
Robinson, D.J. Graham and D.G. Castner, submitted.

Oral Session
Wedensday, 19th SeptemberRoom E

15:00 － 17:00

Session 30: Data Processing and Informatics for SIMS
Chair:

DaeWon Moon（Center for Nano-Bio Technology, Korea Research Institute of Standards and Science, Korea）

S30-1540
15:40 － 16:00

Multivariate analysis application to ToF-SIMS data of organic layers

Yasuko Kajiwara1,2, Satoka Aoyagi2,3
1

MGC Inc., Tokyo, Japan, 2Tottori University, Tottori, Japan, 3Shimane University. Shimane, Japan

Keywords:
ToF-SIMS, Multivariate analysis, Organic layers
Novel aspects:
Effectiveness of multivariate analysis techniques such as multivariate curve resolution for complex Tof-SIMS data was investigated.
Abstract:
Time-of-flight secondary ion mass spectrometry（ToF-SIMS）has been widely used in the analysis of surface chemical structures oforganic materials due to its high surface sensitivity and high chemical specificity［ 1 ］. However, it is often difficult to interpretcomplicated ToF-SIMS data of complex samples which include polymers. Recently, multivariate analysis（MVA）techniques havebeen applied to ToF-SIMS data analysis because they provide helpful information for interpreting ToF-SIMS spectra and images ofcomplex samples［2, 3 ］.
In this study, principal component analysis（PCA）and multivariate curve resolution（MCR）after being preprocessed with Poissonscaling were compared to evaluate a cross-section surface of organic layers. Organic layers were composed of three different
polymers and the cross-section surface was mirror-polished with an ultra-microtome. ToF-SIMS spectra and images were acquiredwith a Ga ion source, and 216 peaks of secondary ions ranging from m/z 10 to 200 were employed to MVA.
In PCA, principal components could not properly extract the information of each polymer. Even though data with auto-scaling andno-scaling were also evaluated by PCA, the polymers were not clearly classified either. It was assumed that PCA missed subtle
differences between similar materials when there were other dominant factors such as sample topography.
On the other hand, images of the three polymers were clearly separated by MCR and the spectra of these three components
were similar to the reference spectra of the three polymers. Moreover, in the comparison with no-scaling, Poisson-scaled data
results provided clearer images than no-scaled data results since Poisson-scaling relatively enhanced weak peaks which werecharacteristic to each polymer.
In conclusion, it is indicated that MCR is useful to extract pure components from mixed ToF-SIMS spectra of the organic
composites. Moreover, minor differences such as functional groups among similar materials can be distinguished by MCR.
［ 1 ］M. S. Wagner, D. J. Graham, B. D. Ratner and D. G. Castner, Surf. Sci. 570（2004）78-97［ 2 ］M. S. Wagner, D. J. Graham and D. G. Castner, App. Surf. Sci. 252（2006）6575-6581［ 3 ］J. L. S. Lee, I. S. Gilmore, I. W. Fletcher and M. P. Seah, Surf. Interface Anal. 41（2009）653-665

Oral Session
Wedensday, 19th SeptemberRoom E

15:00 － 17:00

Session 30: Data Processing and Informatics for SIMS
Chair:

DaeWon Moon（Center for Nano-Bio Technology, Korea Research Institute of Standards and Science, Korea）

S30-1600
16:00 － 16:20

The Full Spectrum protocol: how to provide quantitative analysis formatrix elements in heterogeneous nanometer scale layers with ToF-SIMS

Matthieu Py1, Jean-Paul Barnes2, Jiro Matsuo1,31

Kyoto University, Uji, Japan, 2CEA-Leti, MINATEC Campus, Grenoble, France, 3JST-CREST

Keywords:
ToF-SIMS, matrix effect, depth profiling, data treatment, full spectrum
Novel aspects:
The proposed Full Spectrum data treatment enables accurate, quantitative depth profile of matrix elements in heterogeneous
matrix layers of nanometer scale with a commercial ToF-SIMS, which is usually impossible.
Abstract:
We propose to present on a theoretical and practical point of view a data treatment protocol for Time of Flight Secondary Ion
Mass Spectrometry（ToF-SIMS）that we recently updated, the so-called Full Spectrum protocol [1-4]. The latter, although relatively
unknown and scarcely represented in the literature, is interesting because it capitalises on the specificity of ToF-SIMS（parallel
monitoring of numerous secondary ions in one large range mass spectra）. It indeed consists in taking into account as muchinformation as possible from the ToF-SIMS mass spectra by following all of the secondary ions containing the element of interest,
in contrast to all other approaches which usually focus on no more than one or two secondary ions. Precisely, for a given element
A, one will sum the contributions of all AxRy - secondary ions normalised by x and by their isotopic abundances to obtain a newquantity corresponding to the atomic quantity of element A in the secondary ion beam. Instead of depth distributions ofsecondary ions intensities, the depth profiles are transformed into depth distributions of normalised sums of secondary ion
intensities, somewhat equivalent to envelope functions, which are then calibrated to give quantitative depth profiles.
As a result, this approach shows better statistics and is found to be more representative of matrix element depth distribution inheterogeneous stacks. Another feature of this approach is that the signals it yields for matrix elements is directly proportional（although not equal）to the material composition. This approach presents therefore the advantage of enabling quantification for
all matrix elements in one depth profile. Furthermore, since this ability only results from data treatment and not from instrumental
or experimental conditions changes, one simultaneously keeps the ability of ToF-SIMS to quantify impurities or dopants if present.
Due to the increased statistics, the quantitative profiles obtained through this protocol are also more reliable, reproducible andless noisy. Furthermore, for the particular case of very thin（<10 nm）samples, it was shown that this protocol enabled acquisitionof quantitative profiles without resorting to reference samples（through cross-characterisation with X-ray Photoelectron
Spectroscopy measurements）.
In this work, the different aspects of the specific data acquisition and treatment for the Full Spectrum protocol will be discussed.
First the theory behind the data treatment will be addressed. The improvements it brings in terms of reduction of undesired
effects such as surface transients or matrix effects will be presented through simple, model samples as well as through structures
for advanced microelectronics. Practical implementation issues will also be treated. Finally, limitations of the protocol will bequickly overviewed.
［ 1 ］M. Py, J. P. Barnes, J. M. Hartmann, Surf. Interf. Anal. 2011, 43, 539.
［ 2 ］M. Py, J. P. Barnes, D. Lafond, J. M. Hartmann, Rap. Comm. Mass Spec. 2011, 25, 629.
［ 3 ］M. Py, J.-P. Barnes, R. Boujamaa, M. Gros-Jean, K. Nakajima, K. Kimura, C. Roukoss, B. Pelissier, N. Gambacorti, J. Vac. Sc.
Technol. B. 2011, 29, 032208.
［ 4 ］M. Py, E. Saracco, J. F. Damlencourt, J. P. Barnes, J. M. Fabbri, J. M. Hartmann, Appl. Surf. Sc. 2011, 257, 9414.

Oral Session
Wedensday, 19th SeptemberRoom E

15:00 － 17:00

Session 30: Data Processing and Informatics for SIMS
Chair:

DaeWon Moon（Center for Nano-Bio Technology, Korea Research Institute of Standards and Science, Korea）

S30-1620
16:20 － 16:40

Evaluation of secondary ions from lysozyme and peptides using G-SIMS
and g-ogram

Satoka Aoyagi1, Ian S Gilmore2, Ichiro Mihara3
1

Shimane University, Matsue, Japan, 2National Physical Laboratory, UK, 3Kuraray Co., Ltd., Kurashiki, Japan

Keywords:
G-SIMS, g-ogram, protein fragmentation, lysozyme
Novel aspects:
Complex fragment ions from a protein on ToF-SIMS spectra were characterized and clearly separated from secondary ions related
to other organic materials by G-SIMS and g-ogram.
Abstract:
The characterization of proteins and peptides at surfaces is crucial for developing medical and diagnostic devices, bio-sensors anda tissue imaging technique. Time-of-flight secondary ion mass spectrometry（ToF-SIMS）is powerful for such purposes owing to its
femto mole sensitivity, specificity between proteins, sub-micron imaging and 3 D imaging. Since the surface information depth（approximately 1 nm）is smaller than the typical dimension of a protein it has been shown to give orientation specificinformation. Fragments from proteins relating to the functional group of each amino acid are mainly observed because it is difficult
to generate intact protein ions, which makes interpretation of ToF-SIMS spectra of complex samples including proteins andpeptides difficult. In addition, it is also difficult to characterize secondary ions and to distinguish target peaks from peaks fromsubstrate materials and contaminants especially at a high mass range due to overlapped peaks.
Gentle-SIMS（G-SIMS）and g-ogram provide information on fragmentation of secondary ions based on ratio of static-SIMS spectra
obtained with two primary ion conditions causing different amounts of fragmentation. The g-ogram is based on G-SIMS which is aneffective method to simplify spectra based on the comparison between spectra having lower fragmentation and those having
higher fragmentation. When the low fragmentation spectrum is multiplied by the ratio of the lower fragmentation spectra tohigher ones then the peak intensities of the more intact, structurally significant, fragments are enhanced and the more degradedfragments suppressed in a G-SIMS spectrum. The g-ogram is an image representation for the G-SIMS spectrum normalized to the
maximum intensity and suggests the fragmentation energetics of the ion formation like a traditional chromatogram（where theseparation parameter here is the fragmentation energy）.
In this study, G-SIMS and g-ogram were applied to protein and peptide samples in order to investigate effectiveness of G-SIMS on
characterization of proteins or peptides. Lysozymeor（des-tyr）-enkephalin on silicon wafer samples were measured using TOFSIMS with Bi+ and Mn+ primary ion sources, and then peaks of secondary ions were analyzed by G-SIMS and g-ogram. As a result,molecular ions of secondary ions of（des-tyr）-enkephalin and secondary ions specific to lysozyme were clearly separated fromother secondary ions related to substrates or organic contaminants by G-SIMS and g-ogram analysis. Some of the indicatedprotein-related secondary ions could be from dipeptide or tripeptide parts though it is generally difficult to find dipeptide or
tripeptide peaks because they are often very weak and hidden by dominant peaks. Thus, G-SIMS and g-ogram analysis unveils
intricate relationships between secondary ions to identifyt hem appropriately, and is useful to identify unknown peptides with coexisting organic materials and to characterize orientation or surface structure of a protein.

Oral Session
Wedensday, 19th SeptemberRoom E

15:00 － 17:00

Session 30: Data Processing and Informatics for SIMS
Chair:

DaeWon Moon（Center for Nano-Bio Technology, Korea Research Institute of Standards and Science, Korea）

S30-1640
16:40 － 17:00

Ab initio peak identification for SRM/MRM data

Ken Aoshima1, Koikegami Shigeru3, Fukuda Mitsuru3, Takahashi Kentaro1, Matsuura Kentaro1,
Watanabe Hideki1, Sato Yoshiaki1, Uehara Taisuke1, Kimura Takayuki1, Nakamura Tatsuji1,
Parry Howell2, Tanaka Satoshi2, Utsunomiya Shin-ichi2, Kajihara Shigeki2, Tanaka Koichi2, Oda Yoshiya1
1

Eisai Co. Ltd., Tsukuba, Japan, 2Shimadzu Corp., Kyoto, Japan, 3Ibio-Tech

Keywords:
Targeted quantitative proteomics, MRM, peak detection, peak alignment
Novel aspects:
High accurate and large scale quantitation for SRM/MRM data
Abstract:
Introduction
Selected Reaction Monitoring（SRM）or Multiple Reaction Monitoring（MRM）is a powerful technique for proteomics analysis； it
can selectively quantify each specific fragment pair of precursor and product. There are several software tools already developed
such as Skyline 1 , MRMer 2 and Pinpoint software 3 for MRM based targeting quantitative proteomics analysis. However, some
software tools can only quantitate SRM/MRM data based on MS/MS spectral libraries that were built in-house or publicly
available, or can only perform relative quantitation using stable isotope labeling. In order to fully take advantage of SRM/MRM it ishighly desired to develop an application tool which can perform absolute quantitation without the help of mass spectral libraries.
MethodOur application called ’EMS’ derived from freeware software ’Mass++’ is now equipped with a set of functions for ab initio peakidentification of SRM/MRM data. These functions have been developed from those used in liquid chromatography mass
spectrometry（LC/MS）data analysis：
1 ）The peak detection function finds a two-dimensional mixed Gaussian function which best fits to an MS chromatogram after theconvergence process by expectation-maximization（EM）algorithm or by Levenberg Marquardt（LM）algorithm（also known as thedamped least-squares method）. Before starting the convergence process, the function guesses the expected values of every
component of the mixed Gaussian function as initial parameters based on k-means clustering. The number of components of themixed Gaussian function should be selected by the Bayesian information criterion（BIC, also known as Schwarz criterion）or
Akaike information criterion（AIC）to avoid over-fitting.
2 ）Dynamic programming（DP）and locally weighted scatterplot smoothing（LOWESS）are exploited in the peak alignment
function.
ResultWe have evaluated a variety of peak shapes of peptides obtained from different separation systems, using different MS data
formats. Our peak detection algorithm can not only recognize the peaks identified by other software tools with help of mass
spectral database/libraries, but can also properly detect the peaks which could not be extracted from mass spectral databases/
libraries. This means that our algorithm can be useful for quantitative analysis of peptides which have not been registered inexisting spectral databases/libraries. We have successfully analyzed over 80 complex proteomics data sets using our ab initio peakdetection and alignment algorithms. All data sets were manually confirmed, proving that this algorithm was capable of analyzinglarge scale complex biological samples. Our ab inito peak detection and alignment algorithms will be integrated as one of theplug-ins for Mass++ and will be freely available on the Mass++ distribution site；
http：//www.first-ms 3 d.jp/english/achievement/software
References
1 ）MacLean B, et al, Bioinformatics. 2010；26：966.
2 ）Martin DB, et al. Mol. Cell. Proteomics. 2008； 7 ：22702278.
3 ）http：//www.thermoscientific.com/

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 21: Platform Technology for Metabolomics

PWe-001
11:10 － 12:20

Negative ion tandem mass spectrometry of prenylated fungal metabolites
and their derivatives

Ramona Heinke, Norbert Arnold, Ludger A Wessjohann, Juergen Schmidt
Leibniz Institute of Plant Biochemistry, Halle, Germany

Keywords:
meroterpenoids, boviquinones, fungi, Basidiomycetes, LC-（-）-ESI-MS/MS
Novel aspects:
characterization of prenylated fungal metabolites and their derivatives by liquid chromatography negative ion electrospray tandem
mass spectrometry
Abstract:
Meroterpenoid quinones occur widespread in various living organisms including macromycetes. They are natural products ofcombined biosynthetic origin which are partially derived from terpenoids［ 1 ］. A group of structurally related 2,5-dihydroxy-1,4benzoquinones with prenyl side chains has been isolated from different fungal fruiting bodies belonging to the Boletales s.l.
（Basidiomycetes）such as boviquinone- 3 and boviquinone- 4 including the related tridentoquinone formed by an unusual
intramolecular ring closure［2, 3 ］. These metabolites were investigated by liquid chromatography electrospray tandem mass
spectrometry under negative ionization. A detailed study of the fragmentation behavior of various prenylated benzoquinones andphenols from different fungal species was carried out by using an ion trap system.
The results obtained can be summarized as following：· Prenylated benzoquinones and phenols exhibit a characteristic fragmentation pattern in negative ion electrospray tandem mass
spectrometry.
· The mass spectral decomposition of both prenylated phenols and boviquinones under negative ion ESI conditions is mainly
characterized by typical successive losses of the isoprene units. This is in agreement with their corresponding EI mass spectra［4,5 ］.
· The loss of the isoprenoid side chain of the boviquinones leads to a common radical anion comprising the quinoid moiety.
· In contrast to the linear boviquinone-4, the cyclic tridentoquinone shows a quite different fragmentation pattern.
· In case of the prenylated phenols, the loss of the complete isoprenoid side chain gives also information about the substitution
pattern of the benzyl type moiety.
In conclusion, the loss of the whole side chain as well as the characteristic neutral loss of a single isoprene unit provides useful
information both structural features of the meroterpenoids.
The obtained mass spectral data were applied to a classification of several fungal species of different genera belonging to
Boletales s.l. by a principal component analysis（PCA）.
References［ 1 ］Geris, R. and Simpson, T.J., Nat. Prod. Rep.26, 2009, 1063-1094.
［ 2 ］Muehlbauer, A., Beyer, J., Steglich, W., Tetrahedron Letters39, 1998, 5167-5170.
［ 3 ］Lang, M., Muehlbauer, A., Graef, C., Beyer, J., Lang-Fugmann, S., Polborn, K., Steglich, W., Eur. J. Org. Chem.5, 2008, 816-825.
［ 4 ］Besl, H., Hoefle, G., Jendrny, B., Jaegers, E., Steglich, W., Chem. Ber.110, 1977, 3770-3776.
［ 5 ］Beaumont, P.C., Edwards, R.L., J. Chem. Soc. (C) 19, 1969, 2398-2403.

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 21: Platform Technology for Metabolomics

PWe-002
13:30 － 14:40

Metabolome profiling of human embryonic stem cells by gas
chromatography-mass spectrometry

Takashi Suzuki1, Masahiro Miyoshi2, Katsuhiro Nakagawa1, Hirofumi Suemori3, Shinichiro Chuma3,
Norio Nakatsuji3

Shimadzu Co., Kyoto, Japan, 2Shimadzu Analytical and Measuring Center Inc., Kyoto, Japan, 3Kyoto University, Kyoto,
Japan

1

Keywords:
Biomarker, GC-MS, hESCs, metabolomics
Novel aspects:
This is the first study to perform metabolome profiling of human embryonic stem cells by gas chromatography-mass spectrometry
Abstract:
Human embryonic stem cells（hESCs）have the potential not only to proliferate indefinitely but also to differentiate into any cell
type in the body. These properties make hESCs promising for applications such as drug testing, disease modeling, and cell
replacement therapy. Development of techniques for their quality control and stable supply is indispensable for the above
applications. For this purpose, effective biomarkers that are able to evaluate the quality of hESCs should be established. Although
there are many studies of characterization of pluripotent stem cells in genomics, transcriptomics, and proteomics, a few efforts
have been made in metabolomics. The aims of our study are to establish effective metabolite biomarkers for quality evaluation of
hESCs and to develop a simple and quantitative method for measurement of the biomarkers using mass spectrometry（MS）. In
this study, we analyzed differences of metabolic signature of hESCs in the undifferentiated and differentiated state using gas
chromatography-MS（GC-MS）. Two lines of hESCs, KhES- 1 and KhES-3, were analyzed. We observed about 50 peaks in the
spectra of oxime-trimethylsilyl derivatives of cellular metabolites. We observed about 10 metabolites which show more than twofold difference between differentiated and undifferentiated hESCs. We also analyzed total cellular fatty acids. GC-MS profile inundifferentiated hESCs showed differences from that in differentiated counterparts. These results suggest that metabolomic
technique is one of the effective strategies to discover biomarkers for evaluating the undifferentiated state of hESCs.

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 21: Platform Technology for Metabolomics

PWe-003
11:10 － 12:20

Metabolic characterization by mass spectrometry of Cordyceps bassiana
mycelium cultivated under various culture medium and light conditions

Sun-Hee Hyun1, Seok-Young Lee1, So-Hyun Kim1, Gi-Ho Sung2, Seong Hwan Kim3, Hyung-Kyoon Choi1

1
College of Pharmacy, Chung-Ang University, Seoul, Korea, 2Mushroom Research Division, Department of Herbal Crop
Research, National Institute of Horticultural & Herbal Science, Suwon, Korea, 3Department of Microbiology, Dankook
University, Cheonan, Korea

Keywords:
Cordyceps bassiana mycelium, Metabolic characterization
Novel aspects:
Metabolic characterization of C. bassiana mycelium cultivated under various conditions and optimum culture conditions for the
production of target metabolites.
Abstract:
Metabolic alterations of Cordyceps bassianamycelium were investigated under the following culture medium and light conditions：dextrose agar supplemented with 0.5 % yeast extract（SDAY）medium with light（SL）, SDAY medium without light（SD）, nut
medium without light（ND）, and iron-supplemented SDAY medium without light（FD）. The growth of mycelia was retarded
under the ND condition compared to the other conditions. The levels of asparagine, aspartic acid, glutamic acid, glutamine,
histidine, lysine, ornithine, and proline were significantly higher under SD and SL conditions. The levels of most of the alcohols,
saturated fatty acids, unsaturated fatty acids, fatty acid esters, sterols, and terpenes were higher under the ND condition than in
the other conditions, but beauvericin was not detectable under the ND condition. The FD condition was favorable for theenhanced production of aminomalonic acid, malic acid, mannonic acid, and erythritol. Thus, the metabolic characteristics of C.
bassiana can be manipulated by varying the cultivation conditions, rendering this fungus potentially favorable as a nutraceuticaland medicinal resource.

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 21: Platform Technology for Metabolomics

PWe-004
13:30 － 14:40

Electrochemical simulation of Phase I and II Drug Metabolism, A Powerful
Technique for Metabolite Characterization and Synthesis

Jean-Pierre Chervet1, Agnieszka Kraj1, Hendrik-Jan Brouwer1, Nico Reinhoud1, Martin Eysberg1,
Uwe Karst2

1
Antec, Zoeterwoude, the Netherlands, 2Institut fur Anorganische und Analytische Chemie, Westfalische WilhelmsUniversitat, Munster, Germany

Keywords:
Metabolite synthesis, drug metabolism, electrochemistry
Novel aspects:
A long-lasting metabolite synthesis by applying square wave pulses
Abstract:
Electrochemistry（EC）in combination with mass spectrometry creates a powerful platform to simulate various oxidation andreduction processes in life sciences. Electrochemistry is a complementary technique to traditional invivo or in vitro metabolism
studies, and delivers the oxidative metabolic fingerprint of a（drug）molecule in a very short time. Mass spectrometry delivers
selective and sensitive detection and allows for unambiguous identification of all products generated in the electrochemical cell.
Additionally, automated data analysis by use of data bases（proteomics）or e.g., mass spectral trees（metabolomics）can shorten
considerably the total time needed for the experiment.
In this poster a dedicated electrochemical system will be presented for fast screening of drugs to obtain their metabolic
fingerprint. The easy and fast electrochemical conversion of Amodiaquine into its major phase I metabolites will be presented inboth analytical and preparative EC cells. In a second step Glutathione（GSH）is added to the electrochemically generated
metabolites to form the appropriate GSH-metabolite adducts, mimicking phase II reactions. All known adducts were successfully
formed and identified with MS. Investigation of drug-protein adducts by conventional techniques（microsomal incubation, in-vivo
studies）are very laborious and time-consuming. With the application of EC, it is possible to activate proteins and drugs within
seconds to undergo covalent drug-protein binding. The conjugation of ß-Lactalbumin with Amodiaquine metabolites will beshown.
A new method for highly efficient metabolite synthesis based on a square-wave potential pulses will be presented. The stable
oxidation conditions were obtained without the need of any cell maintenance for a prolonged period of time.
Verapamil was chosen as model drug and its oxidation products and metabolites were investigated. A highly concentrated sample（250uM）of the Verapamil was used for the experiments and electrochemically synthesized metabolites are collected off-line.
Mass spectra are measured to confirm the presence of Verapamil metabolites in the collected samples.
Additionally, the applicability of on-line EC/MS to oxidize other drugs and xenobiotics（e.g., acetaminophen, irinotecanetc.）will bepresented. The data demonstrate that hyphenation of electrochemistry with ESI/MS provides a versatile and user-friendly platform
for rapid and cost efficient screening of target compounds（drugs, xenobiotics, etc.）in phase I and phase II metabolism studies.

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 21: Platform Technology for Metabolomics

PWe-005
11:10 － 12:20

Identification of a new plasma biomarker of Alzheimer Disease using
metabolomics technology

YOSHIAKI SATO1, Ikumi Suzuki1, Tatsuji Nakamura1, Francois Bernier1, Ken Aoshima1, Yoshiya Oda2
1

Eisai Product Creation Systems, Tsukuba, Japan, 2Eisai Product Creation Systems, Andover, MA 01810

Keywords:
Alzheimer’s disease, Biomarker, LC/APCI-MS, Desmosterol
Novel aspects:
Plasma desmosterol could be a powerful new specific biomarker for the early and easy Alzheimer Disease diagnosis.
Abstract:
Alzheimer’s disease（AD）is a neurodegenerative disorder of the central nervous system（CNS）characterized by a progressive lossof short-term memory accompanied by a gradual loss of cognitive functions. Biomarkers are very useful for diagnosing andmonitoring disease progression and are important for patient selection, monitoring side-effects, aiding to select appropriate
patient treatment and helping new drug discovery. For the clinical studies of AD therapeutics, there is an increasing need for
diagnostic markers to ensure that therapies are targeted at the right patient population, to initiate early treatment when diseasemodifying drugs will be available and to monitor disease progression. Several studies have investigated AD biochemicalbiomarkers in various tissues including blood and CSF. Of those approaches, CSF Ab x-42 and tau protein phosphorylation are
currently considered the most useful biomarkers, though they cannot predict conversion from MCI to AD accurately and are notuseful for guiding drug treatment. In addition, analyzing those markers requires performing delicate CSF collection from patients.
Hence, access to less invasive biomarkers found in easy to acquire fluids such as plasma would accelerate and reduce the cost of
AD diagnosis and offer windows of opportunity for selecting and treating patients with disease-modifying drugs once they areavailable.
Herein, we performed unbiased analysis of steroids related compounds to identify novel AD plasma biomarkers using LC/APCI-MSand Mass ++ data analysis software 1) . The analysis revealed that Desmosterol, a cholesterol precursor, was found to be
decreased in AD plasma vs healthy elderly controls plasma（n=10）with a fold change and p value of 0.36 and less than 0.01,
respectively. In order to precisely quantify variations in desmosterol between normal controls and AD patients, we established ananalytical method to measure desmosterol and cholesterol concentration using LC/APCI-MS system that allowed separating 5
endogenous main isomers and interfering peaks. Using this LC-based method, we discovered that desmosterol and desmosterol/cholesterol ratio are significantly decreased in AD patients using this LC-based method. Those changes were not observed insamples from Parkinson’s disease（PD）and schizophrenia patients samples, suggesting the specific association of desmosterol
with Alzheimer’s disease and or with cognitive decline. Next, the validation of this assay using 109 clinical samples confirmed thedecrease of desmosterol in AD patients as well as a change in desmosterol/ cholesterol ratio in AD patient’s plasma. Interestingly,
we could also observe a difference between control and Mild Cognitive Impairment patients（MCI）. In addition, the decrease ofdesmosterol was somewhat more significant in female patients. Receiver operating-characteristic（ROC）analysis between controls
and AD, using plasma desmosterol shows a score of 0.80, indicating a good discrimination power of this marker in the two
reference populations and confirms the potential usefulness of measuring plasma desmosterol levels for diagnosing AD. For
control versus MCI, the ROC value using plasma desmosterol is 0.71, suggesting a more moderate discrimination power. Moreovera significant correlation of plasma desmosterol with CSF Ab x-42/Ab x-40 and Mini-Mental State Examination（MMSE）scores,suggesting that desmosterol/cholesterol ratio might be reflective of disease progression.
Our studies demonstrate a sensitive and accurate method of detecting plasma desmosterol concentration and suggest thatplasma desmosterol could be a powerful new specific biomarker for the early and easy AD diagnosis.
Reference1 ）Sato, Y. et al, Identification of a new plasma biomarker of Alzheimer’s disease using metabolomics technology, J. Lipid. Res.
53： 567-576, 2012

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 21: Platform Technology for Metabolomics

PWe-006
13:30 － 14:40

Metabolic profiling of mouse brain with MALDI-FT-ICR imaging mass
spectrometry techniqueKazunori Saito1, Daisuke Miura2, Fujimura Yoshinori2, Nirasawa Takashi1, Wariishi Hiroyuki2
1

Bruker Daltonics K.K., 2Kyushu University

Keywords:
Metabolomics, Imaging MS, FT-ICRMS
Novel aspects:
MALDI-FT-ICR imaging MS can reveal the distribution of the various metabolites without tandem MS technique in tissue sample
Abstract:
Matrix assisted laser desorption ionization（MALDI）-based imaging mass spectrometry（MS）is a superb technique to understandthe distribution of the various molecules in tissues. Fourier transform ion cyclotron resonance mass spectrometry（FT-ICR MS）isthought to be one of suitable detection technique for the small molecules, such as drugs and metabolites, due to its ultra-highresolution. This ultra-high resolution（typically ~500,000 FWHM in small molecules）of FT-ICR MS can resolve not only for separationof the closed mass of different molecules but also so-called resolved isotopic fine structures, which can allow more confident
identification of the elemental composition. Here we apply MALDI-FT-ICR imaging MS to detect metabolites in ischemia mousebrain.
Ischemia-induced mice were used as a model animal. Normal and ischemia mouse brain tissue were sectioned at 10um thickness
and placed on ITO-coated glass slides. A matrix-solution of 5 mg/mL of 9 -aminoacridine in MeOH was applied to the sample byan airbrush. A Bruker Daltonics FT-ICR MS solariX was used for imaging mass spectrometry experiment. The instrument equippedwith the 7 tesla superconductive magnet and ESI/MALDI dual ion source. The ion source has a dual ion funnel and a quadrupole.
The generated ions were accumulated in a hexapole to enhance the sensitivity. Ions were injected into ICR cell through thehexapole ion guide. Image was generated on flexImaging software and spectra were analyzed on DataAnalysis software.
MALDI with 9 -aminoacridine matrix can detect many metabolites such as nucleotides and phosphorylated sugars. Those species
were revealed in MALDI-imaging MS as well. Ultrahigh spectral resolution of FT-ICR MS could resolve closed peaks, e.g. only 13
mDa differentiated peaks of deprotonated cAMP anion（328.04524）and sodium-adducted glutathione
anion（328.05847）. In addition, two mass imaging for these two compounds could be generated, and spatial distribution of bothcompounds in the tissue was separately shown. These facts show that mass spectra acquired in MALDI-imaging MS werepotentially intricate and this level of high resolution is essential for imaging MS of the metabolites or any other small
molecules from the tissue sample. Furthermore, high resolution spectra can allow direct identification of the metabolites without
tandem MS technique, and elemental compositions were clearly identified from the mass spectra. The formula
identification was strength by comparing the obtained spectrum with the theoretical one which showed isotopic fine structures.
In this study, spatial distribution of the metabolites were analyzed by FT-ICR imaging MS without tandem MS. More than 20 of themetabolites were detected and analyzed by one acquisition of the imaging MS. Ultrahigh spectral resolution was required toseparate complicated peaks and peak assignments. It was concluded that FT-ICR MS is one of suitable platform for imaging MS of
the metabolites from the tissue sample.

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 21: Platform Technology for Metabolomics

PWe-007
11:10 － 12:20

Ultra Fast Analysis of Amino Acids in Cultured Cell Extracts Using UHPLC/
MS/MS

Taku TSUKAMOTO1, Yuki SATO2, Satoshi YAMAKI1
1

Shimdzu Co., Kyoto, Japan, 2Shimadzu GLC Ltd., Tokyo, Japan

Keywords:
amino acids, fast analysis, UHPLC/MS/MS, cell extracts, dipeptide
Novel aspects:
Ultra fast analysis of amino acids using UHPLC/MS/MS was applied to cultured cell extracts. As a result, low concentrations ofamino acids were detected without severe interference from the matrix.
Abstract:
An essential aspect for the research of metabolic behavior involves the analysis of amino acids, which is typically carried out using
ion-exchange high performance liquid chromatography（HPLC）with post-column derivatization using o-phthalaldehyde or
ninhydrin. Recently, not only relatively high concentrations of amino acids like proteinogenic amino acids but only relatively low
concentrations of amino acids and related compounds like non-proteinogenic amino acids and dipeptides are also monitored as
significant targets for this purpose. In case of biological samples, however, interferences by other compounds which involve highlevels of amino acids significantly affect the sensitivity for low concentrations of amino acids due to low selectivity of this analyticalmethod. Moreover this method requires over two hours for the trace analysis of amino acids including related compounds.
Because of these problems of traditional method, many kinds of methodology which aimed at higher selectivity or faster analysis
time have been investigated. Highly selective and sensitive analysis of amino acids can be achieved using mass spectrometry.
Using reversed-phase ultra high performance liquid chromatography（RP-UHPLC）techniques remarkably developed in recent years
with pre-column derivatization can shorten analysis time drastically. Herein, we describe the trace analysis of amino acids andrelated compounds in biological samples using UHPLC/MS/MS with pre-column derivatization, along with its application for the
analysis of cultured cell extracts.
Samples were prepared and derivatized using the EZ：faast amino acid kit（Phenomenex）and analyzed using a reversed-phasecore-shell type column Kinetex C18（Phenomenex）and a UHPLC system（Shimadzu Nexera）coupled with a triple quadrupole typemass spectrometer（Shimadzu LCMS-8030）. Using our analytical methodology, 36 amino acids and related compounds weredetected selectively with MRM and LOQ were ranged from 2 to 500 pmol/mL. Moreover the total analysis time using this method
was significantly shorter than that of traditional HPLC methods - our method requires 7 minutes for sample cleanup /derivatization, and 7 minutes for the chromatographic analysis.
The applicability of our methods for biological samples was demonstrated using cultured cell extracts of human colon cancer celland human fibroblast cell. As a result, pmol/mL range of amino acids like prolylhydroxyproline, carnosine and aminoadipic acid
were detected from human colon cancer cells as isolated peaks with the selectivity of triple quadrupole mass spectrometer
besides coexistence of nmol/mL range of amino acids like glutamine, glycine and alanine.Also in the case of human fibroblast
cells, any severe interference in peaks of amino acids which exist at relatively low concentrations was not confirmed. Recovery
test was also carried out for 8 amino acids which were detected at low concentration in each sample. Recovery ratio for thoseamino acids were in the range from 80 to 120 percent about both human colon cancer cell and human fibroblast cell and it
indicates the matrix in cultured cell extracts didn’t cause severe suppression effect nor enhancement effect.
Those satisfactory results show that the UHPLC/MS/MS with pre-column derivatization can be applied to biological samples as
ultra fast analysis in particular for low concentrations of amino acids and related compounds.
Investigations are currently underway to expand our methodology using other biological samples.

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 21: Platform Technology for Metabolomics

PWe-008
13:30 － 14:40

Untargeted screening of pesticides metabolites by LC-HRMS: a tool forhuman exposure evaluation?

Emilien L JAMIN1,2, Nathalie BONVALLOT1,2,3,4, Marie TREMBLAY-FRANCO1,2, Jean-Pierre CRAVEDI1,2,
Cecile CHEVRIER4, Sylvaine CORDIER4, Laurent DEBRAUWER1,2

INRA, UMR1331, Toxalim, Research Centre in Food Toxicology, F-31027 Toulouse, France, 2Toulouse University, INPT,UPS, UMR1331, F- 31062 Toulouse, France, 3EHESP School of public health, Rennes Sorbonne Paris Cite, France,
4
INSERM UMR 1085 IRSET Institut de recherche en sante, environnement, travail, Rennes, France
1

Keywords:
Pesticides, screening, untargeted, HRMS, metabolomics
Novel aspects:
The semi-targeted method developed in this work allowed to distinguish various groups of individuals according to their exposure
to pesticides, on the basis of several urinary metabolites.
Abstract:
The estimation of human exposure to pesticides still represents a challenge since sample amounts available from a human cohort
study are often very low, and the search for possible compounds has to be as thorough as possible. From urine samples,
pesticides are generally detected as metabolites whereas all possible metabolites structures could be unknown. In this context,
this work aimed at the assessment of an untargeted approach using UHPLC-HRMS to characterize pesticides metabolites in urine
samples from the“PELAGIE“（endocrine disruptors： longitudinal study on pregnancy anomalies, infertility and childhood）humancohort study. The PELAGIE study was drawn to evaluate the consequences of the exposure to multiple contaminants, andparticularly to determine the influence of the exposure to pesticides on pregnancy, birth and psychomotor growth of the child.
This study was conducted on a representative cohort of 3421 pregnant women living in a French rural area（Brittany）.
In our work, 40 samples were randomly selected from 4 groups of individuals variously exposed to pesticides on the basis of theirenvironment： urban population versus rural population surrounded by more or less cereal cultures. These samples were directly
analyzed by UHPLC-HRMS（stationary phase C18, Electrospray ionization in the positive and negative mode, LTQ-Orbitrap mass
spectrometer）. Obtained data were processed with the MetWorks software（Thermo Scientific）to extract and integrate HRMS
signals of 47 pesticides and their known or theoretical metabolites. Moreover, a major advantage of this approach is the possible
detection of compounds which are not present in the initial metabolites list. Up to now, almost 450 substances（pesticides +putative urinary metabolites）were monitored by this way. Following their detection by UHPLC-HRMS, MS n experiments were
performed to confirm or not, the detected compounds as potential or probable metabolites. Some of them have also been
confirmed by comparison with metabolites generated during a parallel animal experimentation.
From human samples, 24 metabolites were identified using ESI in the negative mode and integrated. Data obtained by ESI in thepositive mode could only confirm the identification of the metabolites detected in the negative mode, but did not allow thecharacterization of new compounds. Data were then processed by PLS-DA after an OSC filtration. The best separation of samples
groups was obtained by the data normalization with the use of an internal standard during UHPLC-HRMS experiments. According
to the model generated by this way, the separation of individuals was explained by 6 variables representing 3 pesticides, among
which 2 fungicides classically used in cereal cultures were found.
The semi-targeted method developed in this work allowed to distinguish various groups of individuals according to their exposure
to pesticides, on the basis of several urinary metabolites. The acquisition of full scan HRMS signals allows having complete
datasets available to setup targeted MS/MS experiments for the structural identification of the metabolites detected in a firstattempt.

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 21: Platform Technology for Metabolomics

PWe-009
11:10 － 12:20

Metabolite profiling of Makgeolli with Different Wild Yeast Strains
Saccharomyces cerevisiae 98-5 and Pichia anomala 197-13 During
Fermentation

HyeRyun Kim, JeoungMae Son, JaeHo Kim, ByungHak Ahn
Korea Food Research Institute, Gyeonggido, Korea

Keywords:
metabolite, makgeolli, Saccharomyces cerevisiae, Pichia anomala
Novel aspects:
Metabolite profiling of Makgeolli（traditional Korean alcoholic beverage）is reported for the first time.
Abstract:
Makgeolli, which is also called takju, is a traditional Korean alcoholic beverage. It is made by steaming glutinous rice, nonglutinous rice, barley, or flour with nuruk, yeast and water. After fermentation for 7 days, it contains about 15-16% alcohol content.
When drinking, water is added so that the alcohol content of makgeolli is lowered to around 6 - 7 % . In this study, temporalchanges in the metabolites of makgeolli by using wild yeast strains Saccharomyces cerevisiae 98- 5 and Pichia anomala 197-13
during fermentation were analysed by ultra performance liquid chromatography-quadrupole-time of flight mass spectrometry（UPLC-Q-TOF MS）and gas chromatography. The resultant data were statistically processed by partial least squares-discriminant
analysis（PLS-DA）and principal component analysis（PCA）. Various metabolites, including amino acids, organic acids, fatty acids,
small peptides, and urea cycle intermediates were obviously altered by increasing the fermentation period. Changes in thesemetabolites permitted discrimination among makgeolli samples with different wild yeast strains and fermentation periods（1, 2, 3,
6, 7, and 8 d）on a PCA score plot. The sensory attributes of the makgeolli used in this research had higher scores than 5 -point of
9 -point hedonic scale with anchors（ 1 -extremely dislike, 5 -neither dislike or like, 9 -extremely like）.

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 21: Platform Technology for Metabolomics

PWe-010
13:30 － 14:40

Plant metabolite analysis using comprehensive two dimensional gas
chromatograph quadruple mass spectrometer

Shuichi Kawana, Katsuhiro Nakagawa, Yuki Sakamoto, Riki Kitano, Haruhiko Miyagawa
Shimdzu Co., Kyoto, Japan

Keywords:
GCxGC-QMS, High scan speed, 20,000u/sec, Metabolomics, Plant
Novel aspects:
We developed GCxGC coupled with a quadruple mass spectrometer（QMS）featuring high scanning speed up to 20,000 u/sec andutilized for the analysis of plant metabolites.
Abstract:
Introduction
The various metabolites at different concentration levels are analyzed simultaneously in metabolomics field. GC-MS is one of themost widely used techniques. However, the separation capability is not sufficient. Comprehensive two dimensional gas
chromatograph mass spectrometer（GCxGC-MS）is promising method for improving the separation.
Even though time of flight mass spectrometer（TOFMS）is commonly used as the mass spectrometer of GCxGC-MS which
requires high scan speed, the dynamic range of TOFMS is insufficient to cover a wide concentration range for plant metabolites.
We developed GCxGC coupled with a quadruple mass spectrometer（QMS）featuring high scanning speed up to 20,000 u/sec andutilized for the analysis of plant metabolites.
Methods
The work present here was performed by GCMS-QP2010 Ultra（Shimadzu, Japan）equipped with GCxGC modulator（Zoex, US）.
A 30 m long, 0.25 mm id capillary with a 0.25 μm thick stationary phase of 5 % phenyl-95%-methylpolysiloxane（DB-5, J&W, USA）
was used as the first-dimension column and a 2.5 m long, 0.10 mm id capillary with 0.10μm thick stationary phase of 50% phenyl
polysilphenylene-siloxane（SGE, Australia）was used as the second-dimension column. For compound identification, NIST mass
spectral library and GC/MS metabolite mass spectral database（Shimadzu）were used.
Grained rice, Japanese millet and foxtail millet were prepared as evaluation samples. An extraction solvent consisting of a mixtureof water/ methanol/ chloroform（ 1 /2.5/ 1 ）and the heptadecane as internal standard were added to 30mg of the crushed sample.
After thorough mixing and centrifugation, 900 µL of the liquid phase was withdrawn. Milli-Q water（400 µL）was added to theretained liquid phase to separate the mixture into 2 phases （water / methanol phase and chloroform phase）. Aftercentrifugation, 400 µL of the water / methanol phase was removed. Next, application of partial vacuum and centrifugation wereconducted to remove the methanol from solution, and the remaining liquid was freeze-dried. The freeze-dried residue wassubjected to trimethylsilyl（TMS）derivatization；and this derivatized sample was used for GC/MS analysis.
Results and Discussion
The samples were analyzed at scan speed of 20,000 and 10,000 u/sec, separately. The width of peaks was from 300 to 400 ms. The
number of the sampling points across the peaks was around 5 to 6 at 10,000 u/sec and 10 at 20,000 u/sec. Since at least eightsampling points are necessary for precise chromatographic peak shapes for each peak, the scan speed of MS should be set to
20,000 u/sec. However, at higher scan speed, ion transmission is remarkably decreased in QMS. In order to overcome this problem
relating to QMS, we optimized a DC voltage to be imposed on quadruple rods（Advanced Scanning Speed Protocol）. Using thistechnology, the sensitivity and quality of mass spectra at 20,000 u/sec were improved and the dynamic range was expanded to
1,000 without compromising the sensitivity. Using this system, the metabolites extracted from rice, Japanese millet and foxtail
millet were analyzed. The number of detected metabolites was from 1950 to 2147 compounds after separating the over-lapping
peaks in 1 st dimensional chromatograph. In rice sample, 2147 compounds were detected and 1988 compounds showed more than
600 similarity score by mass spectral library search. These results demonstrated the GCxGC-QMS can be effectively used to detect
plant metabolites with a wide concentration range.

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 21: Platform Technology for Metabolomics

PWe-011
11:10 － 12:20

Differentiation of German and Roman Chamomiles by Comprehensive
Chemical Fingerprinting using Ultra Performance Supercritical Fluid
Chromatography Coupled

Shinnosuke Horie1, Motoji Oshikata1, Michael D Jones2, Bharathi Avula3, Kate Yu2, Yan-Hong Wang3,
Mei Wang3, Dominic Moore2, Warren Potts2, Ikhlas Khan3
1

Nihon Waters, Osaka, Japan, 2Waters Corporation, Milford, MA, 3University of Mississippi, Oxford, MS

Keywords:
Comprehensive chemical fingerprinting, UPSFC/MS
Novel aspects:
Comprehensive chemical fingerprinting of chamomiles using UPSFC/MS for the first time with pros/cons relative to other mature
analytical techniques investigated.
Abstract:
Introduction
Natural product ingredient profiling is a challenging task due to sample complexity. The analyte diversity requires the utility ofmultiple analytical techniques to provide orthogonality which can best provide comprehensive knowledge of the analytecomposition. Presently, a variety of analytical techniques exist（such as HPTLC, UPLC, GC, NMR etc）which cover a range ofchemical space. Here, we use Chamomile as an application example to investigate and explore the advantages and limitations of
a novel technology, the Ultra Performance SFC（UPSFC）with the end goal of obtaining a comprehensive ingredient profiling for
Chamomile and gaining chemical understanding of the key ingredients that differentiated different Chamomiles（such as German,
and Roman）. This UPSFC/UV/MS method was compared with our previous studies.
Methods
The Chamomile samples were extracted with methanol（UPLC/UV/MS, UPSFC/UV/MS）, and hexane（UPSFC/UV/MS）separately.
For UPLC/UV/MS, a Waters ACQUITY UPLC with PDA and Xevo Qtof MS was used. The mobile phase was water with 0.05 %formic acid（A）, and acetonitrile（B）. Data acquisition was ESI+. For UPSFC, a Waters ACQUITY UPSFC/UV/SQ detector wasused. A VIRIDIS Hybrid UPSFC column with 2.1 x 150 mm was used with carbon dioxide as mobile phase A, and methanol as
mobile phase B.
Preliminary Data
Chamomile is often used for relieve symptom of sleeplessness, anxiety, and gastrointestinal conditions. The flowering tops of thechamomile plant are used to make teas, liquid extracts etc. Normally, Chamomile refers to either German chamomile or Romanchamomile, which are from the same family（Asteraceae）but belong to different genera. The main components described inchamomile flowers belong to the classes of volatile derivatives and flavonoid components. The analytical methods can be used forplant comparison studies including GC-MS, LC-UV-MS, and UPSFC/UV/MS, all have unique capabilities in identification andauthentication of plant samples and commercial products. Use of all these techniques will be helpful for chromatographic
fingerprint analysis of chamomile samples. Information obtained represents a comprehensive qualitative approach for the purposeof species authentication, quality control, ensuring the consistency and evaluation of their related commercial products. For
UPSFC analysis, we took a systematic screening approach showing the impact of the column, mobile phase modifier, pressure,and flow rate on the packed column supercritical fluid separation. A simple 10 minute gradient method ramping 2 -40% co-solvent（s）was used to screen columns of cyano, flourophenyl, 2 -EP, C18 and hybrid silica stationary phases. A flow rate of 2 mL/min
was used. The final chromatographic conditions are presently being optimized and will be presented. Coupling to the mass
spectrometer was achieved by splitting the flow prior to entry to the photodiode array detector. Different methods optimizing theMS signal were explored. The compared approaches include direct flow to the MS versus the addition of a make-up flow toincrease ionization versus applying heat to the flow stream prior to entry into the MS stimulating a phase change increasing
solubility of the sample with the co-solvent. The UPSFC result will also compared with our previous results obtained fromanalytical technique（UPLC-UV-MS）.

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 21: Platform Technology for Metabolomics

PWe-012
13:30 － 14:40

Synthesis of 13C6- and 13C12-dabsyl chloride to perform amine-targetedclinical profiling

Tetsuo Ishida, Hiroyuki Tanaka, Kihachiro Horiike
Shiga University of Medical Science, Shiga, Japan

Keywords:
dabsyl chloride, stable isotope, amine, amino acid, plasma
Novel aspects:
We have prepared dabsyl chloride with three diffrent mass to develop a method to perform accurate profiling of amines in clinicalsamples.
Abstract:
Dabsyl chloride（ 4 -dimethylaminoazobenzene- 4 ’-sulfonyl chloride）has a visible chromophore with very high extinction
coefficient. Therefore, this reagent has been used to accurately quantify amines in various biological samples. To obtain thequantitative profiles of amines in clinical samples such as urine and plasma, it is necessary to perform high performance liquid
chromatography coupled with UV-vis detection and mass spectrometry（HPLC-vis-MS）. However, due to the limitation of themobile phase suitable for MS, base-line resolution of chromatography is difficult to attain. To overcome this problem, in thepresent study, we successfully synthesized 13C 6 - and 13C12-dabsyl chloride in gram order from 13C 6 -aniline. First, sulfanilic acid13C 6 and N,N-dimethylaniline-13C 6 were synthesized from aniline-13C 6 by treatment with sulfuric acid and methylp-toluenesulfonate, respectively. Second, 13C 6 - and 13C12-methyl orange were produced by diazocoupling of sulfanilic acid-13C 6with N,N-dimethylaniline. Finally, methyl orange was treated with PCl5. By labeling sample of interest, control sample, and a
mixture of standards with 13C 0 -, 13C 6 - and 13C12-dabsyl chloride, respectively, each amines is expected be detected as triplet
peaks mutually separated by 6 mass. To confirm this, we derivatized a mixture of 18 amino acids by incubation with 13C 0 -, 13C 6 , and 13C12-dabsyl chloride, respectively, at 70C for 10 min under vigorous stirring. These three labeled samples were mixed andthen analyzed by HPLC-vis-MS）using a capillary C18 column and electrospray ionization. All of the amino acids could be detected
as characteristic triplet peaks, and the completeness of the derivatization could be evaluated quantitatively.

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 21: Platform Technology for Metabolomics

PWe-013
11:10 － 12:20

Use of culture media fingerprinting by LTQ FT Ultra MS to predict humanembryonic implantation potential

Eduardo Morgado Schmidt1,2, Elaine C Cabral1, Sylvia Sanches Cortezzi3, Marcello Garcia Trevisan4,
Christina Ramires Ferreira1, Edson J Borges5, Marcos Nogueira Eberlin1

1
UNICAMP/ ThoMSon Mass Spectrometry Laboratory, Campinas, Brazil., 2Nova Analitica Importacao e Exportacao
LTDA, Sao Paulo, Brazil., 3Sapientiae Institute Educational and Research Center in Assisted Reproduction, Sao Paulo,
Brazil., 4Institute of Exact Sciences, Federal University of Alfenas, Alfenas, Brazil., 5Fertility Assisted Fertilization Center,
Sao Paulo, Brazil.

Keywords:
Fingerprinting, metabolites, culture media, reproductive
Novel aspects:
This MS metabolites profile could help the selection of the most viable embryo, improving single embryo transfer and thus
eliminating the risk and undesirable outcomes of multiple pregnancies.
Abstract:
Embryo selection methods based on morphologic parameters are usually associated with the Assisted Reproductive Technologies（ART）routine. However, most studies suggest that morphology evaluation is not enough to predict a genetically normal embryoand successful implantation. As the number of ART cycles increases worldwide, improvements in the ability to perform a noninvasive embryo viability prediction has become a crucial target for reproductive medicine. Mass spectrometry（MS）fingerprinting
is a global chemical screening approach to compare and classify samples based on metabolite patterns, with the ultimate goal toidentify discriminating ions, usually corresponding to metabolites. MS-fingerprinting uses direct sample infusion in the ionization
source and usually just sample dilution is performed. This case-control study evaluated mass spectrometric（MS）fingerprinting
patterns of culture media samples used for human embryo culture of patients undergo ingintracy to plasmic sperm injection（ICSI）
with minimal sample preparation and minute-analysis to predict embryo implantation potential.
Culture medium used for the incubation of 85 embryos from 38 patients undergoing ICSI was collected after embryo transfer at
Day 5. The remaining culture media were collected and samples were split into groups according to their implantation outcomes：
100 % of implantation（n=15, 8 patients）, 66.7 % of implantation, when 3 embryos were transferred but 2 embryos implanted（n=12, 4 patients）, 50 % of implantation, when 2 embryos were transferred and 1 implanted（n=16, 8 patients）, 33.3 % of
implantation, when 3 embryos were transferred and only 1 implanted（n=21, 7 patients）and 0 % of implantation（n=21, 11
patients）. Samples were individually diluted and injected directly to the ESI source coupled to a MS. The samples（ 2 µL）were
dissolved in 200 µL of methanol/water 4 ： 1 （v/v）containing 0.1 % ammonium hydroxide for analysis in the negative ion mode.
Mass spectra fingerprinting were acquired using a 7.2T LTQ FT Ultra-MS equipped with a chip-based direct infusion
nanoelectrospray ionization source operating in the negative ion mode at the follow conditions： a 200 nL/min flow rate, 0.3 psi
backing pressure, and 1.5 to 2.0 kV electrospray voltages during 120 s, controlled by ChipSoft software. Mass resolution was fixedat 100,000（defined for an ion of m/z 400）throughout. Data were obtained as transient files（scans recorded in the time domain）
along the 100-1000 m/z range. MS datasets were exported using the Xcalibur 2.0 and organised by Markerlynx XS, where the ionsrelative intensities of each spectrum were considered. Data analysis was conducted in MatLab 7. 0 version using Partial Least
Squares Discriminant Analysis（PLS-DA）toolbox.
There were 6,476 observed ions in the m/z range of 100 to 1000 after spectral noise reduction. The samples were divided into
calibration and validation subsets. MS spectra from samples of 0 % and 100% of implantation were organize into calibration set（n=36）and spectra from 66.7%, 50% and 33.3% were mount into validation set（n=49）. The PLS-DA model described more than
92.2 %（y-block）of the data variance and was able to classify all of the calibration samples correctly. In the validation process,samples of the 66.7% group were correctly identified with 87.8% average probability； samples of the 50% and 33.3% groups were
accurately classified with 100% average probability in this multivariate model.
The preliminary results show that implantation success of human embryos can be correlated with specific biochemical profile intheir culture media. MS-fingerprinting by LTQ FT Ultra-MS is a fast, simple and non-invasive way to detect these patterns, whichallied to the multivariate statistical model, was able to predict with a high confidence the potential of individually embryo
implantation. This biochemical profile could help the selection of the most viable embryo, improving single embryo transfer andthus eliminating the risk and undesirable outcomes of multiple pregnancies.

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 21: Platform Technology for Metabolomics

PWe-014
13:30 － 14:40

Ionic liquid-based microextraction for sensitive determination of urinary
aromatic amines by liquid chromatography-mass spectrometry

Jingyueh Jeng, Cheng-Yuan Shih

Chia nan University of Pharmacy & Science, Tainan, Taiwan

Keywords:
Room temperature ionic liquids, liquid chromatography-mass spectrometry, urinary aromatic amines, dermal exposure
Novel aspects:
Room temperature ionic liquid as a novel media to extract the urinary aromatic amines and sensitive determination of amines byliquid chromatography-mass spectrometry
Abstract:
Room temperature ionic liquids（RTIL）were used as the novel media for extraction and preconcentration of aromatic amine in rat
urea. A suitable mixture of extraction solvent（250µL, 1 -butyl- 3 -methylimidazolium hexafluorophoshate,［C 4 MIM］［PF 6 ］）anddispersive solvent（500µL, methanol）were injected into the aqueous samples（ 4 mL）, forming a cloudy solution. After centrifuging,enriched analytes in the sediment phase were determined by HPLC-MS/MS. Some important parameters, such as the kind andvolume of extraction solvent and dispersive solvent, sample pH, sample volume and salt effect were investigated and optimized.
Under optimum conditions, enrichment factors for 2,4- and 2,6-toluenediamine were above 50 and the limits of detection（LODs）
were 2.7 and 3.4 ng/mL, respectively. Relative standard deviations（RSDs）were below 5.0%. Furthermore, the estimated percentageof recovery from amine-spiked urine samples ranged from 77 to 112%. The method presented here is simple and fast, and does not
involve the use of volatile organic extractants.

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 21: Platform Technology for Metabolomics

PWe-015
11:10 － 12:20

Adjustment of total cellular metabolite concentration by flow-injection
analysis mass spectrometer for metabolomic study

Guan-yuan Chen1, Ching-hua Kuo1,2

School of Pharmacy, College of Medicine, National Taiwan University, Taipei, Taiwan, 2The Metabolomics Core
Laboratory, Center of Genomic Medicine, National Taiwan University, Taiwan

1

Keywords:
FIA-MS, metabolomics, cellular metabolites, calibration, adjustment
Novel aspects:
This is the first method using FIA-MS to adjust the cellular metabolite concentration prior to metabolomic analysis
Abstract:
Metabolomic studies on cell biology have shown huge impact on drug discovery, cancer therapy, toxicology, and stem cellresearch in recent years. A general approach for genomics and proteomics study is to adjust the total amount of nucleic acid andprotein for a fair comparison. However, in metabolomic studies, only cell number was controlled regardless of the difference incell volume or cell types which could lead to a biased comparison. This study proposed a novel and reliable method to determine
the total amount of cellular metabolite concentration through the evaluation of total ion chromatograms（TIC）obtained by flow
injection analysis -mass spectrometry（FIA-MS）. Area under TIC was used to estimate cellular metabolite concentration.
Parameters including data acquisition rate, FIA mobile phase composition, flow rate, injection volume, and MS parameter such as
capillary voltage, sheath gas temperature and flow in Jet Stream electrospray ionization（ESI）, vaporizer temperature and corona
voltage in atmosphere pressure chemical ionization（APCI）, were investigated for their effects on TIC of FIA-MS. Both ESI and
APCI were compared for their performance on calibrating cellular metabolite concentration. Serial dilution of cell extracts wasused to construct the calibration curve, and it was used to calibrate cellular metabolites concentrations in metabolomic studies.
Our data indicated that using FIA-MS is effective to adjust cellular metabolite concentration before metabolic profiling of cell
extracts.

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 21: Platform Technology for Metabolomics

PWe-016
13:30 － 14:40

Metabolomics of Curcuma: approaching by studying constituents of wide
range polarity using LC-DAD-ECD-MS

Paramitha Messayu, Kuo-Lung Ku

National Chiayi University, Chiayi, Taiwan

Keywords:
metabolomics, Curcuma, on-line assay, electrochemical detector, mass spectrometry
Novel aspects:
We developed a special ECD coupled diode array detection and electrospray ionization mass spectrometry for detecting
molecules eluted from gradient HPLC mobile phase, involve from aqueous to non-aqueous.
Abstract:
The characteristic information, such as structure and antioxidation property of curcuminoids and polyphenolic compounds in
different Curcuma species, or in the same species collected from different cultivation regions is critically important forunderstanding the metabolomics of the plant and which can be applied in medicine, new species breeding, or even quality
control. However, there is no suitable on-line antioxidant assay method for analyzing the relatively non-polar and polar
constituents in Curcuma. A lot of researchers have studied antioxidant activities and structure of antioxidant compounds.
Meanwhile, variety of analytical methods was developed to meet the need of the activity and structure identification
simultaneously. The devices designed for simultaneous antioxidant assay and structure identification are currently most promisingfor high throughput analysis. However, the application that published in activity assay of antioxidant compounds is still have many
problems. Electrochemical detector（ECD）is one of simple and widely used method for antioxidant analysis. Application of ECD
in detecting molecules of wide range polarity eluted from high performance liquid chromatography（HPLC）is limited by the poor
solubility of electrolytes in non-aqueous mobile phase. In the present study, we developed a special ECD coupled diode array
detection and electrospray ionization mass spectrometry for detecting molecules eluted from gradient HPLC mobile phase, involvefrom aqueous to non-aqueous. The device was applied to inquired antioxidants, including polar and nonpolar ingredientssimultaneously in Curcuma species. We got signals of antioxidant activity response by applied of the ECD for qualitative analysis,
chromophore absorbance of corresponding curcuminoid compounds in wavelength 425 nm and polyphenolic compounds inwavelength 280 nm by applied of diode array detector, and mass spectrometry for identifying curcuminoids and polyphenolic
compounds from four Curcuma species in the eight different cultivation regions. The rhizomes of Curcuma xanthorrhiza, Curcuma
domestica, Curcuma aureginosa, and Curcuma heyneana were analyzed, three major components： curcuminoid（ 1 ）,demetoxycurcuminoid（ 2 ）, and bisdemetoxycurcuminoid（ 3 ）have been identified, based on their electrochemical spectra, UV
spectra, and mass spectra. The method was successfully in characterizing curcuminoids and polyphenolic compounds, theirpolarity are essentially opposing to each other. Hence, the present high throughput method can be used for studying
metabolomics of Curcuma and other similar species, that containing secondary metabolites of diverse polarity.

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 21: Platform Technology for Metabolomics

PWe-017
11:10 － 12:20

Effect of glucose addition against ion suppression by sodium chloride

Megumi Wakimoto1, Yui Okamura1, Hajime Mizuno1, Naohiro Tsuyama1, Sachiko Date2,
Tsutomu Masujima1,2

Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima, Japan, 2Quantitative Biology Center (QBiC),
RIKEN, Japan

1

Keywords:
Ion suppression, ESI, nano ESI
Novel aspects:
We investigated ion suppression effect of NaCl and found moderating effect of glucose against suppression effect induced by
NaCl.
Abstract:［Introduction］
In the process of electrospray ionization, matrix molecules cause signal increase or decrease of target analytes, which is referred
as“matrix effect“. For example, salts, which are included in biological samples a lot, cause signal suppression.
We have developed a nano ESI-based mass spectrometric method called“Live Single-cell Mass Spectrometry“, which cananalyze small amounts of subcellular contents directly by a nanospray tip. Therefore, ion suppression effect can be a serious
problem if it occurs in Live Single-cell MS even though nano ESI is one of the methods that enables to decrease the ion
suppression effect.
In this study, by comparing offline nano ESI with conventional ESI, we investigated if nano ESI could prevent ion suppression
effect. Also we examined how we could improve this problem.
［Method］
To analyze the ion suppression, sodium chloride（ 1 -1000µM）or D-glucose（ 1 -1000µM）was added to a mixture of nine aminoacids（Ser, Thr, Leu, Asp, Glu, Met, His, Phe, Arg, 1 µM each）dissolved in 50% MeOH. We analyzed these samples by offline nano
ESI and conventional ESI at positive ion mode. Spray voltage was 1.0kV in nano ESI and 4.5kV in conventional ESI. Flow rate inconventional ESI was 10.0µL/min. The experiments（n= 6 ）were perfomed on the LTQ Orbitrap XL（Thermo Fisher Scientific）at theresolution power of 100,000. Nanospray emitters we used for these experiments were Cellomix Tips（HUMANIX, i.d. 3 µm）. We
compared the peak intensities of amino acids among different condition. Furthermore we used RBL- 2 H 3 cell lysate（5.0x106 cells/mL）to confirm glucose effect on ion suppression.
［Result］
By adding 1 mM NaCl to the amino acids mixture, protonated ion peaks of most amino acids disappeared in conventional ESI
spectra, while they decreased to 50-80 % in nano ESI. This result showed that the application of nano ESI caused less ion
suppression than conventional ESI. It seemed that nano ESI produced smaller droplets owing to its small internal diametercompared with conventional ESI. Size of droplets was one of the determinants of suppression effect by NaCl. Nano ESI could
improve suppression effect but not completely.
In contrast, peak intensities of amino acids gradually increased by adding glucose. We hypothesized that glucose could remove
sodium ion in the nanospray tip because of its high affinity to sodium ion.
Next, in order to remove suppression effect of NaCl we added glucose to sample solution. The peak intensities of amino acids inthe presence of both NaCl and glucose were about twice as high as only with NaCl. On the other hand, sodium ion adductedamino acids peaks were hardly detected with glucose while it could be highly observed without glucose. It suggested that
increasing sodium ion adducted glucose resulted in decreasing ion suppression because of sodium ion trapped by glucose
molecules.
Moreover, we investigated this glucose effect was available to actual biological samples on enhancement of peak intensities ofanalytes. We measured RBL- 2 H 3 lysate added with 1 µM glucose by nano ESI. As a result, peak intensities of most amino acids
increased by glucose addition. For example, peak intensities of His and Arg increased about five times. Thus, glucose could
increase peak intensities of analytes even in biological samples with complex composition.
Now, we examine if glucose addition enables sensitive detection of various molecules in Live Single-cell Mass Spectrometry.

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 21: Platform Technology for Metabolomics

PWe-018
13:30 － 14:40

Discriminant analysis of Curcuma species grown in different origins usingmass spectrometry

Jueun Lee1,2, Youngae Jung1, Geum Sook Hwang1,31
Korea Basic Science Institute, Seoul, Republic of Korea, 2Sungkyunkwan Univ. Natural Sciences Campus, Suwon,
Republic of Korea, 3Graduate School of Analytical Science and Technology, Chungnam University, Daejeon, Republic of
Korea

Keywords:
Discriminant analysis, Metabolite profiling, Mass spectrometry, Multivariate statistical analysis
Novel aspects:
There was a considerable change of the levels of metabolites such as essential oils and phenolic pigments depending on two
Curcuma species grown in different origins.
Abstract:
Curcuma species is a rhizomatous herbaceous perennial plant of the ginger family, Zingiberaceae. It has been used as a spice incurries and natural dye and investigated the effect as herbal medicines to treat some diseases such as Alzheimer, cancer andother clinical disorders. It has been also known that secondary metabolites such as essential oils and phenolic pigments in
Curcuma species can have therapeutic actions in humans and which can be refined to produce drugs.
In order to investigate the metabolic difference of two Curcuma species in Jeju-do and Jin-do of South Korea, four groups ofsampleswere grown for 5 months. Quantitative analysis of essential oils and phenolic pigments like curcuminoids in Curcuma
species was performed using GC-TOF-MS and UPLC-TOF-MS, respectively.
Metabolite profiling coupled with multivariate statistical analysis was carried out to characterize the differences between species（C. aromatica and C. longa）or origins（Jeju-do and Jin-do）. PCA score plot showed significant differentiation among two
Curcuma species, whereas there is no difference between Jeju-do and Jin-do. A t-test was performed to statistically certify themetabolite difference in metabolite levels. Differenciation between two Curcuma species was due to higher levels of fifteen
metabolites such as eucalyptol, gemacrenediones and elemene and lower levels of seventeen metabolites shch as cedrene,
curcumene, germacrone, tumerone and zingiberene in C. aromatica. The levels of Curcuminoids were also changed in two
Curcuma species grown in different origins.
This study indicated that there was a considerable change of the metabolic pattern depending on two Curcuma species grown in
different origins and mass spectrometry coupled with multivariate analysis is an efficient method to discriminate Curcuma speciesgrown in different origins.

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 21: Platform Technology for Metabolomics

PWe-019
11:10 － 12:20

Copper Stress Induced Global Metabolites Profiling of Burdock Roots by
1H NMR, GC-MS and UPLC-QTOF MS Analysis

Youngae Jung1, Miyoung Ha1,2, Geum-Sook Hwang1,31
Korea Basic Science Institute, Soeul, Korea, 2Sungkyunkwan University, Suwon, Korea, 3Graduate School of Analytical
Science and Technology, Chungnam National University, Daejoen, Korea

Keywords:
Burdock Roots, Copper Stress, Metabolomics, GC-MS, UPLC-QTOF MS
Novel aspects:
GC-MS analysis showed the increase of fatty acids such as linoleic acid and linolenic acid in copper stress-induced burdock roots.
Abstract:
Arctium lappa L.（Asteracease）, known as burdock, has long been cultivated as a popular vegetable for dietary use and folk
medicine worldwide, as a diuretic and antipyretic tea as well as hypertension, gout, arteriosclerosis, hepatitis and otherinflammatory disorders. In the literature, many health benefits associated to occurrence of different classes of bioactive secondary
metabolites have been reported. These classes include, among others, flavonoids and lignans, and burdock is an important natural
source of compounds from both families.
Stress in plants could be defined as any change in growth condition（s）that disrupts metabolic homeostasis and requires anadjustment of metabolic pathways in a process that is usually referred to as acclimation.
Phytoalexins are low molecular weight antimicrobial compounds that are synthesized de novo and accumulate in plants underabiotic stress. Phytoalexins have been well documented in the field of plant defense. Primary bioactive compounds accumulated
under abiotic stress may possess various health promoting benefits including antioxidant activity, anti-inflammation activity,cholesterol-lowering ability, and even anticancer activity.
Copper, one of the heavy metals, is a microelement necessary for plant growth. Generally, heavy metals activate thephenylpropanoid pathway and increase lignin synthesis in many plant species.
Metabolomics could significantly contribute to the studies of biological stress responses in plants and other organisms byidentifying different compounds, such as by-products of stress metabolism, stress signal transduction molecules or molecules thatare part of the acclimation response of plants.
In the present study, 1 H NMR, GC-MS and UPLC-QTOF MS have been developed for the detection of copper stress-induced
metabolites in burdock roots. We could observe the changes of these metabolites by global metabolite profiling using 1H NMR
and UPLC-QTOF MS analysis. In particular, GC-MS analysis showed the increase of fatty acids such as linoleic acid and linolenicacid in copper stress-induced burdock roots. These data suggest that metabolomic approach using combined 1H NMR, GC-MSand UPLC-QTOF MS analysis is an effective analytical method to understand metabolism about copper stress-induced metabolites
in burdock roots.

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 21: Platform Technology for Metabolomics

PWe-020
13:30 － 14:40

The impact of high-resolution MS techniques on the discovery of novel
natural products from myxobacteria

Yasuhiko Maekawa1, Daniel Krug2,3, Thomas Hoffmann2,3, Aiko Barsch4, Gabriela Zurek4, Rolf Mueller2,3
1
3Bruker Daltonics K.K., Yokohama, Japan, 2Helmholtz Institute for Pharmaceutical Research, Saarbrucken, Germany,
Saarland University, Saarbrucken, Germany, 4Bruker Daltonik GmbH, Bremen, Germany

Keywords:
Identification of new econdary metabolites, high-resolution TOF mass spectrometry, combining targeted and untargeted profiling,metabolome-mining
Novel aspects:
Myxobacteria as showcase： a survey of current developments in high-resolution MS and database-assisted strategies enhancing
the natural products discovery workflow.
Abstract:
Introduction
Myxobacteria represent an important source of novel natural products exhibiting a wide range of biological activities. Some ofthese so-called secondary metabolites are investigated as potential leads for novel drugs. Traditional approaches to discovering
natural products mainly employ bioassays and activity-guided isolation, but genomics-based strategies become increasingly
successful to reveal additional compounds. These“metabolome-mining“ approaches hold great promise for uncovering novel
secondary metabolites from myxobacterial strains, as the number of known compounds identified to date is often significantly
lower than expected from genome sequence information. High-resolution mass spectrometry plays a crucial role for bridging theapparent gap between genomic potential and secondary metabolome reality, by enabling a powerful combination of targeted anduntargeted analysis.
Methods
Myxobacteria were cultivated in complex media and extracts were separated by RP chromatography using an U-HPLC system. MS
measurements were performed in ESI positive and negative mode using an ultra high resolution Q-TOF mass spectrometer（scanrange 100-2000 m/z）. A specialized database system -Myxobase- was developed to support targeted screening. Identification of
known compounds was accomplished using precise EICs and considering exact mass, retention time and isotope pattern forincreased confidence. Myxobase was also designed to host comprehensive spectral information following processing of raw databy molecular feature extraction. In addition, feature-extracted HR-MS data were used for untargeted profiling by Principle
Component Analysis. MS/MS information confirmed compound identity or enabled evaluation of chemical novelty for compounds
differentiating bacterial strains.
Results
We present here high-resolution MS applications which are aimed at the discovery of novel secondary metabolites frommyxobacteria. The challenges encountered in these studies comprise the need for sensitive and highly reproducible analysis incombination with high resolution, accuracy and extended sample throughput. Using data from an up-to-date ESI-UHR-Q-TOF MS
platform, the development of methods and tools which are key to success is discussed.
Targeted profiling：
Full scan spectra enable the targeted screening for known myxobacterial compounds based on highly selective EIC traces, using
Myxobase as analyte database. Profiling reports are parsed into Myxobase with all relevant analytical performance qualifiers,
enabling collaborative evaluation of results and crosslinking with bioactivity assays and strain-related data. Our screening
campaign comprising more than 1000 samples allows for the first time the in-depth comparison of high numbers of myxobacterial
secondary metabilte profiles and the evaluation of metabolite diversity across all myxobacterial species, based on extracts from aworld-wide collection of myxobacteria.
Metabolome-mining：Molecular feature extraction facilitates the deposition of comprehensive metabolite profiles inside Myxobase. Thereby, thesecondary metabolomes of all analyzed myxobacteria become backwards-searchable for the presence of novel natural products.
This feature enables the straight-forward identification of alternative producers, an important achievement since newly identified
secondary metabolites are frequently produced in low yields only. Moreover, the comparison of feature-extracted secondary
metabolomes using statistical tools has the potential to uncover previously“hidden“ natural products. This untargeted“metabolome-mining“ approach is exemplified by the recent discovery of myxoprincomides, an intriguing family of novel
secondary metabolites from Myxococcus.
Identification：
Spectral matching to a reference library of high-resolution MS 2 data from myxobacterial compounds is used to confirm theidentity of known metabolites. In addition, high-resolution tandem-MS measurements are a crucial prerequisite for assessment of
putative chemical novelty and serve as a valuable starting point for structural elucidation.

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 21: Platform Technology for Metabolomics

PWe-021
11:10 － 12:20

Differential analysis of fermented beverage using fast polarity switching
TOFMS acquisition with high mass accuracy and multivariate analysis

Satoshi Yamaki, Manami Kobayashi, Takahiro Goda, Tsutomu Nishine
Shimdzu Co., Kyoto, Japan

Keywords:
Profiling study, Electrospray, Time of Flight, MSn, Structure Determination
Novel aspects:
Profiling study using polarity switching and MSn data acquisition without compromising mass accuracy on fermentation productresearch
Abstract:
The metabolomics technique can rapidly bring information about the similarities and differences within a chromatographic
dataset. A metabolomic based approach has been established for metabolite profiling and biomarker discovery. However, it isequally applicable to other research fields including industrial chemical product characterization, food analysis and natural
product research. In the case of fermented product research, sample profiling is often a significant challenge due to the intrinsic
differences between samples influenced by the combination of ingredients, production processes, and storage conditions. In thisstudy, we developed an LC-based approach to determine metabolite profiles including polar metabolites and to identify specificendogenous components, aiming at high throughput and comprehensive methods using TOFMS acquisition.
Commercially available beers were used as a test sample for the polar metabolite analysis. All UHPLC units are consisted of
Nexera LC-30A series（Shimadzu Corporation）with a pressure range up to 130 MPa. The chromatographic system used a binary
solvent system delivered as a gradient of water and acetonitrile containing 0.1% formic acid, and used a Synergi Hydro-RP column（Phenomenex）. The accurate full-scan MS measurement was carried out on a Shimadzu LCMS-IT-TOF mass spectrometer with
rapid polarity switching. Profiling Solution software（Shimadzu）was used to create a data array for all sample data； this tool wasused to highlight specific components that were statistically different and to export data to SIMCA-P+（Umetrics）for PCA and PLSDA analysis.
Beer is a beverage made by fermentation from malted barley and flavored with hops, and the existence of many organic
compounds in it is known. These compounds show ion signals in both positive and negative ionization（organic acid preferentiallyionizes in negative mode, whilst nucleoside shows higher signal intensities in positive mode）. To obtain the complete profile froma sample, it is necessary to run the LC/MS analysis both positive and negative modes. In preliminary experiment, seventeen
products of commercial beer and related beverage were analyzed in triplicate by LCMS-IT-TOF systems using ESI with a scan
range of m/z 80 to 1000. Synergi Hydro-RP column could separate the polar metabolites such as organic acid, nucleobase andnucleoside under water-rich conditions. Profiling Solution software was used to transform LC/MS raw data into an aligned dataarray of retention time and m/z pairs. 912 peaks were detected by peak extraction function from positive mode and 528 peaks innegative mode, within 20 min of LC separation. To determine metabolite profiles, principal component analysis was performed
with thousands of unique retention time and m/z pairs. By performing the PCA, four experimental groups classified according to
the malt ratio（classification by Liquor Tax Law of Japan： beer, low-malt beer and the third beer）were roughly classified into
three clusters. Profiling software and PCA was used not only to establish the differences between the two conditions but also tohighlight specific components that could account for the PCA data. The ion of m/z 191.0199 in negative mode and m/z 205.0976 inpositive mode was extracted as a characteristic peak of high-malt beer. These peaks were tentatively assigned as citric acid
（C6H8O7）and tryptophane（C11H12N2O2）respectively, using formula prediction software that takes into account mass accuracy,
isotope modeling and MS n information. These were further identified by comparison with authentic standards. When massaccuracy was checked with the known compound such as malic acid and adenosine, it turned out that MS measurement was
performed in the accuracy of less than 3 ppm（using external calibration）acquired with fast polarity switching.
In conclusion, PCA of LCMS peak profiles of beers resulted in the separation of samples according to some of their different
compositional properties.

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 21: Platform Technology for Metabolomics

PWe-022
13:30 － 14:40

Simultaneous Quantitative and Qualitative measurements for Primary
Metabolism Investigations using a Quadrupole-Time-of-Flight Mass
Spectrometer

Noriyuki Iwasaki1, Don Richards3, Mark Savage4, Angus Nedderman4, Carsten Baessmann2

1
Bruker Daltonics K.K., 2Bruker Daltonik GmbH, Bremen, Germany, 3Bruker UK Ltd, Coventry, UK, 4Unilabs Bioanalytical
Solutions, Sandwich, UK

Keywords:
DMPK, QTOF, metabolite, quantitation
Novel aspects:
Demonstration of simultaneous quantitative and qualitative DMPK analyses using Q-ToF technology at 1 uM drug concentrations.
Abstract:
Recently there has been considerable interest in simultaneously performing both quantitative and qualitative DMPK analyses insupport of small molecule drug discovery. This work describes an investigation into the possibility of using a Quadrupole-Time-ofFlight mass spectrometer to obtain clearance data, metabolite identification, structure elucidation and metabolite profiles from
P450 microsomal incubations at a drug concentration of 1 mM from a single sample set.
P450 microsomal incubations of commercially available drug substances including Pindolol, Verapamil and Haloperidol wereprepared at 1 mM concentration. The incubations were sampled and quenched, at intervals to provide a time course over a
period of 60 minutes. The analysis of the samples was carried out by LC-MS using a Bruker Maxis Impact Quadrupole-Time-ofFlight mass spectrometer to obtain data suitable for measuring clearance and plotting metabolic profiles. Data dependent MSMS
spectra were collected in order to identify and elucidate the structures of the observed metabolites. The chromatographic
method used a generic gradient, reverse phase method based on a Kinetex C18 column and 0.1 % formic acid and acetonitrile
mobile phases at a flow rate of 300 ml /min.
Preliminary data showed that Pindolol was not cleared. The clearance values obtained for Verapamil and Haloperidol were t1/2 =7 and 47 minutes respectively. These were as expected and comparable to the values obtained on the same sample set using a
triple quadrupole mass spectrometer.
In the case of Verapamil two metabolites, Norverapamil and an N-dealkylation product were easily observed by comparison of
base-peak chromatograms and their structures determined through examination of the MSMS data. A third metabolite was
uncovered using metabolite detection software and although the response was considerably weaker, it’s structure wasdetermined in the same manner. The LC-MS data were re-processed to plot the profiles of the three metabolites.
No metabolites of Haloperidol were immediately apparent but two were uncovered using the metabolite detection software.
These were found to be oxidation of the piperidine moiety to a pyridine and an N-dealkylation product. Again, the LC-MS data
were re-processed to plot the profiles of these metabolites.
The ability to carry out all of these analyses at 1 mM concentrations and under generic conditions indicates that improved
workflows for simultaneous DMPK measurements can be developed to greatly enhance the information yield and productivity ofboth quantitative and qualitative determinations. A refined methodology and work flow will be fully described in the poster
presentation together with further examples.

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 21: Platform Technology for Metabolomics

PWe-023
11:10 － 12:20

Development of accelerate quantification analysis for hydrophilic
metabolites using ionparing chromatography with a high-speed triple
quadrupole mass spectrometer

Hashim Zanariah2, Yudai Denpo2, Tairo Ogura1, Ichiro Hirano1, Takeshi Bamba2, Eiichiro Fukusaki2
1

Shimadzu Co., Kyoto, Japan, 2Osaka University, Osaka, Japan

Keywords:
Targeted metabolomics, Non-volatile compounds, Hydrophilic metabolites
Novel aspects:
High throughput analysis technique of hydrophilic metabolites using ion-pare chromatography with a high-speed MRM analysis
were developed.
Abstract:
Metabolomics is the comprehensive study of metabolites, and it allows for detailed phenotypic analysis through a combination of
metabolite information. Among the many kinds of metabolic reactions, central pathway of energy metabolism is one of biologicallyimportant pathway, and it includes around a hundred of hydrophilic compounds such as sugar phosphates, organic acids, andnucleotides. Ion-pare chromatography coupled to a triple quadrupole mass spectrometer is one of the techniques to analyze
these hydrophilic metabolites. On the other hand, in order to improve throughput of analysis, the mass spectrometer which
combines high-speed with a quantitative capability is needed. In this study, we report a developed analytical system forhydrophilic metabolite using ion-pare chromatography with a high-speed triple quadrupole mass spectrometer.
Standard samples of each metabolite were analyzed to optimize conditions of liquid chromatography and mass spectrometer.
And the cellular extracts were analyzed as test samples. Samples were measured by electrospray triple quadrupole mass
spectrometer（LCMS-8030, Shimadzu）coupled to UHPLC system（Nexera series, Shimadzu）. Analytical conditions were as follows,column： L-Column ODS（ 2 mmI.D. x 150 mmL., 3 micrometer）； flow rate： 0.3 mL/min； column temperature： 40C； mobile
phase A： water containing 10 mmol/L tri-butyl ammonium acetic acid； mobile phase B： methanol； gradient program： 0 %B（ 0 -0.5min）- 25%B（7.5 min）-90%B（11-11.5 min）- 0 %B（11.6-15min）； sample cycle time： 15 min. The mass spectrometric parameters
were optimized automatically by flow injection analysis.
LCMS-8030 provides a high throughput quantitative analysis with the high chromatographic resolving power of UHPLC systems. In
this study, we developed the quantification system for metabolites included in central pathway of energy metabolism with cycle
time 15 min by ion-pare and reversed-phase chromatography coupled to LCMS-8030 with modification for improving its sensitivity.
Most metabolites in central pathway of energy metabolism show high hydrophilic property and have an acidic functional group.
All compounds were detected as deprotonated molecules（［M-H］-）in ESI negative ion mode, and mass spectrometricparameters for MRM analysis such as monitoring ion and collision energy were optimized by automatic optimization procedure. In
comparison of several brands of ODS column, we chose L-Column ODS in terms of separation of sugar phosphates.
As the results of optimization for chromatographic and mass spectrometric parameters, over 80 metabolites could be analyzed
simultaneously in injection cycle time 15 min. The metabolites range from m/z 73 to m/z 866 and include ribitol as an internal
standard. The analytical method we developed could also analyze non-volatile compounds such as nucleotides which could notanalyzed by GCMS, and stability of retention time was satisfactory. We analyzed both of standard samples and cellular extracts toevaluate the qualitative capability such as limit of detection, dynamic range, carry over, and stability.
Yeast extract was analyzed as model samples to confirm applicability of developed method to biological samples. 75 components
were detected successfully. Furthermore, we analyzed yeast extract with serial dilution to confirm linearity of metabolites quantity
observed by this method. These results show that this method could detect change of a metabolite quantitatively.
In this study, we developed and evaluate comprehensive, ragged, and rapid quantitative system for metabolites in central
pathway of energy metabolism. The result suggests that this system could be broadly applicable as a tool for widely targeted
metabolomics.

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 21: Platform Technology for Metabolomics

PWe-024
13:30 － 14:40

Metabolic changes during aging complementary GC-EI-MS and GC-APCITOF-MS analysis of a short-lived mitochondrial knockdown of
Caenorhabditis elegans

Jouji Seta1, Verena Tellstroem3, Aiko Barsch3, Gabriele Zurek3, Carsten Jaeger2, Bernd Kammerer2

Bruker Daltonics K.K., 2Center for Biological Systems Analysis (ZBSA), University Freiburg, Germany, 3Bruker Daltonik
GmbH, Bremen, Germany

1

Keywords:
GC/MS, GC-APCI, QTOF, metabolite, c. elegans
Novel aspects:
Metabolic profiling in Caenorhabditis elegans using GC-EI-MS and GC-APCI-TOF-MS
Abstract:
Aging is a highly complex biological process involving a multitude of alterations at the metabolic, cellular and organismal level.
According to the free radical theory of aging, mitochondria are the major source of oxidative stress within the cell and a maincause of aging. Mitochondria may become increasingly dysfunctional with age and fundamental impacts on cellular energy
metabolism may occur. The simple nematode worm Caenorhabditis elegans is a well-established model system for aging； several
mitochondrial mutations significantly reduce life span. However, metabolic alterations associated with life span reduction arelargely unknown.
We compared whole-organism methanolic extracts of short-lived C. elegans depeleted in mitochondrial prohibition to those of
non-treated C. elegans with normal life span. Non-targeted metabolite profiling of methoximated and trimethylsilylated
compounds was performed with gas chromatography-mass spectrometry（GC-MS）with electron impact（EI）ionization. Peak
areas were normalized to internal standard and sample dry weight, peak identification was achieved by database-assisted mass
spectral/retention index matching（AMDIS, FiehnLib/NIST05）. In addition, gas chromatography coupled to high resolution TOF-MS
with softer atmospheric ionization（APCI）was used to analyze the same methanolic extracts both for metabolic profiling andsubsequent identification of known and unknown compounds. Molecular formulae were derived from the accurate mass andisotopic pattern data and searched in public databases（PubChem, KEGG）.
Initial profiling with GC-EI-MS yielded 212 reconstructed mass spectra present in at least 40 of 48 chromatograms. Of these 212
mass spectra, 134 could be assigned structures at a match factor of 800, while the rest remained unidentified. Principal component
analysis（PCA）and partial least squares discriminant analysis（PLS-DA）both allowed clear differentiation between metabolic
phenotypes of short-lived and normal-lived worms. PLS-DA indicated that a set of 17 compounds was particularly relevant forphenotype differentiation； however, five of these relevant metabolites could not be identified by database search.
In contrast to GC-EI-MS, GC-APCI-TOF-MS analysis readily yields pseudomolecular ions facilitating the identification of unknowns.
Feature extraction from the GC-APCI-TOF-MS analyses for statistical analysis resulted in approx. 650 features/chromatogram.
Differentiation using pattern recognition methods between RNAi-treated and non-treated animals was similarly possible based on
the APCI data. Sum formula generation from accurate mass and isotopic pattern successfully confirmed identities of primarymetabolites from GC-EI-MS analysis such as glycine or alanine. The identification of the unknown 5 phenotype related metabolites
is currently ongoing.

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 21: Platform Technology for Metabolomics

PWe-025
11:10 － 12:20

Development of automated data processing tool for large scale MRM
experiment in metabolomics study

Hiroshi Tsugawa1, Mitsuhiro Kanazawa2, Atsushi Ogiwara2, Takeshi Bamba1, Eiichiro Fukusaki1
1Osaka University, Osaka, Japan, 2Reifycs Inc., Tokyo, Japan

Keywords:
Metabolomcis, Data analysis, Multiple reaction monitoring
Novel aspects:
Development of an automated data processing tool to accurately and objectively identify the detected peaks by multiple reaction
monitoring method
Abstract:
Multiple reaction monitoring（MRM）is a well-known mass spectrometric method to detect and quantify the target metabolites athigh sensitivity and selectivity in complex samples. Triple quadrupole mass spectrometer capable of high-speed scanning has
been increasingly developed to obtain a huge amount of compounds, and total number of transitions is more than 1000 in a single
run. This large scale MRM method is frequently used for“widely targeted metabolomics“ as a trend methodology to obtain a lotof metabolite information from 50 to 100 compounds.
MS instruments and their related assays have been remarkably improved, but data analysis tools are less developed accompany
with large scale data sets. This is the reason why MRM measurement data are mostly evaluated by manual verifications. Manualverifications for MRM data are not only laborious tasks especially peak identification, but it causes more severely situations due tosubjective and irreproducible approach.
To address this situation, we developed an automated data analysis tool for“widely targeted metabolomics“. It is easy to
perform large scale MRM data especially for LC-MS/MS. Our challenges are algorithm developments to detect peaks without any
subjects, and to accurately identify targeted compounds by LC-MS/MS. Existing peak detection algorithms are based on eluted
shape of target compound. This tool utilizes retention time alignment among comparative samples and transitions along with theexisting algorithm. As for accurate target compound identification, we constructed a reference library that has intensity ratio
regarding multiple transitions for every target compound. Intensity ratios are referred to identify target compounds, and it
differentiates target compounds that have same precursor and fragment m/z. Scores of the retention time similarity, peak shapesimilarity and correlation with the reference transition data are calculated, and total score is utilized to check whether thedetected peak is the real target or not. In addition to the peak identification algorithm, our data analysis tool offers an organizeddata matrix from large amount of data sets for further statistical analysis directly.
We performed three experiments in order to demonstrate the main features of our tool. In this study, we utilized the ion-pair LCMS/MS system which is used for sensitive analysis of central metabolites such as sugar phosphates and cofactors. The firstexperiment validated the accuracy of peak identification by analyzing a defined mixture of 72 standard compounds and the yeast
extract spiked the mixture. The second experiment determined the quantitative performance by analyzing a defined mixture of 10
standard compounds measured at different concentrations. Finally, we validated the usefulness and versatility by using real
biological samples. Our platform has been applied to perform widely targeted metabolomics of a lot of ion-pair LC-MS/MS
chromatogram datasets of Saccharomyces cerevisiae cells sampled at different time points during the diauxic shift which is awellknown phenomenon in the yeast cell.

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 21: Platform Technology for Metabolomics

PWe-027
11:10 － 12:20

LC-MS/MS-based plasma metabonomics: finding potential biomarkers forlung cancer

Wei Yang1, Yanhua Chen1, Cong Xi1, Ruiping Zhang1, Yongmei Song2, Jiuming He1, Jinfa Bai1,
Qimin Zhan2, Lvhua Wang2, Nan Bi2, Zeper Abliz*1

Institute of Materia Medica, Chinese Academy of Medical Sciences and PekingUnionMedicalCollege, Beijing, P. R.
China, 2Cancer Institute and Cancer Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College,
Beijing, P. R. China

1

Keywords:
Metabonomics； LC-MS/MS； Lung Cancer； Plasma
Novel aspects:
In the study, the influence range and influence extent of the solvent effect has been studied in detail so that more accurate data
could be got.
Abstract:
Introduction
Metabonomics is an important platform for understanding of the metabolites of integrated living systems and its dynamic
responses to the changes caused by both endogenous and exogenous factors. Our laboratory has developed the method of
metabonomics inprevious study. However, we found that re-dissolution solvents of dried plasma extracts lead to much different
chromatographic behavior. In this research, the re-dissolution solvents have been optimized systematically for plasma
metabonomics study.
The developed method of plasma metabonomics based on LC-MS/MS, multivariate statistics and metabolic correlation networks
has been implemented to find biologically significant metabolite biomarkers in lung cancer. This strategy not only helps identify
potential biomarkers for prognoses of early-stage disease, but also provides biochemical insights into changes in lung cancer.
Methods
232 lung cancer patients, including adenocarcinoma（n= 65）, quamous cell carcinoma（n=32）, small cell carcinoma（n=49）andblind samples of lung carcinoma（n=86）, and 156 healthy volunteers were recruited from the Cancer Institute and Hospital of theChinese Academy of Medical Sciences, Beijing, China. Proteins of plasma samples were removed with ice-cold acetonitrile. Thenthe supernatant was collected and completely evaporated to dryness in a SpeedVac concentrator（Thermo Savant）. The sample
residues were dissolved in initial mobile phase and mixed by a vortex-mixer on ice. Then the samples were filtered with 96-wellplate filter and injected with 96-well collection plate（Agilent, Captiva ND）. Rapid resolution liquid chromatography coupled to
Q-TOFMS/MS system with ESI, were employed to detect the prepared plasma samples.
Raw LC-MS data files were converted into mzData format using Wiff to mzData utility（Applied Biosystems/MDS Sciex）and
directly processed by open-source XCMS package under R statistical software（version 2.10.0）to carry out peak discrimination,filtering and alignment. The matrices were introduced into SIMCA-P software（Umetrics AB, Umeå, Sweden）for multivariate
analysis.
Preliminary data
The samples that have been dried in a SpeedVac Concentrator were redissolved in different ratios of acetonitrile. It was
manifested that the solvent effect could be presented when the solution is different from the initial mobile phase, even though
there is only a tiny change. It was noting that the compounds eluted before 10 min could not be monitored when the samples
were redissolved in 100 % acetonitrile. The larger deviation of the acetonitrile ratio between the solution and the initial mobile
phase, the wider influence range and stronger influence degree of the solvent effect could occur. Now the influence range andinfluence extent of the solvent effect has been studied in detail.
The acquired LC-MS data of squamous carcinoma were imported into XCMS, yielding 1064 peaks of positive ions from 0 to 30 minof retention time. The resulting data was exported into SIMCA-P software for subsequent processing by multivariate data analysis.
And 35 metabolites have been regarded as potential biomarkers. Until now, 15 potential biomarkers have been identified, including
glucose, amino acids, carnitines and lysophosphatidylcholines. Next we will identify the potential biomarkers of adenocarcinoma
and small cell lung cancer. And the potential biomarkers will be validated with the blind samples of lung carcinoma.
Acknowledgement
The study has been supported by National Natural Science Foundation of China（21175154）.

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 21: Platform Technology for Metabolomics

PWe-028
13:30 － 14:40

Metabolomics and its application to high resolution phenotype analysis

Eiichiro Fukusaki

Osaka University, Osaka, Japan

Keywords:
Metabolomis, Metabolic fingerprinting, Metabolic turnover analysis, PCA, PLS
Novel aspects:
Power of metabolome as a high resolution phenotype analysis is proved. Early developmental stage of vertebrate was firstpredicted by metablome. Strategic lifespan extension was performed using metabolome information.
Abstract:
Introduction
Metabolomics is applicable without genome sequence information. Therefore, Metabolomics would be powerful analytical toolfor commercially available plant and microorganisms. Among the operation of Metabolomics, metabolic fingerprinting, in whichmetabolic profile is used as fingerprint for samples’ performance prediction and classification, is thought to be a possible tactics.
On this occasion, practical operation of Metabolomics for high resolution phenotype analysis would be presented.
Metabolome analysis of early developmental stage of experimental animal
Metabolome analysis of early developmental embryo was conducted to prove metabolome can express early developmental
stage quantitatively. Time course sampling of early developmental embryo, which were prepared by artificial insemination, wasperformed . The obtained early developmental embryos were subjected metabolome analysis. Principal component analysis（PCA）revealed that 1 st component clearly indicated developmental stage of embryo. Partial least square projection on latent
structure（PLS）was carried out using ’metabolome’ as explanatory variable and ’time after fertilization’ as responsible variable. PLS
regression successfully indicated developmental stage using one embryo with only 30 minutes error. Similar experiment can beavailable for nematode（C.elegans）development.
Metabolomics based semi-rational prediction of yeast life span and its application to seeking of lifespan related mutants.
First, possibility of Metabolomics based prediction of“Life span“, which is highly integrated and complicated quantitative
phenotype, was studied to find strong relationship between metabolome and lifespan by means of PCA. Orthogonal projection on
latent structure（OPLS）based model can predict yeast life span using metabolome as fingerprint. Using information of principal
component vector in PCA life span related metabolites were nominated and life span related genes were speculated.
Dynamic metabolome analysis by stable isotope dilution based time course sampling
Metabolic profiling usually imply a static analysis based on multiple snapshot of metabolome. In some case, dynamic information
would be necessary. Metabolic flux analysis is one of the most important tactics for dynamic metabolic analysis. But the method israther difficult for person who is not familiar with metabolic engineering. Time course sampling after labeling with stable
isotopomer metabolites would be useful for apparent metabolic turn over analysis. Some preliminary experimental data are also
discussed.

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 21: Platform Technology for Metabolomics

PWe-029
11:10 － 12:20

Implementation of a Data Independent MS/MS Acquisition Strategy for
Metabolites Identification in a Metabolomics MS-based Workflow

Emmanuel Varesio, Gerard Hopfgartner
University of Geneva, Geneva, Switzerland

Keywords:
LC-MS/MS, Metabolites Identification, SWATH, High Resolution Mass Spectrometry, Human Urinary Metabolites
Novel aspects:
Alternative MS/MS acquisition strategy for metabolites identification in metabolomics MS-based workflows.
Abstract:
Mass spectrometry has emerged in metabolomic studies due to its high sensitivity and throughput, as well as its direct
hyphenation to liquid chromatography when analyzing complex samples. High-resolution mass spectrometers are used inunsupervised LC-MS based assays to propose elemental formulae for the biomarker candidates. Following a search inmetabolomics databases（e.g. KEGG, HMDB, PubChem）, MS/MS experiments are usually performed for structural investigations.
MS/MS acquisition can be done either“on-the-fly“ as an information-dependent acquisition（i.e.IDA）mode, which results in abiased information since the less abundant candidates are usually not submitted to collision-induced fragmentation, or as targeted
experiments that require the re-analysis of the samples. We propose an alternative MS/MS acquisition strategy： the use of
Sequential acquisition Windows of All Theoretical precursor ions（SWATH）approach or Global Precursor ions Scans（GPS）tooffer unbiased information during metabolomics assays.
In this study, a set of compounds relevant as urinary metabolites was selected. Analytes structures were downloaded from the
PubChem Compound database and molecular properties（e.g., pKa, logD, logP）were calculated by using the ACD Labs software
suite. The standard mixture（100 ng/ml）was first injected in reversed phase chromatography to characterize the analytes retention
as well as the LC peak width to determine the optimal MS duty cycle. Typical LC peak width at the base was of 6 s that allows amaximum MS duty cycle of ca. 500ms. In IDA mode, the MS duty cycle could reach a maximum of 450 ms in order to get sufficient
data point to define the LC peak accurately in TOF mode； MS/MS spectra were acquired in dynamic background subtraction
mode to improve spectra quality. Contrary to the IDA approach the SWATH or GPS acquisition mode is an alternative solution
with several advantages in metabolites identification： i）some selectivity is put in the precursor ion selection（i.e.windows of100u）, ii）potentially all the precursor ions undergo fragmentation since MS/MS spectra are acquired with a CE ramp, and iii）the
precursor ion is present in the same spectrum as its fragments, which enables biomarkers quantification. These different strategies
will be applied to identify metabolites in diluted or extracted urine samples using database search and elemental formulae
assignment.

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 21: Platform Technology for Metabolomics

PWe-030
13:30 － 14:40

Alkyl chloroformate - mediated derivatization - extraction platform forparallel GC-MS and LC-MS based metabolomics

Petr Simek, Petr Husek, Helena Zahradnickova, Jana Cimlova, Lucie Rimnacova
Biology Centre, Czech Academy of Sciences

Keywords:
alkyl chloroformate-mediated derivatization - extraction, GC-MS, LC-MS, metabolomics
Novel aspects:
Alkyl chloroformate derivatization - extraction has been an efficient analytical platform complement to the current GC-MS & LC-MS
based metabolomic analytical strategies
Abstract:
Comprehensive metabolomic analysis by GC-MS requires a preceding derivatization step relying mostly on the established（oximation-）silylation procedure［ 1 ］. However, metabolites are found mainly in aqueous environments with high concentrationsof salts and polymeric components and usually are maintained in biochemical buffers while the silylation reaction must be
performed under strictly anhydrous conditions. In this context, derivatization with an alkyl chloroformate（RCF）and/or thecorresponding alcohol（ROH）, and coupled with simultaneous liquid liquid extraction, has been an attractive option in multi-classmass spectrometric metabolite analysis. The RCF-ROH reaction proceeds smoothly with most protic groups in complex aqueous
media under physiological conditions, even when the samples are treated/stored with most common biochemical buffers at molar
concentration levels. Moreover, the typical arising carbamates-carbonate-ester derivatives are efficiently extractable into an
immiscible organic phase and have moderate polarities. They are much easier prepared and considerably more stable than theirsilylated counterparts. Availability of the reagents with various（fluoro）alkyls, including those labeled with stable isotopes, makethe RCF-mediated derivatization-extraction platform attractive in chiral GC-MS and parallel GC-MS & LC-MS based metabolomic
analysis.
In this presentation, some issues important for the maturity of the RCF methodology will be briefly discussed, including：（i）the effect of deproteination on the RCF-mediated derivatization-extraction［ 2 ］,（ii）the combined release of the disulfide-bound metabolites and its use in metabolomic analysis［ 3 ］,（iii）potential of fluoroalkyl chloroformate reagents including the chiral analysis［ 4 ］,（iv）the RCF labeling for the identification and analysis of metabolites and xenometabolites including the potential of the RCF
reagents labeled with stable isotopes［ 5 ］,（v）matrix effects in LC-MS analysis［ 6 ］,（vi）application of the RCF-mediated derivatization - extraction platform in parallel GC-MS and LC-MS based insect metabolomics［ 7 - 9 ］leading to the discovery of a simple strategy which makes an intolerant insect organism freeze tolerant［10］.
References［ 1 ］Koek M M, Jellema R H, van der Greef J, Tas A C, Hankemeier T, Metabolomics 2011, 7 ： 307-328, doi： 10.1007/s11306-01-02543.
［ 2 ］Husek P, Svagera Z, Hanzlikova Z., Simek P, Survey of several methods deproteinizing human plasma before and within thechloroformate-mediated treatment of amino/carboxylic acids quantitated by gas chromatography. J Pharm Biomed Anal 2012, inpress.
［ 3 ］Svagera Z, Husek P and Simek P, Anal Bioanal Chem 2012, 402： 29532963, doi： 10.1007/s00216-012-5727-y.
［ 4 ］Simek P, Zahradnickova H, Husek P, Methods in Molecular Biology 2012, 828： 137-152. Heptafluoro-butyl chloroformate based sample preparation protocol for nonchiral and chiral analysis of amino acids by gas chromatography. Amino Acid
Handbook, Chapter 13, Springer Media & Bussiness Media, New York 2012, doi： 10.1007/978- 1 -61779-445- 2 _13.
［ 5 ］Rimnacova L, Simek P, Mraz J, Husek P, Simultaneous GC-MS profiling of sixteen acidic biomarkers of occupational exposureand endogenous metabolites in urine. J Chromatogr. A 2012, submitted.
［ 6 ］Cimlova J, Kruzberska P, Svagera Z, Husek P and Simek P, J Mass Spectrom 2012, 47： 294-302,doi：10.1002/jms.2952.
［ 7 ］Kostal V, Korbelova J, Zahradnickova H, Cimlova J, Tomcala A, Simek P,PlosOne 2011, 6 ： e25025, doi： 10.1371/journal.
pone.0025025.
［ 8 ］Kostal V, Simek P, Zahradnickova H, Cimlova J, Stetina T, Proceedings of the National Academy of Sciences of the USA,109：2012,370-3274, doi：10.1073/pnas.1119986109.
［ 9 ］Kostal V, Zahradnickova H, Simek P, Proceedings of the National Academy of Sciences of the USA 2011, 108： 13041-13046,
doi： 10.1073/pnas.1107060108.
［10］Two steps help Drosophila melanogaster survive freezing conditions. The Scientist, http：//the-scientist.com/2012/02/13/
freeze-tolerant-flies/.

Financial support. Czech Science Foundation, project No.P206/10/2401（PH, HZ）and Ministry of Health of the Czech Republic, No.
NT11513- 5 （PS, JC, LR）.

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 21: Platform Technology for Metabolomics

PWe-031
11:10 － 12:20

Aberrant Metabolism and Its Response to Treatment in Esophageal
Squamous Cell Carcinoma Revealed by Plasma Metabolomics Study

Jing Xu1, Yanhua Chen1, Ruiping Zhang1, Yongmei Song2, Nan Bi2, Jiuming He1, Jinfa Bai1, Lvhua Wang2,
Qimin Zhan2, Zeper Abliz1
Institute of Materia Medica, Peking Union Medical College, Beijing, China, 2Cancer Institute & Cancer Hospital, Peking
Union Medical College, Beijing

1

Keywords:
metabolomics； esophageal carcinoma； potential diagnostic biomarkers； therapeutic biomarkers；TCA cycle
Novel aspects:
Some potential diagnostic and therapeutic biomarkers by analyzing global changes in an individual’s metabolic profile in response
to pathophysiological stimuli or treatment intervention were discoverd.
Abstract:
Introduction
Metabolomics provides a powerful approach to discover diagnostic and therapeutic biomarkers by analyzing global changes in an
individual’s metabolic profile in response to pathophysiological stimuli or treatment intervention. At present, a plasma
metabolomics study on esophageal squamous cell carcinoma（ESCC）and patients with treatment have been investigated by
rapid resolution liquid chromatography -quadrupole time-of-flight mass spectrometry（RRLC-QTOFMS）in conjunction withmultivariate statistics analysis（MVDA）. The metabolic profiles between pre-treatment patients and controls were performed tofind the potential biomarkers closely associated with the disease. The metabolic profiles between pre- and post-treatment ESCC
patients combined with the therapeutic evaluation was carried out to discover the major metabolites contributing to thediscrimination which might be potential therapeutic biomarkers. The metabolite variations in post-treatment ESCC patients were
further discussed and might be used to monitor treatment progress and presume outcome.
Methods
Pre-treatment samples： 44 males diagnosed with ESCC were recruited from the Cancer Institute and Hospital of the Chinese
Academy of Medical Sciences. Mid- and Post-treatment samples： Plasma samples were collected after the first course of 5 -FU/
CDDP based CRT（ESCC-M group）and the completion of CRT（ESCC-P group）from 32 ESCC male patients with different responseto treatment（overall responders, OR group, n=21； non overall responders, non-OR group, n=11）. RRLC coupled with Q-TOF MS/
MS system（QSTARTM Elite）in electrospray ionization（ESI）positive and negative ion modes were used for metabolomics analysis
after acetonitrile precipitation as sample preparation. The date treatment was processed by XCMS package, SIMCA-P software
12.0（Umetrics AB, Umeå, Sweden）and SPSS software 17.0.
Preliminary data and Results
Metabolic perturbations under pathological conditions were investigated by metabolic profile between ESCC and NC. Major fiftytwo metabolites contributing to the discrimination were screened and twenty-seven were identified as lipid metabolites（lysophosphatidylcholines（lysoPCs）, fatty acids）, lipid metabolism intermediates（carnitine and acylcarnitines）, amino acids,organic acids, sterol metabolite and monosaccharide. The abnormal levels of these metabolites indicated that the disturbed TCA
cycles and the energy metabolism via b-oxidation occurred in ESCC patients. The AUC of the combined metabolites showed thegood discrimination power. Furthermore, on the basis of these screened metabolites, the correlation network between them wasconstructed and a metabolic pathway related to ESCC was proposed in-depth.
To evaluate the physiological responses to treatment, OPLS-DA was performed to discriminate between the pre- and posttreatment metabolic profiles of ESCC patients. The metabolic alterations in ORs and non-ORs were also evaluated, respectively.
Those significantly altered metabolites in ORs are more likely to be associated with the therapeutic effects. Thirteen discriminating
variables between total pre- and post-treatment groups were identified. Eleven of these selected metabolites were accountable
for the discrimination between pre- and post-treatment metabolic profiles in treatment ORs. Furthermore, after screening between
two groups in treatment non-ORs, five metabolites were picked out and they might be the potential therapeutic biomarkers finally.
In addition, the variations of these five metabolites during pre-, mid- and post-treatment were researched. The result indicatedthat these five metabolites might closely relate with treatment effect and might be utlized to monitor treatment progress andpresume outcome.
Acknowledgments
National Instrumentation Program（2011YQ170067）

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 22: Instrumentation Developments in Mass Spectrometric Imaging

PWe-032
13:30 － 14:40

Imaging mass spectrometry and structural analysis of lipids directly on
tissue specimens using a high resolution MALDI-TOF-TOF tandem mass
spectrometer

Shuichi Shimma2, Ayumi Kubo3, Takaya Satoh3, Hisanao Hazama1, Kunio Awazu1, Michisato Toyoda1
Osaka University, Osaka, Japan, aaa, 2National Cancer Center Research Institute, Tokyo, Japan, 3JEOL Ltd., Tokyo,
Japan

1

Keywords:
Imaging mass spectrometry, high resolution, tandem mass spectrometry
Novel aspects:
Direct tissue analysis and imagingmass spectrometry（IMS）was performed by using a high mass resolution tandem time-of-flightmass spectrometer
Abstract:
Introduction
Direct tissue analysis and imaging mass spectrometry（IMS）using a high mass resolution tandem time-of-flight（TOF）mass
spectrometer is described. This system mainly consists of a spiral ion trajectory TOF mass spectrometer“SpiralTOF（STOF）“ andan offset parabolic reflectron（RTOF）. Due to 17-m flight length in STOF, high mass resolution and high precursor selectivity areavailable. Elimination of metastable ions in STOF allowed observation of the product ions inherent to the high energy collisioninduced dissociation（HE-CID）process. By using this TOF-TOF instrument, the product ion spectrum of lipids provided detailed
structural information of fatty acid residues. In this presentation, we will report advantages of high mass resolution IMS andstructural analysis using HE-CID for lipid analysis.
Methods
This study used 6 -week-old male C57BL/ 6 J mice. The mice were anesthetized with isoflurane, sacrificed, and dissected. The brain
block was immediately frozen in powdered dry ice to minimize degradation and kept at -80 degrees C. The tissue sections weresliced in 10 um-thick sections using a cryostat and thaw-mounted on a ITO slide glass sample plate（25 mm x 75 mm and thicknessof 1.1 mm）. The samples were dehydrated in a vacuum chamber and the plate was mounted on a custom-built sample holder.
The instrument was equipped with a 349 nm Nd：YLF laser for matrix-assisted laser desorption/ionization（MALDI）. In IMS
experiment, the interval of data points was 80 um and numbers of laser irradiation in spot were 250 shots.
Preliminary Data
In both positive and negative ion modes, typical mass resolution of approximately 40,000 were obtained. Even in tissue analysis,
the high mass resolution was sufficient to separate monoisotopic ions easily for the subsequent MS/MS experiment.
Positive ion detection mode provided a product ion peak of m/z 798, arising from the potassium ion adduct of phosphatidylcholine（PC）（34： 1 ）. In product ion spectrum obtained using our system, several intense peaks were derived from the polar headgroup. Furthermore, a regular pattern of peaks was obtained above m/z 500. By detailed analysis of the high resolution mass
spectrum, we found several doublet peaks which mass differed 0.1-0.2 u each other. For example, the［M+ 2 ］of m/z 866 and［M］
of m/z 868 were observed in 0.18 u difference. By describing the mass image for each peak, former and latter ions arecharacteristically distributed on gray and white matters in mouse brain tissue section, respectively. The clear separation of IMS
images is advantage of high resolution STOF that could not achieved by conventional reflectron type instrument.
Detail structural analysis showed product ions derived from dissociation of both sn- 1 and sn- 2 fatty acids. The CFR fragment ion
peaks are clearly observed in product ion spectrum from m/z 798, which was expected as PC（34： 1 ）. To validate this result, we
confirmed the pattern of product ion spectra using a standard sample of PC（34： 1 ）. In this presentation, we will discuss
detailed structure of major lipid peaks observed in both positive and negative ion modes.

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 22: Instrumentation Developments in Mass Spectrometric Imaging

PWe-033
11:10 － 12:20

Observation of distributions of Strontium and Cesium stable isotopes intissues sections of medaka by a new mass imaging system

Shinichiro Ikeda, Jun Aoki, Michisato Toyoda
Osaka University, Osaka, Japan

Keywords:
Imaging Mass Spectrometry
Novel aspects:
The distributions of strontium and cesium stable isotopes in tissue sections of small fish was obtained by imaging mass
spectrometry.
Abstract:
Mechanism of biological absorption and accumulation of toxic substance is important matter and well studied. In particular thecase of food, a information about distribution of the bioaccumulative toxins become significant from the aspect of safety forhuman health. Recently, the biological accumulation of radioactive substance became great concern due to the accident of
nuclear power plant in Japan.
Conventionally, the biological distribution of radioactive substance is measured by autoradiography analysis. In the
autoradiography, localization of decay emissions of radioactive substance in the measurement sample is imaged by an x-ray film.
But this method takes up to about 1 week to get images and is impossible to identify the species of radioactive elements. Thus
we developed new observation method to estimate the distribution of elements, for example strontium and cesium, in biologicaltissues by using newly developed imaging mass spectrometer（MULTUM-IMG 2 ）. MULTUM-IMG 2 consisted of LDI ion source,
multi-turn time-of-flight mass spectrometer（MULTUM）and a time and position sensitive detector. The sample surface is irradiatedwith UV laser at a beam diameter of about 0.5 mm. A spatial distribution of molecular on a sample surface is conserved opticallythrough ionization and mass separation process and formed an image on the detection surface. The distribution information ofmass of substances on the sample surface can be obtained by imaging mass spectrometry. Each elements and isotopes is
identified by difference of mass, so measurement object is not limited to radioactive substance. We used non-radioactive
isotopes, which is easy to handling, for this experiment. The biological activities of isotopes of same element is considered as no
significant difference.
We chose medaka（Oryzias latipes）as measurement test subject and 88Sr and 133Cs as bioaccumulation elements. Medaka bred
in 0.001 mol/l SrCl2 and 0.05 mol/l CsI solution for two weeks absorbed these elements. The length of body is about 20 mm and thewithers height is about 6 mm. For sample preparation, the frozen body was sliced into a thickness of 10 μm using a cryostat
microtome and mounted onto a sample plate. The direction of slicing is from the cephalic region to the caudal region. The sample
tissue of whole body was trimmed one-half from cephalic end fit to the sample plate of 10 mm in width. The sample surface wascoated by a thin gold layer of about 10 nm for electrical conductivity, because electrostatic charge accumulation induced by laser
irradiation and ionization deteriorate the image quality. Sample section was installed into the imaging mass spectrometer after
acquisition of an optical microscope image. The view observed in one measurement is 500 μm diameter and we measured 50
times to observe the whole cephalic region.
Biological structure shown in the ion image was verified to be consistent with those in the optical microscope image. The
resulting image showed that distribution profile was different in elements belonging to the same family, for example Na, K and Cs.
Mass resolving power of m/Δm 3450 were obtained for the main peak of 133 Cs at m/z = 132.9. Spatial resolution is enough to
identify a biological structure of about 6 μm size. As the next step, this result is expected to be analyzed in detail with additional
anatomical knowledge.

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 23: Gas Phase Fragmentation Mechanisms of Biomolecular Radicals

PWe-034
13:30 － 14:40

Investigation of fragmentation routes of steroid ethers via MIKES andB2*E-linked-scan techniques.

Heiko Bannick, Juergen Grotemeyer

Institut of Physical Chemistry University of Kiel, Kiel, Germany

Keywords:
B 2 *E-linked-scan, MIKES
Novel aspects:
Fragmentation routes of steroid ethers have been investigated.
Abstract:
Introduction：
Metastable ions have been studied using MIKE- and B2*E-linked-scan techniques to investigate the fragmentation of estradiol- andestriolethers appearing in the EI- or CI-mass spectra. The aim was to find out how a fragment is built： during a normal elimination
process, following a rearrangement or during another consecutive process. Using this spectral data it is possible to draw a“fragmentation tree“ were fragmentation routes are shown.
Methods：
In this work a ZAB 2 F build by Vaccuum Generators was used. Its sector fields are assembled in reversed Nier-Johnson geometry.
The EI-ions were generated at 70 eV and ammonia was used as reagent gas in the CI-process.
To obtain the MIKE spectra a fragment ion was selected by the magnet and the E-field was scanned. In B2*E modus the B- andE-fields were scanned at a constant ratio to point out the precursor ions.
Preliminary data：
The MIKE spectrum of the Estriol- 3 -benzylether radical cation shows the following eliminations： -18, -28, -36, -41, -55, -59, -91, -109,-116, -127 and -181 Da. Using MIKES and B 2 *E-linked scans it is possible to check if these fragments are directly formed byelimination, rearrangements or in a consecutive elimination of two or more precursors.
An example for this is the elimination of 109 Da from Estriol- 3 -benzylether radical cation. This is an elimination of the ether groupas a radical and the elimination of a water molecule. Following a consecutive reaction mechanism beginning with the loss ofwater, the MIKE spectrum of the［M+.-18］should show an elimination of 91 Da which rather appears with quite low intensity. In
addition the B2*E-linked spectrum of the［M+.-109］should show the［M+.-18］as precursor, it does not. Instead it shows the［M+.91］.
Looking at the MIKE spectrum of the［M+.-91］the loss of a water molecule is the most intensive elimination.
This indicates that first the ether group is eliminated and adjacent the water molecule.
Following this example the methyl-, ethyl-, propyl-, allyl-, and benzylethers of estradiol and estriol have been examinated and thefragmentation routes have been determined.

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 23: Gas Phase Fragmentation Mechanisms of Biomolecular Radicals

PWe-035
11:10 － 12:20

Marked difference in fragmentation pathway between chemi-excitationand collision-induced excitation of CT-induced decomposition products ofdioxetanes in negative-mode MALDI-TOF-MS/MS

Hisako K Ijuin, Mamoru Ohashi, Masatoshi Tanimura, Nobuko Watanabe, Masakatsu Matsumoto
Kanagawa University, Hiratsuka, Japan

Keywords:
Dioxetane, CTID, MALDI-TOF-CID-MS/MS, Collision-induced-excitation, Electronically-chemi-excitation
Novel aspects:
We found that molecule-related ion underwent different types of fragmentation depending on the excitation node, i.e. chemi
excitation or vibrational excitation.
Abstract:
A high-energy molecule, dioxetane, bearing an aromatic electron donor displays chemiluminescence by intramolecular chargetransfer（CT）-induced decomposition（CTID）mechanism, in which CT occurs from the electron donor to dioxetane O-O toproduce excited species.
On the other hand, we have very recently reported that electron-transfer ionization takes place for dioxetanes bearing ahydroxyphenyl moiety substituted further with an aromatic ring that acts as an antenna to capture an electron from a matrix, suchas poly（ 3 -octylthiophene-2,5-diyl）, with low oxidation potential in negative-mode MALDI-TOF-MS.   
Herein, we disclose that dioxetane 1 bearing a 3 -hydroxyphenyl moiety substituted with benzothiazol- 2 -yl group undergoes
intramolecular CT-induced decomposition to give keto ester 2 in the electronic excited state through the ionization in MS1 mode
described above, while authentic 2 gives a species in the vibrational excited state in MS2 mode.
Dioxetanes 1 produced characteristic fragment ions depending on the substituent at the 5 -position in MS1 mode. Thus, dioxetane,
1 -［ 4 -（benzothiazol- 2 -yl）- 3 -hydroxyphenyl］- 5 -tert-butyl-4,4-dimethyl-2,6,7-trioxabicyclo［3.2.0］heptane 1a producedfragment
ions due to the elimination of 2 -methyl- 1 -propene（56u）or pivalaldehyde（86u）（by α-cleavage of ketone moiety）from moleculerelated ions. These fragment ions could not form directly from a dioxetane if it retained the four-membered peroxide ringframework. One strong candidate capable of causing such fragmentation is keto ester, dimethyl- 3 -oxobutyl- 4 （benzothiazol2 -yl）
- 3 -hydroxybenzenecarboxylate 2a which is generated by the CT-induced decomposition of dioxetane 1a. Thus, the ionization ofauthentic keto ester 2a was investigated in MS1 mode. The observed molecule-related ions from 2a resembled those of dioxetane
1a. However, the expected fragment ions could hardly be detected. These results in MS1 spectrum strongly suggested that ketoester produced by the decomposition of high-energy dioxetane through ionization in MS would be energetically different fromauthentic keto ester 2a. Thus, we performed further in MS2 mode measurements focusing on deprotonated ion  for dioxetane 1a.
In the MS2 spectrum of deprotonated ion  from 1a as the precursor ion, we observed the fragment ions generated by the loss of
56u or 86u from the precursor ion. On the other hand, by MS2 measurement of the precursor ion［M-H］-   from 2a, we observed thesame product ions as in the case of 1a. Next, we carried out MS1 and MS2 of dioxetanes 1b-d which were 1a analogs in place oftert-butyl group at the 5 -position, bearing isopropyl, ethyl or methyl, and their related keto esters 2b-2d. In contrast to dioxetane
1a andketo ester 2a,dioxetane 1b-d and the corresponding keto ester 2b-d exhibited marked difference in the fragmentation
between MS1 and MS2 spectra. In the MS1 spectrum, 1b-d gave the fragment ions due to α-cleavage of ketone moiety, while in
the MS2 spectrum, both 1b-d and 2b-d underwent β-cleavage of an aromatic ester moiety to show fragment ions.
In conclusion, the present study showed that dioxetanes 1 underwent intramolecular CT-induced decomposition to afford ketoesters 2 in the electronically excited stateinMS1, while authentic keto esters 2 underwent vibrational excitation in MS2. Keto esters
2 in the vibrational excited state caused α-cleavage of a ketone moiety and/or β-cleavage of an aromatic ester moiety in MS2,
depending on the alkyl ketone moiety. The features of fragmentation for a vibrationally excited molecule may be affected by theease of bond cleavage that was caused by acquiring collisional energy.

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 23: Gas Phase Fragmentation Mechanisms of Biomolecular Radicals

PWe-036
13:30 － 14:40

Fragmentation of the [M + Na]+ and [M + Li]+ Ions of Multiply Benzyloxysubstituted Arenes under ESI/CID conditions

Dietmar Kuck1, Sandra Heitkamp1, Matthias C Letzel1, Ishtiaq Ahmed2, Karsten Krohn2
1

Bielefeld University, 2University of Paderborn

Keywords:
Benzyl phenyl ethers, sodiated arenes, catechin derivatives, ESI-CID mass spectrometry, benzyl radicals
Novel aspects:
Fragmentation of［M + Na］+ and［M + Li］+ ions of di- and multiply benzyloxy-substituted arenes, structure-specific loss of two
benzyl radicals, specific retro-Diels-Alder reaction of［M + Na］+ ions.
Abstract:
The fragmentation of sodiated organic molecules formed under electrospray conditions has become a ubiquitous feature in mass
spectrometry. Representing neither protonated nor radical cationic species, the gas-phase ion（organic）chemistry of such［M +Na］+ ions is not well understood but a rich source of surprise. This holds also true for per-O-benzylated polyphenols derived fromdimeric gallocatechins（prodelphinidins）studied recently. 1 Besides a strikingly specific retro-Diels-Alder reaction of the flavanol
rings, pairwise loss of benzyl radicals（ 2 C7H7 ·, 182 u）was observed. To screen the scope of this unusual fragmentation behaviour,
the ESI-CID mass spectra of a series of multiple benzyl ethers of lower di- and oligophenolic compounds were analysed withrespect to the fragmentation of the［M + Na］+ as well as［M + Li］+ quasi-molecular ions.
The successive loss of two C 7 H 7 · radicals from the［M + Na］+ and［M + Li］+ ions under CID has been found to be thepredominating or even exclusive fragmentation channel of simple di- and tribenzyloxy-substituted aromatics, provided that the
C6H5CH2O groups are oriented ortho（or para）to each other. Additional functionalities, such as CO2H, CHO, CH2OH or OH groups
at the central phenol ring do not interfere in most cases. The MS 3 spectra of the［M + Na - C 7 H 7 ］+  ·   ions and further MS nexperiments reveal that only the second loss of C 7H 7 · occurs with particular ease. Pyrogallol derivatives show high abundance
ratios r = {［M + Na - 2 C7H7］+} ： {［M + Na - C7H7］+  ·} > 1 due to the presence of at least two ortho-benzyloxy groups, whereas
phloroglucinol derivatives mostly give values r << 1.This also holds true for higher analogues, such as hydroxychalcone and（gallo）
catechin derivatives bearing up to six benzyloxy groups. It appears that twofold loss of two C 7 H 7 · radicals becomes more
predominant with increasing size of the［M + Na］+ ions and that it is due to the favourable formation of ortho-quinoid structuresin the fragment adduct ions.
1. K. Krohn, I. Ahmed, M. John, M. C. Letzel, D. Kuck, Eur. J. Org. Chem. 2010, 2544-2554.

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 23: Gas Phase Fragmentation Mechanisms of Biomolecular Radicals

PWe-037
11:10 － 12:20

A functional group substitution effect on the free radical initiated peptide
sequencing (FRIPS) method

Han Bin Oh1, Jihye Lee1, Hyeyeon Park1, Kun Cho1,2, Bongjin Moon1
1

Sogang University, Seoul, Korea, 2Korea Basic Science Institute, Ochang, Korea

Keywords:
FRIPS, Peptide fragmentation, Radical, functional group substitution
Novel aspects:
When a new functional group is introduced to the o-TEMPO-Bz-NHS, new radical-induced peptide fragmentation patterns wereshown.
Abstract:
Last decade, we have witnessed the development of various radical-based peptide sequencing methods. Representative examples
include electron capture dissociation, electron transfer dissociation, photodissociation of photolabile group, UV photodissociation,
collisional homolytic cleavage of radical precursor, and so on. In particular, we have paid attention to the collisional homolytic
cleavage of radical precursor molecule, o-TEMPO-Bz-NHS. When collisional activation is applied to o-TEMPO-Bz-peptides, facile
homolytic cleavage occurs between the benzyl carbon and the oxygen of TEMPO moiety, thus producing a benzyl radical peptide
cations. Upon the additional collisional activation on these benzyl radical peptide cations, this localized radical site further induces
peptide backbone fragmentations. These processes are refered to as ’free radical initiated peptide sequencing（FRIPS）’. In thecurrent study, we added a functional group to the benzene ring in order to shed light on how a functional group substitution
affects the radical induced peptide sequencing. The added functional group included NO 2, Br, OMe, and CN. Interestingly, thesubstituted o-TEMPO-benzyl group affected the FRIPS process, yielding MS 3 spectra with different fragmentation patterns. The
precursor survival yields were also obtained as a function of the applied collision energy in order to shed light on the mechanistic
aspect of the substitution effect on the FRIPS process. In the conference, the details of the observed mass spectra will bepresented.

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 24: Regulated Bioanalysis

PWe-038
13:30 － 14:40

Evaluation of peptide fractionation strategies used in proteome analysis

Chih-Wei Chiu, Chun-Lun Chang, Sung-Fang Chen*
National Taiwan Normal University, Taipei, Taiwan

Keywords:
Two-dimensional HPLC, Peptide fractionation
Novel aspects:
Peptide fractionation using SCX, HILIC, alkaline-RP and s-IEF were evaluated. The salt content of a sample lowers separation
efficiency of SCX. Combining complementary separation schemes can increase protein sequence coverage.
Abstract:
Peptide fractionation is extremely important for the comprehensive analysis of complex protein mixtures. Although a few in which
the relative separation efficiencies of 2 D methodologies using complex biological samples are compared, a systematic evaluation
was conducted in this study. Four different fractionation methods, including SCX, HILIC, alkaline-RP and solution IEF prior to LCMS/MS analysis were compared. SCX x RP-LC, using desalted samples, permitted the greatest number of proteins to be identified（96.54 % of the total proteins were identified）. The result was followed closely by HILIC x RP-LC and alkaline-RP x RP-LC. It isnoteworthy that, when SCX x RP-LC was used after desalting the sample, significantly more proteins were identified, comparedwith the non-desalted sample（1990 and 1375,respectively）. We also found that the use of a combination of analytical methods
resulted in a dramatic increase in the number of unique peptides that were identified, compared with only a small increase inprotein levels. The increased number of distinct peptides that can be identied is especially beneficial, not only for unequivocally
identifying proteins but also for proteomic studies involving post-translational modifications and peptide-based quantication
approaches using stable isotope labeling. The identication and quantication of more peptides per protein provide valuable
information that improves both the quantication of, and confidence of protein identification.

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 24: Regulated Bioanalysis

PWe-039
11:10 － 12:20

Simultaneous analysis of 8-oxo-7, 8-dihydroguanine and 8-oxo-7,8-dihydro-2’-deoxyguanosine in plasma by liquid chromatography-tandem
mass spectrometry with automated solid-phase extraction

Yi-Hung Tsai, Mu-Rong Chao

Department of Occupational Safety and Health, Chung Shan Medical University, Taichung 402, Taiwan

Keywords:oxidative stress, plasma, on-line SPE
Novel aspects:
Simultaneous analysis of 8 -oxo-7, 8 -dihydroguanine and 8 -oxo-7, 8 -dihydro- 2 ’-deoxyguanosine in plasma by liquid
chromatography-tandem mass spectrometry with automated solid-phase extraction
Abstract:
Oxidatively damaged DNA has been associated with the development of aging, cancer and some degenerative diseases. 8 -Oxo7,8-dihydro- 2 ’-deoxyguanosine（ 8 -oxodG, so called 8 -OHdG）is the most abundant DNA lesion in cellular DNA, and its repair
products in urine, namely modified base 8 -oxo-7,8-dihydroguanine（ 8 -oxoGua）and modified nucleoside（ 8 -oxodG）, have been
widely studied and used as the biomarkers of oxidative stress. In this study, we developed an isotope-dilution liquid
chromatography-tandem mass spectrometry（LC-MS/MS）with on-line solid-phase extraction（on-line SPE）for rapid andsimultaneous analysis of 8 -oxoGua and 8 -oxodG in plasma.
Two clean-up procedures（i.e., protein precipitation and manual SPE）were used prior to on-line SPE LC-MS/MS analysis. An
aliquot of prepared sample was then directly injected into the on-line SPE LC-MS/MS. The on-line SPE consisted of a switchingvalue（two-position microelectric actuator； Valco）and a C18 trap column（75 × 2.1 mm i.d., 5 μm； Inertsil, ODS- 3 ）. The
switching value function was controlled using PE-SCIEX control software（Analyst； Applied Biosystems）. After automatic sample
cleanup, the sample was automatically transferred onto a C18 column（150 × 2.1 mm i.d., 5 μm； Inertsil, ODS- 3 ）using a Agilent
1100 series HPLC system interfaced with a PE Sciex API 3000 triple quadrupole mass spectrometer with electrospray ion source（ESI）. The mobile phase comprised of various proportions of methanol and ammonium acetate solution. The samples wereanalyzed in the positive ion multiple reaction monitoring（MRM）mode. For all of samples, the［M+H］+ ion was selected by the
first mass filter. After collisional activation, two fragment ions were selected： the most abundant fragment ion was used forquantification（quantifier ion）, and the second most abundant ion was used for qualification（qualifier ion）. Optimal multiple
reaction monitoring conditions were obtained for four channels to different period： 8 -oxoGua（m/z 168 → 140 and 168 → 112）,［15N5］- 8 -oxoGua（m/z 173 → 145 and 173 → 117）, 8 -oxodG（m/z 284 → 168 and 284 → 140）, and［15N5］- 8 -oxodG（m/z 289 →173 and 289 → 145）. The dwell times per channel were set at 100 ms for both the analytes and internal standards.
With the use of isotopic internal standards and on-line SPE, the detection limits of 8 -oxoGua and 8 -oxodG were found to be 17.2
and 5.1 fmol, respectively. This newly developed method was further applied to determine the plasma levels of 8 -oxoGua and8 -oxodG in 20 healthy adults. The results showed that the background plasma levels of 8 -oxoGua and 8 -oxodG were 0.076~0.305
ng/ml and 0.07~0.023 ng/ml, being far lower than those in urine（14~33 ng/ml for 8 -oxoGua and 5 ~13 ng/ml for 8 -oxodG）.
Furthermore, significant correlations were observed between plasma and urine for both 8 -oxoGua and 8 -oxodG concentrations,giving the possibility of using other body fluids in addition to urine specimen for assessing the whole-body burden of oxidative
stress.

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 24: Regulated Bioanalysis

PWe-041
11:10 － 12:20

Quantitative analysis of nine N-nitrosamines in human urine by isotopedilution liquid chromatography- tandem mass spectrometry with on-line
solid-phase extraction

Ming-Yen Hsieh, Chiung-Wen Hu

Department of Public Health, Chung Shan Medical University, Taichung, Taiwan

Keywords:
N-nitrosamines, urine, on-line SPE, LC-MS/MS
Novel aspects:
Quantitative analysis of nine N-nitrosamines in human urine by isotope-dilution liquid chromatography- tandem mass spectrometry
with on-line solid-phase extraction
Abstract:
N-nitrosamines are potentially carcinogenic, mutagenic and teratogenic compounds for animals and humans that are often foundin food, drinking water and environment. In addition to exogenous exposure, N-nitrosamines can also be formed endogenously
inside the body from its precursors, nitrate or nitrite and secondary or tertiary amines. In this study, we developed an isotopedilution liquid chromatography-tandem mass spectrometry（LC-MS/MS）with on-line solid-phase extraction（on-line SPE）for aquantitative analysis of nine N-nitrosamines in human urine, namely N-nitrosodimethylamine（NDMA）, N-nitrosomethylethylamine（NMEA）, N-nitrosopyrrolidine（NPyr）, N-nitrosodiethylamine（NDEA）, N-nitrosopiperidine（NPip）, N-nitrosomorpholine（NMor）,
N-nitrosodi-n-propylamine（NDPA）, N-nitrosodi-n-buthylamine（NDBA）and N-nitrosodiphenylamine（NDPhA）.
A 24 ml of crude urine added with isotopic internal standards, then purified by activated carbon and concentrated to 0.1 ml undera high purity nitrogen stream. A 10 μl aliquot of prepared sample was then directly injected into the on-line SPE LC-MS/MS.
Automatic sample cleanup（on-line SPE）was obtained by using a switching value（two-position microelectric actuator； Valco）
and a C18 trap column（75 × 2.1 mm i.d., 5 μm, ODS-3, Inertsil）that was controlled using PE-SCIEX control software（Analyst；
Applied Biosystems）. After automatic sample cleanup, the sample was automatically transferred onto a C18 column（150 × 2.1
mm i.d., 5 μm, ODS-3, Inertsil）using a Agilent 1100 series HPLC system interfaced with a PE Sciex API 3000 triple quadrupole mass
spectrometer with electrospray ion source（ESI）or atmospheric pressure chemical ionization（APCI）. For a satisfactory sensitivity
or selectivity, the quantifications of NDMA, NMEA, NPyr and NMor were performed in the positive-ion APCI mode, while thequantifications of NDEA, NPip, NDPA, NDBA and NDPhA were performed in the positive-ion ESI mode. For all of the samples, the［M+H］+ ion was selected by the first mass filter. After collisional activation, two fragment ions were selected： the most abundantfragment ion was used for quantification（quantifier ion）, and the second most abundant ion was used for qualification（qualifierion）. Optimal multiple reaction monitoring conditions were obtained for 36 channels： NDMA（m/z 75→43 and 75→58）, NDMA-d6（m/z 81→46）； NMEA（m/z 89→61 and 89→43）, NMEA-d3（m/z 92→64）； NPyr（m/z 101→55 and 101→41）, NPyr-d8（m/z 109→62）；
NDEA（m/z 103→75 and 103→47）, NDEA-d10（m/z 113→81）； NPip（m/z 115→69 and 115→41）, NPip-d10（m/z 125→78）； NMor（m/z117→87 and 117→73）, NMor-d8（m/z 125→95）； NDPA（m/z 131→89 and 131→43）, NDPA-d14（m/z 145→97）； NDBA（m/z 159→103
and 159→57）； NDPhA（m/z 199→169 and 199→66）, NDPhA-d6（m/z 205→175）. The dwell times per channel were set at 50 ms for
all the analytes and internal standards.
With the use of isotopic internal standards and on-line SPE, the detection limits of NDMA, NMEA, NPyr, NMor were 23.9, 6.2, 15.5,
and 0.57 pg in APCI mode, while NDEA, NPip, NDPA, NDBA and NDPhA were 9.3, 1.7, 1.0, 0.34 and 0.14 pg in ESI mode, respectively.
This method was further applied to measure the urinary concentrations of nine N-nitrosamines in nonsmokers. The results showedthat among nine N-nitrosamines only NDMA（~0.34 ng/ml）, NPyr（~1.35 ng/ml）, NDBA（~0.015 ng/ml）and NDPhA（0.025 ng/ml）
were detectable while the others were under LODs of our method. Overall, the present method would be useful to assess thehuman exposure to nitrosamines.

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 24: Regulated Bioanalysis

PWe-042
13:30 － 14:40

Ultra-Performance Liquid Chromatography/Mass Spectrometric Method
for the determination of venlafaxine and its metabolite in plasma:
Application to pharmacokinetic study

Sunil Kumar Dubey1, Hemanth Jangala1, R N Saha1, S Pasha2

1
Birla Institute Of Technology & Science, Pilani, India, 2Institute of Genomics and Integrative Biology, Mall Road, Delhi,
India

Keywords:
Venlafaxine； O-desmethylvenlafaxine； metabolite； UPLC-MS/ESI
Novel aspects:
First（UPLC-MS/ESI）method for simultaneous determination of venlafaxine（VEN）and its metabolites in rat plasma has beendeveloped and validated.Mass Spectrometric conditions were optimised in a unique way.
Abstract:
A new Ultra-performance liquid chromatography-electrospray ionization mass spectrometry（UPLC-MS/ESI）method for
simultaneous determination of venlafaxine（VEN）and its metabolite O-desmethylvenlafaxine（ODV）, in rat plasma has beendeveloped and validated using Venlafaxine d 6 as the internal standard. The compounds and internal standard were extracted
from plasma by solid phase extraction. The UPLC separation of the analytes was performed on ACQUITY UPLC ® BEH Shield RP18
（1.7µm, 100mm×2.1 mm）column, using isocratic elution with mobile phase constituted of water（containing 2 mmol/l ammonium
acetate）： acetonitrile（20：80, v/v）at a flow-rate of 0.3 ml/min. All of the analytes were eluted within 1.5 min. The compounds
were ionized in the electrospray ionization（ESI）ion source of the mass spectrometer, operating in multiple reaction monitoring（MRM）and positive ion mode. The precursor to product ion transitions monitored for VEN, ODV and IS were m/z 278.3→121.08,
264.2→107.1 and 284.4→121.0, respectively. Calibration curves in spiked plasma were linear from 10-2000 ng/ml for VEN and ODV,with coefficients of determination above 0.992 for both. The extraction recoveries for both the analytes were above 76%. The limitsof detection were 2.66ng/ml for VEN and 2.78 ng/ml for ODV. The method was fully validated for its sensitivity, accuracy, precision,
linearity, recovery, matrix effect, and stability studies. The developed method was used for the pharmacokinetic study of VEN in
rats, carried out by administering a single dose（120 mg/kg）of VEN by oral route. The concentration of VEN and ODV, in plasma
was estimated at different time points upto 24 hr post dosing. To 300µl plasma samples, 200µl mobile phase was added and thedrug was eluted by methanol by using solid phase extraction. The eluates were evaporated to dryness at 40 0 C under N 2 gas.
Residues were then reconstituted in 300 μL of mobile phase. Seven microliters of the solution were injected for analysis throughthe auto-injector. Various pharmacokinetic parameters were determined by non compartmental technique using Winnolin. The
AUC and Cmax of VEN, was found to be 2.92 µg.hr/ml and 1.48 µg/ml and those of ODV were 2.33 µg.hr/ml and 0.47 µg/ml. Cl/F and
Vd/F of VEN are 41.03 L/hr/kg and 185.58 L/kg respectively； t1/2 of VEN is 3.13 hr and 2.59 hr for ODV. Elimination rate constant, Ke
for VEN and ODV are 0.221 hr-1 and 0.267hr-1. The elimination half life of both VEN and ODV are found to be same. As Tmax for VEN
was found to be 0.5 hr and that of ODV was 1.0 hr, oral absorption of VEN and conversion to ODV both can be considered as fast.
Based on Cmax and AUC values it is evident that exposure to ODV was less than VEN, this might be due to conversion of more
VEN into other metabolites other than ODV or more formed ODV is metabolized further. The method is accurate, sensitive andreliable for the pharmacokinetic study and therapeutic drug monitoring（TDM）of venlafaxine.

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 24: Regulated Bioanalysis

PWe-043
11:10 － 12:20

Quantitative evaluation of synthase and hemisynthase activity of
glucosamine-6P synthase by matrix-assisted laser desorption/ionization
time-of-flight mass spectrometry

Vincent Guerineau, David Touboul, Florence Wieczorek, Marie-Ange Badet-Denisot, Bernard Badet,
Alain Brunelle, Philippe Durand
ICSN CNRS, Gif sur Yvette, France

Keywords:
Quantification, MALDI-TOF, matrix, 9 -aminoacridine, glucosamine- 6 P
Novel aspects:
Alternative analytical tool for the determination of enzymatic kinetic parameters with minimal sample preparation, rapid analysis,high specificity and high sensitivity
Abstract:
Introduction
Glucosamine- 6 -phosphate synthase（Glms, Gfa or Gfat）is a key in the enzyme metabolic pathway of amino sugar-containing
macromolecules biosynthesis. Main function of Glms is the catalytic conversion of D-glucosamine- 6 P（GlcN- 6 P）and L-glutamate（Glu）into D-fructose- 6 P（Fru- 6 P）and L-glutamine（Gln）, respectively. As Glms plays a crucial role in glucose homeostasis
regulation and toxicity through desensitization of glucose transport system to insulin, it was considered as a possible moleculartarget for the treatment of type II diabetes. Enzyme assays for Glms have been already described for either synthase（Fru- 6 P
converted into Glu- 6 P）or hemisynthase（Gln into Glu）activities using various analytical chemistry methods, such as fluorimetry,
UV spectroscopy, colorimetry or radiometry. Most of these methods showed false-responses due to interferences and can betime- and cost-consuming. In order to overcome these limitations, MALDI was used for the detection and quantification of the two
substrates and the two products in one shot.
Experimental method
Experiments were performed using a MALDI-TOF（Voyager DE-STR, ABSciex）. Prior to analysis, the four analytes were acetylatedusing anhydrous acetic（10%）. This derivation step was introduced due to the encountered difficulty when synthesizing 13C-GlcN6 P internal standard. Buffer solution was optimized and MALDI matrices, i.e. trihydroxyacetophenone （THAP） and9 -aminoacridine（ 9 -AA）, were compared in terms of robustness and sensitivity.By fixing the concentration of either GlcN- 6 P orGlu, Michaelis constants Km for synthase and hemisynthase activities were determined.
Results
In order to avoid signal extinction, TRIS（20 mM, pH 7.2）was chosen as the best buffer solution. THAP matrix appeared not to besuited for reproducible data acquisition because of non-homogeneous crystallization. Moreover, overlapping between isotopic
distributions of［THAP+Na］+ and［N-acetyl-4,4-d 2 -glutamate +H］+ ions makes impossible to monitor the enzyme activity. In thenegative ion mode, an optimized sample preparation with 9 -AA appeared to be more robust and reproducible. This last waschosen for the following experiments.
Determination of Fru- 6 P and Gln Michaelis constants for synthase and hemisynthase activities was performed by combining theresults of two experiments. One consists in fixing the concentration of Glu and the second one in fixing the concentration of GlcN6 P. Km（F 6 P）and Km（Gln）were found to be around 400 and 150 µM, respectively. Vmax was estimated at 15 µM.mL-1. These values
are in excellent agreement with those already published in the literature. MALDI can thus be considered as an alternative
analytical tool for the determination of enzymatic kinetic parameters.

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 24: Regulated Bioanalysis

PWe-044
13:30 － 14:40

Optimization of ESI method for the LC-MS analysis of phosphorylated
peptideIssey Osaka, Mitsuo Takayama
Yokohama City University

Keywords:
ESI, ion yield, phosphorylated peptide
Novel aspects:
The influence of amino acids and phosphorylation on the ion yields of peptide and phosphorylated peptide has been evaluated todetermine the optimized conditions for ESI-MS.
Abstract:
Peptide-mass fingerprinting（PMF）and amino acid sequencing using LC-MS have become common approaches for thecharacterization of proteins and determination of post-translational modifications. Although protein and peptide analysis using ESIMS, which is often coupled to LC, has been performed in positive-ion mode because of its superior ion yields compared to thenegative-ion mode, the use of both positive- and negative-ion mode provides useful information in protein identification and in thescreening for phosphorylated-site sequences in protein digest. If all the peptide peaks of protein digest could be detected in mass
spectra, accurate identification of protein would be achieved. However, the molecular ion yields of peptides and phosphorylated
peptides are strongly dependent on their physicochemical properties, which originate from the individual nature of the side chainsof the constituent amino acids. In order to understand the ion yields in ESI method, here we use a constitutional form where wehave divided the total ionization processes J i into ionization efficiency of analyte molecules I and the rate of desorption orvaporization of molecules Jv as follows：
Ji = IJv ... (1)The efficiency I can be related to thermochemical quantities such as proton affinity, gas-phase basicity, ionization energy orelectron affinity of a given analyte. The quantity Jv represents the ability of analyte molecules to desorb or vaporize from liquid- to
gas-phase in ESI.
Amino acids have a wide variety of physicochemical properties such as acidity, basicity, hydrophobicity and hydrophilicity
originated from the characteristics of the amino acid side chain. It has been reported that the presence of Arg, aliphatic andaromatic amino acid residues enhance the ion yields of peptide in MALDI-MS and FAB. However, the effect originated from Arg
residue and phosphorylation on the ion yields was not evaluated in ESI. Therefore, the relationship between the ion yields andphysicochemical properties remains unclear for LC-MS experiment used ESI, although LC-MS is recognized as an indispensable
analytical tool for proteomics. In this study, we investigated factors governing the molecular ion yields of peptide in ESI, in order to
optimize the ESI method for LC-MS analysis of phosphorylated peptide. The model peptides such as threonine（T）-cluster and
ACTH-based series were used for the evaluation of ion yields.
An enhancement effect of an Arg residue on the ion yields of［M+H］+ in positive-ion MALDI-MS was reported previously. In thepositive-ion ESI-MS mass spectra of T 7 （TTTTTTT）, RT 6 （RTTTTTT）, ACTH22-39（VYPNGAEDESAEAFPLEF）and［Arg22］-ACTH2239（RYPNGAEDESAEAFPLEF）, however, such enhancement effect of Arg residue was not observed. It was found that the numberof basic site was no significant factor in ESI MS.
An enhancement effect of Phe residue on the ion yield of both［M+H］+ and［M-H］- was observed by comparing Phe containingp e p t i d e R T 5 F （ R T T T T T F ） , F T 5 R （ F T T T T T R ） , A C T H 1 8 - 3 5 （ R P V K V Y P N G A E D E S A E A F ） a n d ［ A r g 36 ］ - A C T H 1 8 - 3 6（RPVKVYPNGAEDESAEAFR）. It is likely that the aromatic group of the Phe residue is advantageous for the process of desorption
from liquid-phase of charged droplet to gas-phase, because of its high surface activity. It is understood via the term of the rate ofdesorption of analyte molecule, Jv in equation 1. The ion yield of［Arg36］-ACTH18-36 was poor, although the proton affinity of Arg
residue is the highest. This may be due to the hydrophilic property of Arg in both N- and C-terminal of the peptide. Furthermore,
the effects of phosphorylation and the variety of charged state distributions with sequence of peptide and additives of sample
solution, such as acetate or ammonium acetate, on the ion yields have been evaluated quantitatively. The optimized conditions
for the ESI-MS of peptides and phosphorylated peptide would be presented.

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 24: Regulated Bioanalysis

PWe-045
11:10 － 12:20

Amino acid analysis by LC/MS with LC columns designed for hydrophiliccompounds

Hiroko Arai1, Tsuneaki Kaneko1, Taketoshi Kanda1, Yanli Guo2, Osamu Shirota2
1

Shiseido, 2Shiseido China

Keywords:
Amino acid, LC-MS, hydrophilic interaction chromatography, reversed-phase chromatography, ion-exchnage chromatography
Novel aspects:
Free amino acids were analyzed with LC-MS, by using LC columns specially designed for hydrophilic compounds.
Abstract:
Free amino acids, being polar and electrically charged, are generally considered unsuitable to common high-performance liquid
chromatography（HPLC）with reversed-phase separation and UV detection. Many chemical derivatization techniques have beendeveloped for them to date. This paper will describe simple methods to analyze free amino acids with liquid chromatography（LC）-mass spectrometry（MS）, using several unique LC columns designed for LC-MS of hydrophilic compounds. These are 1 ）CAPCELL PAK C18 AQ, or a column designed for 100% aqueous mobile phase, 2 ）CAPCELL PAK CR, or a column containing bothstrong cation exchanger and reversed phase, and 3 ）PC HILIC, or a column of hydrophilic interaction chromatography（to bereferred as AQ, CR, and PC HILIC, respectively）. Separation characteristics and detection sensitivity of eighteen standard aminoacids were studied by using LC-MS with the three columns.
AQ could retain aliphatic and aromatic amino acids adequately, while it showed small retention for acidic and basic ones. PC
HILIC, on the contrary, showed a relatively large retention for acidic amino acids, and small retention for aliphatic and aromaticones. Retention of basic amino acids on CR was larger than those on other two columns, and CR also could retain aliphatic andaromatic amino acids even under a mobile phase with an increased organic content. Although any single column did not show acomplete separation of eighteen amino acids, LC-MS with these three columns seems simple and useful if one tries to quickly
analyze a particular type of amino acids.

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 24: Regulated Bioanalysis

PWe-046
13:30 － 14:40

Accelerate Method Development using Fast Screening of Mobile Phases
Additives and Solvents for Optimum Sensitivity in LC-MS

Mikael LEVI, Stephane MOREAU
Shimadzu France, Noisiel, France

Keywords:
LC-MS, sensitivity, optimization, mobile phase
Novel aspects:
First proposed methodology for rapid screening of mobile phase reagents for maximum ionization efficiency in LC-MS.
Abstract:
It is now well-known that mobile phase components（i.e. additives and solvents）play a major role in ionization efficiency.
Laboratory facing challenges in fast method development and high sensitivity are often condemned to use generic mobile phases
and to invest in expensive high-end mass spectrometers. Furthermore, recent developments in HPLC columns allow more flexibility
in the use of acidic or basic additives as well as viscous solvents .To the best of our knowledge, no rapid and systematic
methodology are proposed to quickly optimize HPLC mobile phase recipe from a MS sensitivity point of view.
Model compounds representing a wide panel of chemical classes were dissolved in several mobile phase mixtures. Compounds
were chosen with different chemical moieties and hydrophobicity. They were also chosen in order to have both compounds
ionized in positive or negative mode and some of them could only be ionized forming salt adducts. Mobile phase mixtures were
elaborated using a rational combination of solvents with water and several additives including organic acids, bases and salts.
These mixtures were then injected using flow injection analysis and a dummy mobile phase carrier.
Flow injection parameters（e.g. sample volume, flow rate, autosampler parameters）were optimized in order to have an unbiased
overview of the mobile phase component impact on ionization. Conditions were also optimized to reduce the peak intensity
standard deviation, leading to statistically significant differences observed in responses.
The results showed that unexpected or unintuitive mobile phase compositions lead to enhanced ionization efficiencies. This
screening permits to choose mobile phase reagents in less than ten minutes.
While single component analysis allows rapid and clear decisions, in the case of multiclass component analysis（e.g.
environmental analysis）, a methodology is proposed to choose the best compromise.
Results also show that the instrumentation is important to be able to quickly perform this screening. This includes autosampler
performances and mass spectrometer acquisition speed.

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 24: Regulated Bioanalysis

PWe-047
11:10 － 12:20

Effective Strategies for Phospholipid Removal using Supported Liquid
Extraction (SLE) with LC-MS/MS Analysis

Gavin J Jones, Lee Williams, Rhys Jones, Maiko Kaneko, Steve Jordan
Biotage, Cardiff, UK

Keywords:
Sample Preparation, Mass Spectrometry, ion suppression
Novel aspects:
Evaluation of phospholipid content using supported liquid extraction with a comprehensive array of extraction solvent and pH
sample pre-treatment combinations.
Abstract:
Endogenous phospholipids in biological fluids present a major problem in LC-MS/MS analysis. Due to their strong retention
characteristics in reversed phase chromatography phospholipids tend not to elute as discrete peaks and are often very difficult toseparate from analytes of interest. Traditional liquid-liquid extraction（LLE）provides very clean extracts. Supported liquid
extraction（SLE）is analogous to liquid-liquid extraction, however, as a solid supported technique it provides subtle differences inextract cleanliness. This poster compares phospholipid removal from biological fluids using a wide variety of solvent combinations,
pH control and polar extraction solvents on supported liquid extraction plates.
Phospholipid removal was evaluated using 100 µL human plasma pre-treated with： 2 % HCl, 1 % formic acid（aq）, 0.1% formicacid（aq）, 100mM NH4OAc, water and 0.5M ammonium hydroxide to cover the pH range from 1.5-10.5. The use of ion pair reagentswas also investigated. Extraction was performed using various water immiscible extraction solvents eitheralone or in combination（ 1 mL）： 98： 2 hexane： 3 -methylbutanol, heptane, MTBE,DCM, 98：2, 95：5, 90：10（v/v）DCM/IPA, butanol, chlorobutane and
EtOAc. Extracts were evaporated to dryness and reconstituted in mobile phase. Samples were analyzed using a Waters 2795 liquidhandling system coupled to a Quattro Ultima Pt triple quadrupole mass spectrometer. MRM transitions monitoring thephospholipid product ion at 184 Da were acquired using electrospray ionization.
Phospholipid ions were separated into lyso-phospholipids（494-524 Da）and larger molecular weight phospholipids（>701-811 Da）
for comparison. Protein precipitated plasma samples were used as a benchmark to evaluate the degree of phospholipid removal
under the various extraction conditions. Extraction solvents were selected to give varying solvent characteristics. 98： 2 hexane/ 3 methylbutanol, heptane, MTBE and DCM generally resulted in low levels of larger molecular weight phospholipids and lyso PLs in
the final extracts at all pH conditions. More polar extraction solvents such as EtOAc exhibited low levels of lyso PLs but contained
the larger molecular weight PLs in the extract. Increasing percentages of IPA in DCM resulted in increased phospholipid content in
the extracts. Where the use of increasingly polar extraction solvents was required, reductions in load and extraction volumes
resulted in decreased PL content in the final extracts. The use of organic solvents in the load step to help disrupt protein binding
is possible, however, careful selection of extract solvent was required. For example, using IPA at up to 25 % with pre-treatedplasma resulted in low levels of PL in the extract. Substituting IPA with ACN resulted in higher PL levels, which nevertheless was
substantially lower than the corresponding protein crash data.

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 24: Regulated Bioanalysis

PWe-048
13:30 － 14:40

Simultaneous determination seven hydroxyacids by capillaryelectrophoresis and liquid chromatography mass spectrometry

Yen-Ling Chen, Pei-Yu Liu

Kaohsiung Medical University, Kaohsiung , Taiwan

Keywords:
Capillary electrophoresis/LC-MS/ Hydroxy acids/Cosmetics
Novel aspects:
A simpel and rapid analysis method for simultaneous determination seven HAs
Abstract:
A simple cyclodextrin modified capillary electrophoresis（CDCE）method and liquid chromatography mass spectrometry（LC-MS）
have been developed for analyzing seven hydroxyl acids, glycolic acid, malic acid, tartaric acid, latic acid, citric acid, mandelic acidand salicylic acid. The optimized CE conditions were phosphate solution containing CTAB, CD and organic solvent as runningbuffer, sample injection 20 second by 0.5 psi and -15 kV as separation voltage at 25&#8451； with UV wavelength 200 nm. The seven
HAs were separated well in less than 10 min. Compared with CE, LC-MS can offer a significant increase of sensitivity and selectivity.
After optimization and validation, the simple and rapid analysis CE and LC-MS method was established and successfully applied toseveral commercial cosmetic products and biosamples.

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 24: Regulated Bioanalysis

PWe-049
11:10 － 12:20

Fast determination of phosphopeptides by nanoLC-MS/MS in biologicalsamplesRong-Chun Chen, Chi-Yu Lu

Department of Biochemistry, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan

Keywords:
derivatization； phosphopeptide； biological samples
Novel aspects:
Combination of fast digestion and derivatization to detect phosphopeptides in biological samples
Abstract:
Protein phosphorylation is one of the post-translational modifications that plays an important role in signal transduction. This
modification of protein is also related to many chronic diseases, including hypertension, cancer and Alzheimer’s disease. Mass
spectrometry combined database searching is very effective for protein identification and modification site recognition. This study
developed a convenient way for protein digestion by using microwave to shorten the digestion time. After digestion, chemical tag
was introduced to label phosphorylated peptides via beta-elimination and Michael addition. This derivatization protocol could
help phosphopeptides to increase the ionization efficiency and avoid the neutral loss of phosphate group in fragmentation
process. Application of this strategy to fast detect phosphopeptides in biological samples（such as milk, plasma or urine）
demonstrated workable.

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 24: Regulated Bioanalysis

PWe-050
13:30 － 14:40

Derivatization of lipoic acid for analysis by capillary liquid
chromatography and mass spectrometry

Chia-Ju Tsai, Chia-Hsien Feng

Department of Fragrance and Cosmetic Science, College of Pharmacy, Kaohsiung Medical University, Kaohsiung, Taiwan

Keywords:
lipoic acid, derivatization, capillary liquid chromatography, matrix-assisted laser desorption ionization time-of-flight mass
spectrometry
Novel aspects:
Microscale derivatization method to enhance the sensitivity of lipoic acid in UV absorption and ionization ability in mass
spectrometry.
Abstract:
Lipoic acid（LA）is an essential cofactor for mitochondrial enzymes and ideal antioxidant present in prokaryotic and eukaryotic
cells. It is capable of scavenging reactive oxygen species and regenerating other antioxidants such as vitamine C and glutathione.
Direct analysis of LA presents obstacle because its lack of chromophores. This study employed a pre-column derivatizing method
to enhance the sensitivity of LA in UV absorption and ionization ability in matrix-assisted laser desorption ionization time-of-flightmass spectrometry（MALDI-TOF MS）. Dispersive liquid-liquid microextraction（DLLME）method is used as sample pretreatment
method. All important parameters affected the extraction and derivatization of LA were investigated and optimized. The capillaryliquid chromatography（CapLC）separation was carried out by using capillary C18 column with the mobile phase consisting of
acetonitrile-0.1% formic acid. The linear response was ranged from 0.1 to 40 μM and 0.1 to 20 μM in standard solution and urine
respectively, with a correlation coefficient of 0.999. The detection limits was 0.01 μM and 0.03 μM by CapLC-UV, and the detection
limits was 0.008 μM and 0.02 μM by MALDI-TOF MS in standard solution and urine respectively. We successfully applied thesemethods to detect LA in dietary supplements and urine.

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 24: Regulated Bioanalysis

PWe-051
11:10 － 12:20

Highly-Selective Reaction Monitoring (H-SRM) with Automated Mass andResolution Calibration in Testing of Anabolic Steroids in Equine Plasma
and Urine

Harald Oser1, Hans Schweingruber1, Mary L Blackburn1, Shane E Tichy1, Cornelius E Uboh2
1

Thermo Scientific, San Jose, CA USA, 2Pennsylvania Equine Toxicology and Research Laboratory

Keywords:
Automated calibration, H-SRM, Anabolic Steroids
Novel aspects:
Automated calibration using lock-peaks to facilitate the use of highly-selective reaction monitoring（H-SRM）acquisition in theanalysis of anabolic steroids in equine plasma and urine
Abstract:
Use of anabolic steroids in the horse racing industry is under increasing scrutiny and regulation. Numerous regulatory bodies havespecified tolerance limits depending on the sex of the horse. Due to the fact that some steroids are naturally occurring and certain
levels are allowed in stallions（males）as opposed to mares（females）, it is critical to precisely measure and monitor the levels ofsteroids in stallions and mares during competition. The traditional method of steroid testing involves detection, quantification, andconfirmation using a triple quadrupole mass spectrometer. The matrixes monitored include plasma and urine, both of which canprovide significant instrument and data interpretation challenges due to endogenous matrix interferences. Sample preparation andoptimized chromatography can reduce matrix interferences. However, there are occasions when these options are not sufficient.
To reduce matrix interferences, the use of highly-selective reaction monitoring（H-SRM）acquisition is encouraged. H-SRM
acquisition uses a reduced peak width, such as 0.2 FWHM in Q1, and standard peak width of 0.7 FWHM in Q2. Reducing the peakwidth in Q 1 reduces the potential of isobaric interferences. For this reason, we propose the use H-SRM acquisition, utilizing apeak width in Q 1 of 0.2 to 0.1 FWHM as needed to reduce any matrix interferences in the testing of anabolic steroids in horseurine and plasma. The typical triple quadrupole mass spectrometer will lose significant signal intensity when the peak width isbelow 0.7 FWHM rendering H-SRM ineffective in reducing matrix interferences. The triple quadrupole instrument employed in thisstudy utilizes hyperbolic quadrupole rods which enable the use of resolutions of 0.2 or even 0.1 FWHM with minimal decrease insignal intensity.
One potential issue with narrow peak widths in H-SRM is loss of sensitivity or selectivity due to minor mass shifts or resolutionchanges. These long term mass or resolution changes are often due to environmental conditions in the laboratory such astemperature fluctuations. Under unit mass resolution, peak width of 0.7 FWHM, small changes in mass or resolution are
insignificant. However, as mentioned, with H-SRM small mass shifts or resolution changes can cause loss of signal or increase inbackground noise which affect； sensitivity, limits of detection, reproducibility, and ultimately can generate misleading results. To
compensate for changes in mass or resolution we will periodically perform an automated mass and resolution calibration check,and correction, during the analysis sequence. This procedure, using one or multiple lock-peaks, will automatically correct any
mass drift or resolution changes that might occur over time.
Over the course of multiple weeks H-SRM calibration curves for testosterone, boldenone, and stanozolol in equine urine andplasma will be repeatedly analyzed using the method developed at the Pennsylvania Equine Toxicology and Research Laboratory.
Sample analysis using automated re-calibration will be compared to the sample analysis run with a standard calibration performedat the start of the study. Sequential series of samples using standard calibration and automated re-calibration will be analyzed on
the same instrument to eliminate possible instrument variability. The linearity, reproducibility, limit of detection, and accuracy ofquantification for the three anabolic steroids will be used to assess the effectiveness of the automated re-calibration analysis
versus that of standard calibration analysis. We propose that the use of H-SRM analysis with automated re-calibration is a highly
selective, rugged, and reliable method of analyzing anabolic steroids in equine urine and plasma.

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 24: Regulated Bioanalysis

PWe-052
13:30 － 14:40

Selective enrichment of histidine-tagged proteins and peptides on MALDI
plate

shin-yi Huang, Yu-Chie Chen

Department of Applied Chemistry, National Chiao Tung University, Taiwan

Keywords:
histidine-tag, affinity, MALDI MS, Nanoparticles
Novel aspects:
A new type of magnetic nanoparticle-based affinity-mass spectrometric platform for rapid characterization of histidine-tagged
proteins/peptides from complex samples was explored in this study.
Abstract:
Recombinant proteins expressed in bacterial cells are commonly tagged by a polyhistidine for the ease of purification.
Conventionally, recombinant proteins tagged with a polyhistidine, i.e. his-tagged proteins, can be readily purified by metal ions（Ni（II）or Co（II））immobilized affinity columns（IMAC）from complex cell lysates. In this work, we explored that aluminum oxidecoated iron oxide magnetic nanoparticles（Al 2 O 3 @Fe 3 O 4 MNPs）have high affinity toward polyhistidine, leading Al 2 O 3 @Fe 3O 4 MNPs to be suitable trapping probes for his-tagged proteins/peptides. We demonstrated the feasibility of using Al 2O 3@
Fe3O4 MNPs as affinity probes for his-tagged proteins and peptide. Furthermore, taking advantages of the magnetic property of
the affinity probes, the enrichment and isolation of target species can be realized on a matrix-assisted laser desorption/ionization（MALDI）plate with the assistance of a magnet. The species enriched by the affinity probes on the plate can be characterized by
MALDI MS. His-tagged proteins, protein digests, and cell lysates were used as the model samples. High enrichment capability for
his-tagged species can be achieved as high as two orders of magnitude. This approach provides the advantages including
simplicity, high selectivity, high sensitivity（~ nM）, and short extraction time（~ 30 s）. Thus, we believe that this approach shouldbe very suitable for rapid investigation of the expression of his-tagged recombinant proteins in prokaryotic organisms.

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 24: Regulated Bioanalysis

PWe-053
11:10 － 12:20

Determination of Fluconazole and 5-Fluoro Cytosine in human plasmaand cerebrospinal fluid using protein precipitation and LC-MS/MS

Carmen A de Kock, Peter J Smith, Jennifer Norman, Lubbe Wiesner
University of Cape Town, Cape Town, South Africa

Keywords:
fluconazole, 5 -fluoro cytosine
Novel aspects:
Bioanalytical method validation of fluconazole and 5 -fluoro cytosine（ 5 -FC）in human plasma and cerebrospinal fluid
Abstract:
A sensitive and robust method for the detection of fluconazole and 5 -fluoro cytosine（ 5 -FC）in human plasma and cerebrospinal
fluid（CSF）was developed and validated. Stable isotopic internal standards of fluconazole and 5 -FC were used as LC-MS internal
standards. The method was validated over the calibration range of 0.195 µg/ml to 50.0 µg/ml using protein precipitation.
Chromatography was developed on an Atlantis T 3 reverse phase column with a mobile phase of 0.1% formic acid and acetonitrile.
The method was fully validated according to Food and Drug Administration（FDA）recommendations（good selectivity, carry-overless than 20 % , inter-and intra-assay precision and accuracy lower than 15 %）. Stock solutions of fluconazole and its internal
standard were stable for up to 6 hours at room temperature. However stock solutions of 5 -FC as well as its internal standard
were unstable under the same conditions. The stability of both analytes in plasma and CSF for 4 hours was within FDA
recommendations. Recovery, matrix effects and haemolysis were acceptable following FDA recommendations. On-instrument
stability showed that the analytes are stable in extracted matrix for up to 72 hours in plasma and 48 hours in CSF. This method isusable for the routine detection of fluconazole and 5 -FC in human plasma and CSF and may be useful in future clinical studies.

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 25: Glycoanalytical Technology for Systems Glycobiology and Functional
Glycomics

PWe-054
13:30 － 14:40

Studies on the selective binding of divalent metals to cyclodextrins by ESI
mass spectrometry

Xiaodan He, chuanfan Ding

Fudan University,Shanghai,China

Keywords:
divalent metals, cyclodextrins, selective binding,
Novel aspects:
The selective binding of divalent metals to cyclodextrins is firstly studied using electrospray ionization mass spectrometry. The
alkaline-earth di-ions showed different binding strength from transition metal di-ions.
Abstract:
Cyclodextrins（CDs）, composed of α-1,4-coupled D-glucose units, have the ability to form lots of inclusion complexes with a wide
range of molecules in aqueous solution, which allow them to be applied in many areas such as enzyme mimics, catalysis and theencapsulation of drugs 1-4 . However, the study on the complexes of cyclodextrin with inorganic divalent metals by mass
spectrometry is rarely reported.5-7 The electrospray ionization mass spectrometry（ESI-MS）has been recognized as a useful toolfor investigating the general area of molecular recognition and providing a powerful method for the analysis of various host-guest
complexes and other non-covalent complexes in solution.
The most commonly used CDs include α-CD, β-CD, and γ-CD, which consist of 6, 7, and 8 D-glucose units, respectively. In thisreport, the selective binding of divalent metals to cyclodextrins was investigated. In the experiment, theα-,β-,γ-, orhydroxypropyl-β-cyclodextrins, and MgCl 2 ,CaCl 2 ,BaCl 2 , FeCl 2 , CuCl 2 or ZnCl 2 solution were mixed in a molar ratio of 1 ：10,
respectively, and then reacted at room temperature for 12 hours. The evaluation of the binding selectivity of Mass spectrometricexperiments were performed using Thermo Finnigan LTQ ion trap mass spectrometer equipped with an electrospray ionization
source. The capillary voltage was set to 4.0 kV and the capillary temperature was maintained at 350&#8451；. The sample wasintroduced via a syringe pump at a flow rate of 5 µL/min.
The ESI-MS experimental results showed that all of the α-, β-, γ-, and HP-β-CDs can form 1 ： 1 complexes［CD+M］2+（Mrepresents divalent metal）with MgCl2,CaCl2,BaCl2, FeCl2, CuCl2, ZnCl2 in 80%/20% methanol/water solution, but can’t form 1 ： 2complexes［CD+ 2 M］4+, revealing that the ion products is significantly influenced by the Coulomb interaction between charges
rather than the size of cyclodextrin’s interior cavity.
The binding of α, β, and γ-CD to divalent metal ions was further confirmed by collision-induced dissociation（CID）experiment.
The collision energy was 30 eV. The fragmentation pathways for all［CD+M］2+ were similar, which was irrelevant to the D-glucose
units’ number. The fragment ions were mainly［CD+M- n glucose］2+（n =1, 2, 3 ）. It is demonstrated the loss of neutral molecule（D-glucose units of 162 m/z）took place in all fragmentation process, which provides more evidences that the complexes are of
the inclusion type.
For comparison, a competition experiment was also performed. A stoichiometry of cyclodextrin were mixed with Mg2+,Ca2+,Ba2+,
Fe 2+ , Zn 2+ , and Cu 2+ cations respectively. The mixed solutions then incubated at room temperature for 12 hours to reach theequilibrium. From ESI mass spectra, it can be seen that the peak intensity for the complexes of cyclodextrins with Mg2+,Ca2+,Ba2+ is
10 times higher than those of Fe2+, Zn2+ and Cu2+, which indicates that the binding strength for former complexes is stronger thanthe latter. The mechanism for this difference will be further investigated later in this laboratory.
Reference
1. Blair, S.-M.； Brodbelt, J.-S.； Marchand, A.P.； Kumar, K.-A.； Chong, H-S. Anal. Chem., 2000,72,2433
2. Blanda, M.T.； Farmer, D.B.； Brodbelt, J.S.； Goolsby, B., J. Am. Chem. Soc., 2000,122, 1486
3. Reyzer, M.-L.； Brodbelt, J.-S.； Marchand, A.-P.； Chen, Z. Huang, Z.； Namboothiri, I.-N.-N. Int. J.Mass Spectrom., 2001,200,57
4. Chu, Y.-Q.； Dai, X.-H.； Jiang, D.； Jiang, G.-Y.； Fang, X.； Ding, C.-Y.； Rapid Commun. Mass Spectrom., 2010, 24, 2255
5. Choi, S.-S.； Lee, H.-M.； Jang, S.；Shin, J.；Int. J.Mass Spectrom., 2009,279,53
6. Choi, S.-S.； Ha, S.-H.；Bull. Korean Chem. Soc.2006,27,1243
7. Bashir, S.； Derrick, P.-J.； Critchley, P.； Gates, P.-J.； Staunton, J.Eur. J.Mass spectrum, 2003,9,61

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 25: Glycoanalytical Technology for Systems Glycobiology and Functional
Glycomics

PWe-055
11:10 － 12:20

Dual Lectin-based Enrichment Method of N-linked Glycopeptides Using
Asymmetrical Flow Field-Flow Fractionation and Applications to Human
Blood Serum

Jinyong Kim1, Duk Jin Kang2, Myeong Hee Moon1
1

Yonsei University, Seoul, Korea, 2Korea Research Institute of Standards and Science, Daejeon, South Korea

Keywords:
Flow Field-Flow Fractionation, Glycoproteomics, N-linked glycoprotein, nanoLC-ESI-MS-MS
Novel aspects:
Intact forms of targeted N-linked glycopeptides from human blood serum were able to be enriched selectively, with highreproducibility.
Abstract:
Glycosylation of proteins plays an important role in many biological processes related to cell-cell interaction, cell signaling, andcell proliferation. Despite the crucial fact, there are some drawbacks when characterizing glycoproteins in biological samples since
the amount of glycoproteins occupies as significantly low as 2 - 5 % of total amount of proteins. On this account, enrichment ofglycoproteome is a critical step in glycoproteomic study. Among the enrichment methods of glycoproteome, a lectin-based
approach is widely used for its ability to isolate targeted glycoproteins selectively when combined with various analyticaltechniques. In this study, the lectin-based enrichment method was developed for glycoproteomics by using asymmetrical flow
field-flow fractionation（AF 4 ）, a size sorting technique that separates biological components.
Prior to enrichment of glycopeptides in human serum sample, depletion of albumin and IgG, which are abundant proteins inhuman serum, was accomplished to minimize interferences in reaction between lectins and glycopeptides. After the depletion,
raw serum sample was digested in consecutive order using two enzymes（chymotrypsin and trypsin）in order to increase theefficiency of proteolytic cleavage. After a dual enzymatic treatment, proteolytic peptides of serum were blended with a mixture oflectins. Then, with AF4, molecular mass-separation of glycopeptides-bound lectins was carried out. Subsequently, lectinglycopeptide complexes were collected and treated using endoglycosidase（PNGase F）in an attempt to characterize N-linked
glycoproteins using nanoLC-ESI-MS-MS.
To validate the efficiency of selective enrichment of glycopeptides, proteolytic peptides of alpha- 1 -acid glycoprotein（AGP）, a
typical N-linked glycoprotein standard, were used for selective enrichment of glycopeptides containing glycan complex of highmannose type by using Concanavalin A（ConA）. AGP is reported to have five N-linked glycosylated sites and additionally, is posttraslationally modified to glycans, in which 20% was consisted of mannose. Thus five peptides containing the glycosylation site asa deamidation of asparagine（by PNGase F）were detected among tryptic peptides of AGP without detecting other peptides. As aresult, we identified five endoglycosidically digested peptides of ConA fraction using nanoLC-ESI-MS-MS experiments.
The enrichment method was applied to human blood sera from control and lung cancer patients, in order to excavate thebiomarker related to lung cancer. By using the same procedures with two lectins（WGA and SNA）, WGA- and SNA-specificbound glycopeptides from human serum were collected during AF 4 separation and treated by PNGase F in sequence.
Deglycosylated peptides from each lectin-glycopeptide fraction were identified by using nLC-ESI-MS-MS and database search.
Consequently, 71 WGA and 39 SNA of N-linked glycopeptides were identified.

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 25: Glycoanalytical Technology for Systems Glycobiology and Functional
Glycomics

PWe-056
13:30 － 14:40

A nanoprobe-based strategy combined with HILIC enrichment for targeted
glycoprotein profiling

Kai-Yi Wang1,2, Ying-Wei Lu3, Chun-Cheng Lin3, Yu-Ju Chen1,2,4

1
Department of Chemistry, National Taiwan University, Taipei, Taiwan, 2Nano Science and Technology Program, Taiwan
International Graduate Program, Institute of Physics, Academia Sinica, Taipei, Taiwan, 3Department of Chemistry,
National Tsing Hua University , Hsinchu, Taiwan, 4Institute of Chemistry, Academia Sinica, Taipei, Taiwan

Keywords:
nanoprobe, glycopeptide, profiling, HILIC
Novel aspects:
This study demonstrates a sensitive strategy for the discovery of glycosylation pattern of low-abundant serum proteins.
Abstract:
Introduction：
Glycosylation is the most prevalent protein modification and 50 % of proteins are suggested to be glycosylated based on the
genomic information. Increasing evidences have shown that the significant change in protein glycosylation pattern is an importantfeature during oncogenesis. Here we present a sequential enrichment method integrating functionalized nanoprobe withhydrophilic interaction chromatography（HILIC）for glycopeptides characterization using MALDI-TOF MS analysis. Based on thesensitivity of nanoprobe for serum marker purification and the selectivity of HILIC for glycopeptides enrichment, we aim toestablish a simple protocol for the analysis of low-abundant serum glycoprotein bypassing sample fractionation, immuno-depletionof abundant serum proteins and minimum sample loss. To demonstrate the general availability of this approach, prostate-specificantigen（PSA）, the widely used glycoprotein marker for prostate cancer diagnosis, was selected as model system.
Preliminary results:
Taking advantage of efficient affinity extraction by surface-functionalized magnetic nanoparticles（MNPs）and accurate MALDITOF MS readout, we present success on this nanoprobe-based immunoassay for simultaneous enrichment and quantitation of
PSA from human serum. The present results showed the detection limit as low as 5 ng/ml of PSA in human serum, which iscapable of detecting PSA in serum from patient with prostate cancer or benign prostate hyperplasia（> 10 ng/ml）. In addition,
our methodology demonstrated good quantitation linearity of PSA（R 2 &#8786； 0.99）with high precision（<18 %）and accuracy（<10%）with dynamic range of 5 -100ng PSA spiked in healthy female serum. Analysis of the glycosylation pattern was performedby extraction of glycopeptide from tryptic PSA peptides using a homemade HILIC spin column. After the specific enrichment by
HILIC, the removal of the unglycopeptides enhanced the detection sensitivity of heterogeneous glycosylated peptides in MALDITOF MS analysis. This two-step workflow has shown the detection of multi-glycosylation of purified PSA from seminal fluid. The
sensitivity and specificity of this platform shows promise as an alternative tool for the study of glycoprotein. The application of thisapproach to quantitatively compare the PSA glycosylation profiles in benign prostate hyperplasia and patients with prostatecancer is currently ongoing.

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 25: Glycoanalytical Technology for Systems Glycobiology and Functional
Glycomics

PWe-057
11:10 － 12:20

Alteration of glycan profile during early-stage of mesenchymal stem cells
differentiation

Noritaka Hashii, Ryosuke Kuribayashi, Nana Kawasaki
National Institute of Health Sciences, Tokyo, Japan

Keywords:
glycan profile, mesenchymal stem cells, liquid chromatography/mass spectrometry, multivariate analysis
Novel aspects:
Glycan profiles can be used as a molecular index for discrimination of MSCs from differentiated cells.
Abstract:
Human bone marrow mesenchymal stem cells（MSCs）are multipotent cells that are capable of differentiating into several celllineages, including bone, cartilage, and neural-like cells. Cell therapy with MSCs has been recently attracted attention for thetreatment of difficult-to-treat diseases such as neurological disorders. For full-scale practical use of cell therapeutic products from
MSCs, development of a method for distinguishing MSCs from differentiated cells is needed. Most proteins produced in eukaryotic
cells are glycosylated, and the glycan profiles are altered in association with several biological events, including cell differentiationand proliferation. Therefore, the glycan profile can serve as a potential molecular index for distinguishing between MSCs anddifferentiated cells. In this study, we analyzed glycan profile alterations during early-stage of MSC differentiations by using liquid
chromatography/mass spectrometry（LC/MS）and multivariate analysis（MVA）to evaluate the usefulness of the glycan profile as amolecular index.
When MSCs were differentiated neurally, morphological alteration was observed after 2 days, although MSCs remained unalteredby osteogenic differentiation for 2 days. N-linked glycan profiles from MSCs, neural-lineage cells, and osteogenically differentiatedcells showed that the major glycans in these cells were high-mannose-type, pauci-mannose-type, hybrid-type, and complex-typebi-, tri-, and tetra-antennary glycans. By MVA of the MS data, the 3 cells were plotted at different positions, indicating the glycan
profile alterations. Furthermore, the characteristic glycans of the differentiated cells were identified using S-plot analysis, whichallows confirmation of the highest contributing variables for the pre- and post-groups. Significant increase was observed in thelevel of a sialylated complex-type glycan in the neural-lineage cells. The levels of the high-mannose-type, pauci-mannose-type, andnon-fucosylated complex-type glycans were decreased in the neural-lineage cells. In contrast, in the osteogenically differentiatedcells, the levels of fucosylated and sialylated complex-type glycans were increased； however, the levels of high-mannose-type
and pauci-mannose-type glycans were decreased. These results suggest that glycan profiles can be used as a molecular index for
discrimination of MSCs from differentiated cells.

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 25: Glycoanalytical Technology for Systems Glycobiology and Functional
Glycomics

PWe-058
13:30 － 14:40

Data-dependent acquisition system for N-linked glycopeptides using
MALDI-DIT-TOF MS

Masaki Murase, Hidenori Takahashi, Yosihiro Yamada, Sadanori Sekiya, Shigeki Kajihara,
Shinichi Iwamoto, Koichi Tanaka
Koichi Tanaka Laboratory of Advanced Science and Technology, Shimadzu Corp, Kyoto, Japan

Keywords:
N-linked glycopeptide, MALDI, automation, de novo sequencing, database search
Novel aspects:
Development of automatic structural analysis system of glycopeptides integrating data-dependent acquisition and MSn analysis by
MALDI-DIT-TOF MS.
Abstract:
Introduction
Glycosylation is one of the most common protein post-translational modifications, as more than 50% of proteins in humans areglycosylated. Glycosylation has important roles in various biological processes and aberrant glycosylation is associated withnumerous diseases.
Analysis of glycosylation using tandem mass spectrometry at the glycopeptide level can reveal glycosylation sites, glycan
components and peptide sequences. However, the procedure is laborious due to its complexity. To support data analysis of
N-linked glycopeptides, we previously reported the integrated approach, which combines our original de novo sequencing
software with conventional protein database search.
In this study, we developed a data dependent acquisition system called LDIA（Logical Data-Influenced Acquisition）with MALDI
digital-ion-trap（DIT）MS（DIT is a 3 D-quadrupole ion trap driven by a rectangular wave high voltage）. We demonstrate
structural analysis of N-linked glycopeptides with this system.
Methods
Human transferrin glycopeptide GP 1 （［M+H］+：m/z 3682）which contains a disialylated biantennary N-glycan, was prepared withreverse-phase HPLC from its tryptic digest. 2,5-dihydroxybenzoic acid（DHB）was used as a matrix.
Mass spectra were acquired using MALDI-DIT-TOF MS. The MALDI-DIT-TOF MS instrument control software provides functions forautomatic MALDI MS data acquisition such as sweet spot search and laser power tuning.
The LDIA module was implemented as an automatic data acquisition plug-in for the control software. The main functions of theplug-in include（ 1 ）determination of precursor ions using neutral loss of sugars from MS spectra,（ 2 ）determination of MS 3
precursor ions and deduction of glycan components by de novo sequencing from MS2 spectra,（ 3 ）identification of peptides andglycosylation sites from MS 3 spectra. X!Tandem（Global Proteome Machine Organization）was used to identify peptides andglycosylation sites.
Results
An MS 1 spectrum of transferrin-GP 1 was acquired automatically using MALDI-DIT-TOF MS and showed fragmentation of theglycopeptides, especially dominant loss of sialic acids. The LDIA plug-in assigned glycopeptide ion peaks using glycan sequencing
by neutral loss of sugars, and several of the highest intensity peaks were selected as MS 2 precursor ions. Sialylated ions wereautomatically removed so that they were not selected as precursor ions, because CID of such ions results in poor fragmentation.
An MS2 spectrum of［M+ 3 H- 2 Sia］+ was acquired automatically and showed characteristic cleavage for N-linked glycopeptides by
CID as triplet peaks, which were formed by（ 1 ）one HexNAc molecule binding with a peptide（pep + 203Da）,（ 2 ）a peptide
containing ring-cleaved HexNAc（pep + 83 Da）,（ 3 ）a peptide containing no glycans. The LDIA module determined the triplet
peaks as the precursor ions for MS3 to identify the peptide sequence and the glycosylation site. Correct glycan components werealso deduced by de novo sequencing combined with a result from MS1 analysis.
MS3 spectra from the triplet peaks were acquired automatically, and the peptide sequence and glycosylation site were correctly
identified by X!Tandem.
From these results it was shown that the automatic analysis system was useful for structural analysis of N-linked glycopeptides.

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 25: Glycoanalytical Technology for Systems Glycobiology and Functional
Glycomics

PWe-059
11:10 － 12:20

SRM analysis of chondroitin sulfate terminal regions

Harumi Osago1, Tomoko Shibata2, Suguru Kuwata3, Michihaya Kono3, Nobumasa Hara1,
Kazuo Yamada1, Yuji Uchio3, Mikako Tsuchiya11Dept. of Biochem., Faculty of Med., Shimane Univ., Shimane, Japan, 2Cent. for Integ. Res. in Sci., Shimane Univ.,
Shimane, Japan, 3Dept. of Orthopaedic Surgery, Faculty of Med., Shimane Univ., Shimane, Japan

Keywords:
chondroitin sulfate, glycosaminoglycan, selected reaction monitoring（SRM）, free terminal, linkage terminal
Novel aspects:
Sulfated mono- or disaccharides and hexasaccharides derived from free and linkage terminals of CS in cartilage were analyzed
with SRM mode of MS/MS analysis.
Abstract:<Introduction>
The glycosaminoglycan（GAG）consists of repeating disaccharides and shows high heterogeneity generated through modifications
such as sulfation or epimerization along the backbone. GAG family is classified on the disaccharide unit into several groups suchas unsulfated hyaluronan（HA）and the sulfated chondroitin/dermatan sulfate（CS/DS）, heparan sulfate/heparin（HS/HP）andkeratan sulfate（KS）. GAGs are constitutively located on the extracellular matrix of many tissues such as cartilage. GAG lyase
such as chondroitinase ABC liberates unsaturated disaccharides and saturated mono- or disaccharides derived from internal andfree terminal regions of GAG, respectively. Followed byfurther alkaline reduction, unsaturated hexasaccharides are released fromlink protein as linkage terminal region. Generally, the internal unsaturated disaccharides have been target for the analysis of GAGs
and we reported the simultaneous measurement of the saccharides derived from HA, CS/DS, HS/HP and KS by tandem mass（MS/MS）analysis.
Recently, structural changes of the terminal regions of GAGs in cartilage, such as changes of sulfated pattern of free terminal andshortening of the length of CS, have been implied in diseases or aging conditions. In this study, we tried to establish thequantitative analysis conditions of terminal saccharides ofCS, such as GalNAc, GlcA/IdoA-GalNAc andΔUA-GalNAc-GlcA-Gal-GalXyl-ol, with selected reaction monitoring（SRM）mode of MS/MS analysis.
<Abbreviation>
GalNAc： N-acetyl-galactosamine, GlcA： glucuronic acid, IdoA： iduronic acid, ΔUA： 4,5-unsaturated uronic acid, Gal： galactose,
Xyl： xylose<Methods>
Commercially available CS chain, aggrecan and porcine articular cartilage solubilized with collagenase type II were digested withchondroitinase ABC. For linkage terminal analysis, digests were further alkaline reduced.
LC was done onHypercarb（Thermo Scientific）with the ammonium bicarbonate buffer（pH10-11）/ acetonitrile system. MS analysis
was done with ESI-QqQ（AB SCIEX, API3000）in the negative mode.
<Results and discussion>
By LC-MS analysis of the CS digest, the signals corresponding to the free terminal saccharides, mono- or disulfated GalNAc andmonosulfated GlcA/IdoA-GalNAc were detected at m/z 300, 380 and 476, respectively. The MS/MS analysis of these ions detected
m/z 97 ion corresponding to［HSO4］and other product ions matching the fragments of these saccharides. The m/z 97 ion was
selected for their SRM transitions. The signal at m/z 476 had 2 peaks. MS/MS analysis of each peak detected the product ionsknown to be specific to the unsaturated disaccharide containing 4 - or 6 -sulfated GalNAc of CS, indicating the presence of 4 - or
6 - sulfated GalNAc at the free terminal. Unsulfated form of GalNAc and GlcA/IdoA-GalNAc were not detected.
By MS analysis of alkaline-reduced aggrecan digest, the signals corresponding to the linkage terminal saccharides of CS, non- or
mono- or disulfated ΔUA-GalNAc-GlcA-Gal-Gal-Xyl-ol were detected at m/z 506, 545.5 and 585.5, respectively. MS/MS analysis ofthese ions showed fragmentation patterns similar to those of the hexasaccharides reported previously. The product ion for SRM
transition of unsulfated ΔUA-GalNAc-GlcA-Gal-Gal-Xyl-ol was selected at m/z 175 corresponding to glycoside bond cleavage ion
C1. Other two targets generated the product ion at m/z 97, which was selected for their SRM transitions.
By LC-MS/MS analysis of the porcine articular cartilage digest, using SRM mode with the specific product ions selected as
described above, several saccharides derived from both terminals of CS （ 2 monosaccharides, 1 disaccharide and 3
hexasaccharides）were newly detected in addition to the previously detected 8 internal disaccharides.Quantifying CS saccharidesof the both terminals together with internal region may be useful to detect alterations of the GAG in diseases or aging conditions.

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 25: Glycoanalytical Technology for Systems Glycobiology and Functional
Glycomics

PWe-060
13:30 － 14:40

Novel Bioinformatics Tool: Interpretation of Glycan Mass Spectra with
Metal Adducts and Multiple Adduct Combinations

Julian Saba2, Kentaro Takahara1, Ningombam Meitei3, Arun Apte3
1

Thermo Fisher Scientific, Yokohama, Japan, 2Thermo Fisher Scientific, San Jose, CA, 3PREMIER Biosoft, Palo Alto, CA

Keywords:
Glycan, Structual Interpretation
Novel aspects:
Development of bioinformatics tool for support of glycans with metal adducts and multiple adduct combinations.
Abstract:
Introduction
Previously we had presented a bioinformatics tool for automated structural interpretation of glycan MS/MS and MSn data. This
was limited to characterizing glycans with single adduct of H and Na（［M+H］+1,［M-H］-1,［M+ 2 H］+2,（［M+ 2 Na］+2 etc.）. However,
the ionization of glycans by mass spectrometry results in the formation of several different adducts. These adducts maybe present
as single adduct or combination of multiple adducts. Here we expand our bioinformatics tool, SimGlycan, to support Li and K
adducts as well as combination of multiple adducts such as Na + H, Li + H, Na+K etc. In order to demonstrate the utility of thesoftware, a combination of permethylation and MSn are used to characterize glycans derived from bovine fetuin and human IgG.

Methods
Bovine fetuin and human IgG were reduced, alkylated and enzymatically digested. Samples were evaporated to dryness and resuspended in 25 mM ammonium bicarbonate buffer（pH~ 8 ）. Upon re-suspension PNGase F was added to release the glycans.
Released glycans were permethylated as described previously. All MSn experiments were carried out on a Thermo Scientific Velos
Pro linear ion trap mass spectrometer using direct infusion into a nanoelectrospray source. Data analysis was performed using
SimGlycan software from PREMIER Biosoft.

Abstract
Studies have shown that glycans are very susceptible to the effects of salts and other compounds. In most cases small amounts of
sodium and alkali metals are added to improve ionization efficiency. However, introduction of these adducts can result in spectrawith precursors containing multiple adducts in different combinations which complicate interpretation of observed peaks to
identify the structure. We have expanded our bioinformatics tool to support characterization of glycans with Li and K adducts andcombinations of multiple adducts such as Na + H, Li + H, Na+K to name a few. In order to test the performance of the software tohandle multiple adducts, glycans released from bovine fetuin were chosen. This was an ideal system to test the capability ofsoftware because the glycan content of fetuin has been characterized in depth.
Ion trap MS profile was acquired for permethylated glycans of bovine fetuin and specific precursors with different adducts andcombination of adducts were targeted for MS/MS and MS n . Data were imported into SimGlycan software for structuralcharacterization and when needed structural isomer differentiation. SimGlycan characterized glycans were verified using manual
assignment and previously published data. We were able to correctly identify and assign structures to the MS/MS and MSn spectrausing this software confirming what we had assigned previously via manual assignment.
We further extended this to glycans released from Human IgG. This is an area of interest as IgG are involved in human circulationas part of the humoral immune response and the changes in N-glycosylation of IgG associate with various diseases and affect theactivity of therapeutic antibodies and intravenous immunoglobulins. Using a combination of permethylation and MSn more than 30
structures were identified. All of the targeted glycans contain multiple adducts and SimGlyan was used in structural interpretation.

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 25: Glycoanalytical Technology for Systems Glycobiology and Functional
Glycomics

PWe-061
11:10 － 12:20

Determination of Extensive Glycosylation on Glycoproteins andGlycolipids in High-density Lipoprotein using Mass Spectrometry

Jincui Huang, Hyeyoung Lee, Nancy Rivera, Angela M Zivkovic, Jennifer Smilowitz, Bruce J German,
Carlito B Lebrilla
University of California, Davis

Keywords:
Mass Spectrometry, HDL, Sialic Acid, Glycan-microheterogeity
Novel aspects:
Extensive site-specific glycosylation on HDL protein complexes and glycolipid analysis indicate that HDL is potentially a negatively
charged particle.
Abstract:
Introduction
High-density lipoprotein（HDL）is the most abundant lipoprotein that enables lipids to be transported within the water-based
bloodstream. Lipoproteins are macromolecular complexes with a surface envelope of amphipathic phospholipids, free cholesterol,apolipoproteins, and the associated proteins together with an inner core constituting mainly triglycerides（TG）. HDL has thehighest protein content compared with other lipoprotein groups（VLDL, LDL, and IDL）, identified as a strong inverse predictor ofrick of cardiovascular disease. The function of HDL in health and disease is largely relied on the interactions of surface proteins
with cell membrane receptors, enzymes, and lipid-transport proteins. Glycosylation, one of the most common post-translational
modifications, plays important roles in cell-cell recognition and communication. The glycosylation of HDL however has not been
well explored despite the presence of glycoproteins and glycolipids. Using systematic glycomics and glycoproteomics approaches,
we determined for the first time protein and site-specific glycosylation and lipid glycosylation in HDL.
MethodN-linked glycans were released from 60 µL HDL（triplicates）using PNGase F. The released N-glycans were cleaned up via solid
phase extraction（SPE）procedure using graphitized carbon cartridges（GCC）. Pronase E was covalently coupled to CNBr
activated sepharose beads via coupling chemistry, followed by adding 100 µL HDL and incubated at 37 ºC for 18 h. The
glycopeptide digest was desalted and enriched via SPE procedure using GCC. Extraction of gangliosides was performed by mixing
water, methanol and chloroform with 100 µL HDL prior to centrifuging. Supernatant was collected, followed by clean-up procedure
via SPE using C 8 cartridges. MS and MS/MS analyses of glycans, glycopeptides and gangliosides were achieved via nano-LC/
Q-TOF MS with a nano-LC column integrated in a micro-chip packed with porous graphitized carbon for glycan/glycopeptides andreverse phase for gangliosides, respectively.
Preliminary data
HDL proteomic results revealed a number of apolipoproteins, Apo-A 1 as the most abundant one, and the associated plasma
proteins approximately 60% of which are glycosylated.
Glycans were separated with a PGC stationary phase that is incorporated into an HPLC-microchip with high sensitivity andreproducibility. On average, our nano-LC method was able to resolve and identify over 60 N-linked glycan compound peaks withover 20 distinct N-linked glycan compositions from HDL. Each of the identified compositions included two or more peaks
corresponding to structural and/or linkage isomers. Sialylated glycans as Complex type with bi- or tri- antenna were found to be
the predominant glycan type. Interestingly, a bi-antennary glycan with two sialic acids was the most abundant glycan species. The
structures of N-glycans from milk glycoproteins were further elucidated by tandem MS.
Comprehensive glycan-microheterogeneity of HDL glycoproteins was achieved by nano-LC/Q-TOF MS and in-house Glycopeptide
Finder as a bioinformatics tool. Nonspecific protease digestion enabled the representation of all glycans attached to each N- or
O-glycosites with a short peptide tag. In all, over 50 glycopeptides（identified based on composition and including isomers）
corresponding to 20 glycosites were observed and determined in HDL protein mixture. The glycopeptides were a mixture of
N-linked glycopeptides（mainly complex and sialylated glycans）and O-linked glycopeptides（sialylated）. Results werecomprehensive as detailed glycan microheterogeneity information was obtained.
Ganglioside profiles showed that GM 3 （monosialoganglioside, NeuAc23Gal14GlcCer） and GD 3 （disialoganglioside,
NeuAc28NeuAc23Gal14GlcCer）are abundant ions in human HDL. A 75 % GM 3 and 25 % GD 3 distribution was observed. Both
GM 3 and GD 3 are composed of heterogeneous ceramide lipids, including d34：1, d39：1, d40： 1 and d41：1.

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 26: Lipidomics : Recent New Techniques and Applications

PWe-062
13:30 － 14:40

Quantitative analyses of phosphatidylcholine hydroperoxides in oxidized
lipoproteins by LC/MS

Shu-Ping Hui1, Toshihiro Sakurai1,2, Seiji Takeda1, Shigeki Jin1, Hirotoshi Fuda1, Takao Kurosawa3,
Hitoshi Chiba1
1
3

Hokkaido University, Sapporo, Japan, 2Research Fellow of the Japan Society for the Promotion of Science, Japan,
Faculty of Pharmaceutical Sciences, Health Sciences University of Hokkaido, Ishikari-Tobetsu, Hokkaido, Japan

Keywords:
Lipid hydroperoxide, Oxidized lipoproteins, LC/MS
Novel aspects:
Unstable phosphatidylcholine monohydroperoxides in human lipoproteins were measured by liquid chromatography/mass
spectrometry using nonendogenous 1 -palmitoyl- 2 -heptadecenoylphosphatidylcholine monohydroperoxide as an internal
standard.
Abstract:
BackgroundPhosphatidylcholine hydroperoxide（PCOOH）, a primary oxidation product of PC, has been the focus of lipid peroxidation. In
recent years, liquid chromatography（LC）/mass spectrometry（MS）has become a powerful tool for structurally identifying
PCOOH. We previously reported a quantitative LC/MS method for PC-OOH using a synthetic internal standard of hydroperoxide［ 1 ］. Unfortunately, we occasionally observed interference by unspecified substance（s）in plasma samples with this internal
standard. To solve this problem, we established an improved LC/MS method with a new synthetic internal standard and applied itin quantitative analyses of PCOOH in human low-density lipoprotein（LDL）and high-density lipoprotein（HDL）during coppermediated oxidation［ 2 ］.
Methods
1 -Palmitoyl- 2 -linoleoylphosphatidylcholine monohydroperoxide （PC 16： 0 /18： 2 -OOH）, 1 -stearoyl- 2 linoleoylphosphatidylcholine monohydroperoxide（PC 18： 0 /18： 2 -OOH）, and 1 -palmitoyl- 2 -heptadecenoylphosphatidylcholine
monohydroperoxide（PC 16： 0 /17： 1 -OOH as IS）were chemically synthesized. We prepared oxidized LDL（oxLDL）and oxidized（oxHDL）by incubating native LDL（nLDL）and native HDL（nHDL）from human plasma（n=10）with CuSO 4 for up to 4 h. For
quantitative analysis, 2 mL nLDL, nHDL, oxLDL, or oxHDL was extracted with chloroform. The residue was dissolved in 300 μL
methanol and a 10-μL aliquot was injected into our quantitative LC/MS system. Reversed-phase LC separation was conducted ona reversed-phase column（Hypersil Gold C8, 50 mm×2.1-mm inner diameter, 5 μm； Thermo Fisher Scientific, Waltham, MA, USA）
at 60 °C. The mobile phase consisted of three solvents： solvent A was 10.0 mM ammonium acetate aqueous solution, solvent B
was acetonitrile, and solvent C was 2 -propanol. The gradient was programmed as follows： 0.00-2.00 min 100% solvent A； 2.0-5.00
min 10% solvent A and 90% solvent C； 5.0-8.00 min 100% solvent C； 8.0-10.00 min 20% solvent A and 80% solvent B. The flow rate
was 0.3 mL/min. Quantitative analyses were performed using a Prominence LC system（LC-20AD unit and SIL-20A autosampler；
Shimadzu, Kyoto, Japan）and a TSQ Quantum Access MAX triple stage quadrupole mass spectrometer with a heated electrosprayionization probe（Thermo Fisher Scientific, Waltham, MA, USA）. The scan time was 0.333 s in positive ion mode. The electrosprayionization inlet conditions were as follows： ion source voltage ±3.0 kV； vaporizer temperature 350 °C； sheath gas（nitrogen）
pressure 50 psi； auxiliary gas pressure 15 psi； capillary temperature 300 °C； Argon was used as the collision gas at 1.5 mTorr.
Results
The calibration curves for synthetic PC 16： 0 /18： 2 -OOH and PC 18： 0 /18： 2 -OOH, which were obtained by direct injection of
the IS into the LC/MS system, were linear throughout the calibration range（0.8-12.8 pmol）. Within-day and between-day
coefficients of variation were less than 10%, and the recoveries were between 86% and 105%. The limit of detection（LOD）and thelimit of quantification（LOQ）were determined using synthetic standards. The LOD（signal-to-noise ratio 3 ： 1 ）was 0.01 pmol, andthe LOQ（signal-to-noise ratio 6 ： 1 ）was 0.08 pmol for both PC 16： 0 /18： 2 -OOH and PC 18： 0 /18： 2 -OOH. With use of thismethod, the concentrations of PC 16： 0 /18： 2 -OOH and PC 18： 0 /18： 2 -OOH in the lipoprotein fractions during copper
mediated oxidation were determined. The time course of the PC 16： 0 /18： 2 -OOH and PC 18： 0 /18： 2 -OOH levels duringoxidation consisted of three phases. For oxLDL, both compounds exhibited a slow lag phase and a subsequent rapidly increasing
propagation phase, followed by a gradually decreasing degradation phase. In contrast, for oxHDL, both compounds initiallyexhibited a prompt propagation phase with a subsequent plateau phase, followed by a rapid degradation phase. The analytical
LC/MS method for phosphatidylcholine hydroperoxides might be useful for the analysis of biological samples.
References1. Hui SP, et al. J Chromatogr B（2010）878：16771682
2. Hui SP, et al. Anal Bioanal Chem（2012）DOI 10.1007/s00216-012-5833-x

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 26: Lipidomics : Recent New Techniques and Applications

PWe-064
13:30 － 14:40

In situ analysis of soybean and nuts by Probe Electrospray Ionization mass
spectrometry. Sequential ionization of carbohydrates and lipids

Gabriela Petroselli1, Mridul K Mandal2, Lee C Chen2, Kenzo Hiraoka2, Hiroshi Nonami3,
Rosa Erra-Balsells1
1

University of Buenos Aires, 2Yamanashi University, 3Ehime University

Keywords:
Lipids, carbohydrates, soybean, nuts, tissue
Novel aspects:
PESI MS applied to direct analysis of lipids and carbohydrates in intact soybean and nuts. Potential application of PESI MS to nuts
tissue imaging.
Abstract:
The probe electrospray ionization（PESI）is an ESI-based ionization technique that generates electrospray from the tip of a solid
metal needle.1 In the present work, we describe the PESI mass spectra obtained for in situ measurement of soybeans and several
nuts（peanuts, walnuts, cashew nuts, macadamia nuts, pistachios and almonds）using different solid needles as sampling probes.
For comparative study ESI MS and PESI MS analyses were performed for methanolic extracts from the seeds. Because seeds arerich in phospholipids, strong signals of phosphatidylcholine, phosphatidylethanolamine and triacylglycerols of different fatty acidresidues were detected as major ions in the positive ion mode. Lipids were observed mostly as adducts with Na+ or K + . No
significant differences were observed between ESI-MS and PESI-MS. The great advantage of PESI-MS is that no sample preparationis needed and minimum volume of organic solvents is required.
In order to find the optimal experimental conditions for seeds analysis by PESI MS some variables were evaluated.（i）The metalmaterials and needle diameters were evaluated.（ii）A typical protocol for lipids extraction from seed tissues was used and themass spectra obtained for extracts were compared with those obtained in situ PESI analysis.（iii）For direct PESI analysis, several
solvents were used to make the sample surfaces wet for the extraction of the components from the samples.（iv）Two methods
were used to acquire the PESI mass spectra： the single-shot mode and the continuous-shot mode. In the former, the droplet wasleft to be electrosprayed until the electrospray current decreased to zero and in the latter the droplet was periodically renewed.
It was found that PESI MS is a valuable approach for in situ lipid analysis of seeds. Additionally, PESI mass spectrum is not probe
material dependent. However, by modifying the titanium wire surface with pentafluorophenyl-triethoxysilane to be hydrophobic,
time-dependent mass spectra could be obtained when single-shot mode is adopted allowing sequential analyses of carbohydrates
and lipids.
［ 1 ］K. Hiraoka, K. Nishidate, K. Mori, D. Asakawa, S. Suzuki, Rapid Commun. Mass Spectrom. 21, 3139（2007）.

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 26: Lipidomics : Recent New Techniques and Applications

PWe-065
11:10 － 12:20

Comprehensive Lipid Profiling with Multiplexed Precursor Ion Scanning
using the New AB SCIEX QTRAP® 4500 LC/MS/MS System

Tomoko Nembai, Toshiyuki Yamazaki, Noriko Fueki, Kaoru Karasawa
K.K.ABSCIEX

Keywords:
New instrument, Lipid, Profiling, High-throughput
Novel aspects:
As QTRAP® 4500 has fast scan speed and positive/negative switching speed； it took only a few minutes to complete multiple
experiments.This advantage also resulted in saving consumption of samples.
Abstract:［Purpose and Methods］
Cellular lipids play an important role in biological functions which include the formation of cell membranes, regulation, energy
metabolism, and signal transactions.
Lipids have highly structural diversity due to the complex combination of hydrophobic acyl chain molecular species； hence it issignificant to characterize and quantify diverse types of lipids, and also to analyze variability when understanding various aspectsof physiological phenomena.
Mass spectrometry has emerged as a powerful analytical tool for lipidomics research. To detect and identify lipids selectively andcomprehensively, multiple specific precursor ion and neutral loss scan mode experiments which based on lipid classes and fatty
acid chain fragments are widely used. However, it is sometimes difficult to do comprehensive profiling to detect trace amount ofcomponent due to scan speed or sensitivity limitation. Moreover, identification of numerous types of lipids is very complicated.
The new AB SCIEX QTRAP® 4500 System, which offers the very fast quadrupole scanning, up to 12000 Da/s, achieving a wide mass
range survey scans from 5 to 2000 Da in milliseconds and delivering improvements in sensitivity, makes it possible to overcome
the limitations and difficulties in conventional systems. Improved performances enable multiple precursor ion and neutral loss scanmode experiments in both positive and negative polarities with high scan speed and high sensitivity simultaneously. What is more,
post-acquisition data processing can be fully automated using LipidView™ Software which allows lipid investigator the ability toeasily find, identify and interpret lipid species from complicated mass spectrometry data.
In this work, asolectin from soybean（Sigma-Aldrich Inc.）and other lipid mixtures were used as test samples.
A total of 80 combinations of Precursor ion scan（PIS）and Neutral loss scan（NL）experiments were acquired in a single methodwith positive and negative polarity switching.
Samples were diluted with 0.1 % formic acid/ 5 mM ammonium formate in isopropanol and introduced by direct infusion. These
combinations were based on lipid class-specific fragment ions or fatty acid-specific ones up to 22 carbon chain lengths.
For post-acquisition data processing, LipidView™ Software was used for identification and quantification of lipids.
［Results］
As QTRAP® 4500 has 1000 Da/sec scan speed and 50msec positive/negative switching speed, it took only 10 minutes to complete
80 experiments（accumulation cycle 20 times）, whilst conventional LIT system needs 60 minutes.
This speed advantage also resulted in saving consumption of samples. To acquire equal or better quality of data, QTRAP® 4500
needed only 100 uL of sample needs and it was 1 / 5 amount of current system.
The acquired data was processed by the LipidView™ Software.
In asolection, phosphatidyl ethanolamine（PE）34： 2 and phosphatidyl inositol（PI）34： 2 constructed in the fatty acid 16： 0 and
18： 2 were mainly identified.
［Conclusions］
The AB SCIEX QTRAP® 4500 System provides versatility, sensitivity, specificity and speed required for high-throughput lipid profilingof complex biological extracts by direct infusion.
These improvements enable us analysis of scarce sample, because it requires less amount of sample than conventional system.
When combined with LipidView™ Software, this complete hardware and software package offers unique data acquisition
strategies.

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 26: Lipidomics : Recent New Techniques and Applications

PWe-066
13:30 － 14:40

Comprehensive fatty acid profiling using a single lipidomic approach

futoshi sato1, Giorgis Isaac2, Amrita Cheema3, Kieran J Neeson2, Jeff Goshawk2, Jayne Kirk2,
Andrew Baker2, Alan Millar2, Albert J Fornace Jr 3, Jim Langridge2, Giuseppe Astarita2
1

Nihon Waters K.K., Tokyo, Japan, 2Waters Corporation, Milford, US, 3Georgetown University, Washington D.C., US

Keywords:
Lipids, UPLC, Ion mobility
Novel aspects:
Comprehensive screening and fingerprinting of fatty acid composition for phenotypic identification and comparative lipidomic
analysis.
Abstract:
Introduction
Fatty acids are present in biological samples both in unesterified（free）and esterified forms. Their analysis usually requires multistep procedures, which includes enzymatic or chemical hydrolysis and their subsequent derivatization. These laborious procedures
not only affect the sensitivity of detection, but also lead to a loss of information regarding the actual fatty acyl composition ofdifferent complex lipid classes（e.g., glycerolipids, glycerophospholipids, sterol lipids and sphingolipids）. Here we present arobust method for the simultaneous profiling of free and esterified fatty acids from various biological samples using ultra
performance liquid chromatography（LC）coupled with ion mobility mass spectrometry.
Methods
We used - 2 µm particle size LC with an ion mobility TOF to discriminate lipids based on the different cross collision sections.
Within the same chromatographic run, the mass spectrometer was set to apply alternate collision energies to the transfer collisioncell between low and elevated collision energy ramp, with the latter inducing lipid fragmentation. As the fragmentation occurs
post ion mobility separation, the precursor at low energy share drift time with its fragments from the elevated energy scan. Postacquisition, we extracted and visualized the fatty acid information contained in the spectra using novel informatics solutions forneutral loss or precursor ions survey.
Abstract
We analyzed total lipid extracts from various animal tissues using sub- 2 µm particle size LC coupled with ion mobility. The post
ionization separation by ion mobility allowed to increase the peak capacity and the specificity of identification. Lipid ions withdifferent degree of unsaturation and acyl length migrated with characteristic mobility times due to their unique interactions with
the nitrogen gas in the ion mobility cell. By alternating low and elevated collision energy in a data independent acquisition mode,we were able to simultaneously collect both complex lipids and fatty acyl information. Fatty acyl groups were detected as either
neutral loss in positive mode or as charged ions in negative mode. The post-acquisition analysis allowed to differentiate the fatty
acyl content and distribution in various lipid classes according to the different biological samples analyzed. Fatty acyl composition
could be visualized as bidimensional maps（drift time versus retention time）, which translated in molecular fingerprints of thevarious tissues

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 26: Lipidomics : Recent New Techniques and Applications

PWe-067
11:10 － 12:20

MALDI-TOF MS analysis of phosphatidic acid using phosphate capture
molecule, Phos-tag

Mai Urikura1, Yoshino Kondo1, Jun-ichi Morishige1, Tamotsu Tanaka2, Kiyoshi Satouchi1
1

Fukuyama University, Fukuyama, Japan, 2University of Tokushima Graduate School, Tokushima, Japan

Keywords:
MALDI-TOF MS, Phos-tag, phosphatidic acid
Novel aspects:
We developed the method for PA determination using phosphate capture molecule, Phos-tag.
Abstract:［Background］
Liquid chromatography mass spectrometry coupled with electrospray ionization（ESI LC-MS）has been established as a method
for identification and quantification of compounds with low molecular mass, and now it has been prevalent for lipidomics in the
biological system. Another soft-ionizing method, matrix-assisted laser desorption and ionization mass spectrometry coupled with atime-of-flight mass spectrometry（MALDI-TOF MS）has been primarily established for the analysis of compounds with highmolecular mass such as protein. Recently, MALDI-TOF MS have been evaluated as a useful methodology in lipid research due toits simplicity and high performance. Compared with ESI LC-MS, advantage of MALDI-TOF MS is free from carry-over contaminationand deterioration of sensitivity due to dirt of ion source. On the other hand, however, it requires purification step if objective
molecule is a minor component in the samples.
In the present session, we would like to show the MALDI-TOF MS analysis of phosphatidic acid（PA）, a potential anti-ulcer
phospholipid produced by phospholipase D（PLD）in cruciferous vegetables such as cabbage.
［Experimentals］
Cabbage juice was obtained by squeezing row cabbage leaves and Phos-tag was obtained from MANAC Inc.（Fukuyama）.
Cabbage juice was incubated with egg yolk or soybean lecithin. The products were extracted by Bligh and Dyer procedure andanalyzed by MALDI-TOF MS using Phos-tag coordinated with the single isotopic zinc, 68Zn. Phos-tag was coupled chemically to
Toyopearl resin and the bead was used for isolation of PA.
［Results and Discussion］
PA is a minor membrane phospholipid constituting less than one hundredth of total phospholipids. PA is an intermediate ofphospholipid synthesis, but it has been recently recognizing that PA is biologically active phospholipid, such as second messenger
upon cell stimulation and an anti-ulcer protector in digestive tracts. Accordingly, demands to quantify a small and transient
amount of PA have been increasing. PA is monoester type of phospholipid and has two negative charges in phosphate, thus, threepositive charges are necessary to produce a single positively complex ion. As a result, multiple ions due to additions of protons,
sodium and potassium ions, and their combinations are detected in a mass spectrum of PA in positive mode. This is the problem
for analysis of molecular species, where PA comprised several molecular species with many fatty acids. Zinc coordinated complex,
Phos-tag, was synthesized by referring the spatial configuration of zinc metals in the active site of alkaline phospahatase. This
reagent can capture phosphate compound specifically and dose-dependently. Phos-tag can unify adducts of PA to［PA 2-/Phostag3+］+ in positive mode of MS. It contributes not only to the simplification of mass spectra, but also the increase in detection
efficiency.
Using 68Zn Phos-tag, PA formation by the action of PLD from cabbage juice can follow in a short time by MALDI-TOF MS without
purification step. Molecular species of PA produced from egg yolk lecithin were 1 -saturated- 2 -unsaturated species like palmitoyl/oleoyl（16： 0 /18： 1 ）PA, whereas those from soybean lecithin were 1,2-diunsaturated species like linoleoyl/linoleoyl（18： 2 /18： 2 ）PA in addition to 1 -saturated- 2 -unsaturated species.
For a trace amount of PA, suppression effects of co-existing other phospholipids are unavoidable. To overcome, Phos-tag
Toyopearl bead is available. An equal amount of mixture of phospholipids, phosphatidylcholine（PC）and PA was incubated withthe bead in methanol. PC, diester type of phospholipids, is eluted from the bead thoroughly, whereas PA having monoester typeof phosphate interacts with Phos-tag. The retained PA in the bead was recovered by addition of 0.01 M HCl in methanol. Using
Phos-tag Toyopearl for isolation, technique for quantitation of PA by MALDI-TOF MS has been under investigations.

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 26: Lipidomics : Recent New Techniques and Applications

PWe-068
13:30 － 14:40

Novel Non-targeted Lipidomic Quantitation using HILIC-HPLC/MS

Eva Cifkova, Miroslav Lisa, Michal Holcapek

University of Pardubice, Pardubice, Czech Republic

Keywords:
Lipidomics； lipid class； quantitation； HILIC-HPLC/MS
Novel aspects:
Non-targeted lipidomic quantitation of lipid classes using hydrophilic interaction liquid chromatography and mass spectrometry.
Abstract:
The identification and quantitation of all lipids in complex biological tissues is the first step towards the understanding how lipids
function in a biological system and the elucidation of the mechanism of lipid-related diseases including obesity, atherosclerosis,
cancer, cardiovascular diseases, etc. HPLC/MS has the highest potential to obtain comprehensive information about the whole
lipidome, but the reliable quantitation of all lipid classes is a difficult and challenging task. Our optimized HILIC-HPLC/MS enables
the fractionation of 19 lipid classes in a wide range of polarities［J. Chromatogr., A 1218（2011）5146］. The goal of current research
work is the development of new non-targeted method for the quantitation of main lipid classes and also lipid species within theseclasses using a single platform. For this purpose, the novel non-targeted lipidomic quantitation was developed and results werecompared with the conventional approach with multiple selected reaction monitoring（SRM）scans on triple quadrupole analyzer.
Concentrations of individual lipid classes were obtained by the peak integration in the HILIC mode multiplied by response factors
calculated for individual classes and correlated with sphingosyl ethanolamine（d17： 1 /12： 0 ）as an internal standard. In
comparison to conventional SRM approach, our method allows to quantify all separated lipid classes without the definition ofindividual SRM transitions. Subsequently, fractions of individual lipid classes are collected and used for off-line 2 D HILIC x RPHPLC/MS analysis. RP-HPLC separation in the second dimension provides more detailed qualitative and quantitative information
about lipid species within these classes including the isobaric differentiation. Our non-targeted quantitation is an appropriate tool
for the comprehensive lipidomic characterization of multiple lipid classes and the determination of lipid composition differences
between healthy and disease tissues.
This work was supported by the grant project No. 206/11/0022 (Czech Science Foundation).

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 26: Lipidomics : Recent New Techniques and Applications

PWe-069
11:10 － 12:20

Shotgun analysis of lipid extract from mouse brain by using
comprehensive LC/MS

Jisun Yoo, Eun Jung Bae, Taeseong Park, Young Hwan Kim
Korea Basic Science Institute, Ochang, Korea

Keywords:
lipidomics, LC/MS,
Novel aspects:
A method of combined profiling and MS/MS using UPLC coupled with mass spectrometry was developed for the comprehensive
analysis of lipid extract from mouse brain.
Abstract:
Lipidomics is a rapidly expanding research field in which multiple techniques are utilized to quantify the precise chemical
constituents in a cell’s lipidome and identify their cellular organization. The results obtained by lipidomics provide the informationon their biochemical mechanisms through which lipids interact with each other and with crucial membrane-associated proteins.
LC/MS has played an important role in the detection and identification of lipids. Especially, the ultra-performance liquid
chromatography（UPLC）is now widely used and applied to not only lipid analysis and which have a high efficiency, resolving
power, and sensitivity, and rapid analytic ability. In this study, a method for lipid profiling using UPLC coupled with mass
spectrometry has been demonstrated for the analysis of mouse brain. For the analysis of lipids in biological samples, the use of
MS E data results in multiple levels of information which is extremely useful when trying to confirm the identity of a specificcompound. For the determination of the repeatability, linearity and recovery, the ratio of the peak areas of the endogenous lipid
and the corresponding internal standard was calculated. On the other hand, MSE acquires all relevant information in both low
energy and high energy acquisitions during the same analysis, and in the subsequent data processing available information on
fragmentation pathways, diagnostic precursor and neutral losses allowed us to determine the polar head group and fatty acidcomposition in a relatively fast and efficient manner.

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 26: Lipidomics : Recent New Techniques and Applications

PWe-070
13:30 － 14:40

Top-Down Analysis of Lipoproteins by On-Line Field-Flow Fractionationand ESI-MS-MSKi Hun Kim, Ju Yong Lee, Myeong Hee Moon
Yonsei University, Seoul, Korea

Keywords:
lipoprotein, lipid analysis, field-flow fractionation
Novel aspects:
By using on-line cAF 4 -ESI-MS-MS, lipoprotein particles are separated by sizes and various lipids contained in HDL and LDL can bedirectly anayzed with structural identification by top-down approach.
Abstract:
Lipoproteins are globular particles composed of lipids and proteins in blood serum and their roles include transferring fats andcholesterols through blood vessels throughout the body. Especially, it has been known that smaller and denser low density
lipoprotein（LDL）and low levels of high density lipoprotein（HDL）are risk factors of atherosclerosis and other cardiac diseases.
Among lipoprotein components besides cholestrol, phospholipids（PLs）are a major lipid class composed of various molecular
categories differed by the polar head groups and the lengths of acyl chains along with the degrees of unsaturation in acyl chain.
For the analysis of lipoprotein components, it requires an isolation/fractionation of HDL and LDL particles from blood sample andthese are commonly carried out by using density gradient ultracentrifugation（DGU）, gel-based electrophoresis, and size
exclusion chromatography（SEC）. However these techniques have minor drawbacks such as the requirement of large amount ofsample for DGU and the possible sample loss due to the interaction between lipoprotein particles with gel matrix for the last two
methods.
Flow field-flow fractionation（FlFFF）is a technique capable of separating nanoparticles or proteins based on hydrodynamic size
differences in an open channel by the simultaneous movement of a migration flow and a crossflow, where the crossflow is applied
perpendicular to the migration flow. FlFFF has been utilized for the size determination of HDL and LDL from blood serum samplesof CAD patients and the collected HDL and LDL particles can be further analyzed for the profiling of phospholipids by nanoflow
liquid chromatography-electrospray ionization-tandem mass spectrometry（nLC-ESI-MS-MS）. This presentation introduces the topdown analysis of various lipid molecules contained in lipoprotein particles from plasma sample using a direct interface of the chiptype asymmetrical FlFFF（cAF 4 ）, a miniaturized AF 4 channel that has recently been developed to use microflow regime, with
ESI-MS-MS in aqueous phase. HDL and LDL particles from blood sample were separated by sizes in aqueous solution in cAF 4channel and the eluting lipoprotein particles were directly analyzed by direct ESI-MS-MS. By using on-line cAF 4 -ESI-MS-MS,
lipoprotein particles can be desalted on-line during cAF 4 separation which enhances ionization of lipid molecules contained inlipoproteins during ESI-MS. Structural determinations of lipid molecules including phospholipids, lysophospholipids, cholesterylesters, and triacylglycerols were carried out with in-source fragmentation during ESI followed by data dependent CID experiments.

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 26: Lipidomics : Recent New Techniques and Applications

PWe-071
11:10 － 12:20

Potential Biomarkers of Coronary Artery Disease by Flow Field-Flow
Fractionation and Nanoflow LC-ESI-MS-MS

Seul Kee Byeon, Ju Yong Lee, Myeong Hee Moon
Yonsei University, Seoul, Korea

Keywords:
nLC-ESI-MS-MS, CAD, biomarkers, phospholipid, flow field-flow fractionation
Novel aspects:
Potential candidate biomarkers of coronary artery disease can be identified by profiling phospholilpids and lysopholipids fromhuman plasma using nLC-ESI-MS-MS.
Abstract:
Ranking high in the leading causes of cardiovascular diseases, coronary artery disease（CAD）is commonly found well over theworld, ranging from industrialized countries to less-developed countries. Although it has been generally diagnosed in people over
40 years-old, current trend in CAD is starting to be affiliated with younger generation of age under 20 as well. As a disease that canaffect everyone regardless of sex and age, attention on CAD in an attempt to understand and explain the disease has been
increasing high in medical and chemical fields. There are many reported risk factors of this disease including high blood pressure,lack of exercises, obesity, smoking, diabetes, high cholesterol, and high level of low-density lipoprotein（LDL）and low level ofhigh-density lipoprotein（HDL）are highly related to pathogenesis of the diseases. Among different subclasses of lipids, studies on
phospholipids have sparked an interest for the past decades because of their propensity to control various types of cellularresponses. Numerous studies that have been published nowadays engage with lysophospholipids（LPL）, which are phospholipids（PL）with one fatty acid chain instead of two, and PLs because they are reported as biomarkers of human diseases such asbreast, prostate, and ovarian cancer. In this study, LPLs and PLs from plasma of control group and patients with CAD wereidentified and compared in order to discover potential biomarkers of the disease.
First, HDL and LDL from human plasma were separated according to the particle size by using flow field-flow fractionation（FlFFF）. Among different types of FlFFF techniques, multiplexed hollow fiber flow field-flow fractionation（MxHF 5 ）was
specifically chosen in order to inject a substantial volume of plasma sample at a time. The fractionated HDL and LDL werecollected over time, and intact LPLs and PLs were simultaneously extracted while proteins were removed from these collections
using a modified Folch method with methyl-tert-butyl ether（MTBE）and methanol. In order to identify as much LPLs and PLs as
possible, including the species that existed in trace amount, nanoflow liquid chromatography electrospray ionization-tandem mass
spectrometry（nLC-ESI-MS-MS）was employed so that LPLs and PLs could be identified in an order of hydrophobicity, regardless of
their amount. A comprehensive analysis of LPLs and PLs from control and patients was performed both qualitatively andquantitatively, by nLC-ESI-MS-MS. In order to produce a highly-reliable and valid data, a total of ten plasma samples from eachgroup were obtained and analyzed.

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 26: Lipidomics : Recent New Techniques and Applications

PWe-072
13:30 － 14:40

Profiling of the Oxidized Phospholipids in Human Lipoprotein by Nanoflow
LC-ESI-MS-MS

Ju Yong Lee, Myeong Hee Moon
Yonsei University, Seoul, South Korea

Keywords:
nLC-ESI-MS-MS, oxidized LDL, Phospholipid, Lysophospholipid
Novel aspects:
Using collision induced dissociation patterns of the oxidized phospholipids obtained from nLC-ESI-MS-MS, phospholipids fromoxidized LDL can be identified.
Abstract:
Metalloprotein hemoglobins（met-hemoglobin）refer to a complex when the charge of an iron ion from hemoglobin changes from+ 2 to + 3 and function differently. They oxidize lipoprotein, apolipoproteins, and phospholipids to transport the lipid by bloodvessel. According to recent studies, it is known that oxidized low-density lipoproteins（LDL）function differently from intact LDL
and their particle sizes and characteristics as well. Moreover, as macrophages fail to get rid of oxidized LDLs, they travel via bloodstream inside human body and end up causing severe inflammation in arteries, which can lead to one of most common heartdisease, called Coronary Artery Disease（CAD）. Despite the fact that oxidized LDLs play an important role in human body, notmuch studies have been conducted so far, and among a few number of studies that have been published, their investigation is
primarily on oxidized phosphatidylcholine（PC）only. In this study, a systematic analysis of different classes of oxidized
phospholipids（PLs）has been carried out to discover the detailed molecular patterns.
To examine the oxidation pattern of PLs, standard PLs（18： 0 /18： 0 -PC, 16： 0 /22： 6 -PC, 18： 0 /22： 6 -PG, and 18： 0 /22： 6 -PA）
were utilized to make each vesicle first, and then the resulting vesicles were oxidized by adding 10 μM CuSO4. The mixtures werestored at 37&#8451； for two different periods of 1 and 2 hours. In order to promote quenching the oxidation reaction, EDTA wasadded to the samples. For oxidation of LDL particles, CuSO4 was mixed with LDL standard and stored for two hours. Nanoflow
liquid chromatography electrospray ionization tandem-mass spectroscopy（nLC-ESI-MS-MS）was utilized to characterize oxidized
PL molecules.
For PLs with saturated acyl chains, there was not much of a change observed. However, for PLs with poly unsaturated acyl chains,a variety of oxidation in acyl chains was observed regardless of head groups； addition of hydroxyl group to fatty acids andproduction of different types of lysophospholipids（LPLs）. Based on these results, intact LDL and oxidized LDL were analyzed bynLC-ESI-MS-MS under the same conditions and a total of 160 PL species（including oxidized PLs）were identified.

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 26: Lipidomics : Recent New Techniques and Applications

PWe-073
11:10 － 12:20

Discovery of lipid markers by LC-ESI/MS for early detection of low doseradiation exposure

Nai-Chun Huang, Jui-Ping Li, Chia-Hwa Chen, Chung-Shi Yang, Jen-Kun Chen
National Health Research Institutes, Zhunan, Taiwan

Keywords:
lipids, radiation, biological dosimetry, sphingomyelin, ceramide
Novel aspects:
lipid molecules of cell membrane act as biological dosimetry for low dose exposure of radiation
Abstract:
The Japanese nuclear accident in 2011 has awakened us to pay much attention to nuclear safety and health risk for the exposureof radiation. In conventional scope, alteration of genomic DNA was believed the primary and unique molecular marker owing to
its critical role in delivery of genetic information. Radiation undoubtedly influences cell membrane together with DNA； and we try
to use HPLC-ESI/MS for rapid discovery of cell membrane-related markers. The specific aim is to clarify the chemical differences oflipids on cell membrane prior to chromosome aberration. We anticipate to（ 1 ）explore early detection marker for radiation
exposure of citizens and（ 2 ）advance understanding of lipid damage mediated apoptosis for scientists in the field of radiobiology
and radiotherapy. Human glioblastoma U87-MG cells and mouse colon carcinoma CT26 cells were irradiated with X-ray for 0.5 and
5.0 Gy of doses in comparison to untreated cells. Twenty-four hours after X-ray irradiation, cells were lysed with isopropyl alcohol（IPA）followed by centrifugation at 12500 g for 30 min at 10oC. The phospholipids’ extracts were harvested using solid phaseextraction（Ostro (R) 96-well plate, Waters）for further analysis by HPLC（Agilent 1100）conjugated with high resolution ESI/MS（microTOF II focus, Bruker Daltonics）. Chromatograms and mass spectra were displayed and reconstructed using Compass
DataAnalysis（version 4.0, Bruker Daltonics）. The identification of molecular formula was based on the exact mass of
monoisotopic peak with the tolerance of 5 mDa. Sphingomyelin（d18： 1 /16： 0 ）eluted around 27 min with 703.57 m/z of
protonated molecular ion was significantly eliminated by the increase of X-ray dose, which directly correlated with the increase of
signals with 538.52 m/z, eluted at 3.4 min and assigned as ceramide（d18： 1 /16： 0 ）. Several phosphatidylethanolamines（PEs）
and phosphatidylglycerols（PGs）were found and positively correlated with increasing dose of X-ray radiation, whereas
phosphatidylcholines（PCs）represented no significant change. Co-stained with propidium iodide（PI）and YOPRO- 1 demonstrated
the biological property of cell membrane did change after the treatment of radiation. We therefore suggest the breakdown of
sphingomyelin to ceramide could be a potential diagnostic biomarker for low dose exposure（<0.5Gy）of radiation.

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 26: Lipidomics : Recent New Techniques and Applications

PWe-074
13:30 － 14:40

Characterization of moquito cuticular lipids for age, species, and sex
discrimination by MALDI-IT-TOF-MS

Kevin A Schug1, Sheheli Islam1, Jaroslaw Krzywinski2, Seoung Bum Kim3

The University of Texas at Arlington, Arlington TX USA, 2Liverpool School of Tropical Medicine, Liverpool UK, 3Korea
University, Seoul, Korea

1

Keywords:
mosquito, MALDI, cuticular lipids, tandem mass spectrometry
Novel aspects:
MALDI-IT-TOF-MS is used to identify specific cuticular lipid components, which can be used to differentiate species, sex, age, andmating status of malaria-carrying mosquitoes
Abstract:
Malaria is an insidious disease. Millions of people are affected, hospitalized, or killed annually as a consequence of infection. The
malaria parasite is tranferred to humans through the bites older female Anopheles gambiae mosquitoes. Currently, treatment
strategies for malaria are limited, and much effort is given to risk assessment and abatement. To assess the risk of infection for agiven community, it would be ideal to understand the proportion of mosquito population that is capable of conferring the parasite.
Previously, our group has published a MALDI-TOF-MS method（Anal. Chim. Acta2011, 706, 157-163）, which, based on mass
spectral fingerprints generated following chloroform extraction of exoskeletal compounds, can be used to differentiate A. gambiae
mosquitoes based on their age, sex, and mating status. Cuticular lipids and hydrocarbons are well known to help prevent
dessication of insects and contribute to mate recognition. Signals are generated through the combined use of an acenaphthenematrix and a AgNO 3 cationization reagent. Though useful, relatively little information could be obtained regarding the identity ofsignals which differentiate between classes. More recently, we have obtained a Shimadzu Resonance MALDI-ion trap-time of flightMS instrument. Capable of isolation, multi-stage fragmentation, and high resolution measurement of MALDI signals, we have now
been able to pursue identification of important lipids compounds obtained from the extract. Our preliminary results show that a
mix of hydrocarbons（< 500 Da）and lipids（500 - 1200 Da）are extracted and contribute to the fingerprint. Lipids are believed toinclude different chain length variants of ceramides and sphingomyelins. Work is ongoing to unequivocally identify these species.
Additionally, based on the increased sensitivity and resolution of the new instrument, we are able to perform extracts of single
mosquitoes, whereas three mosquitoes were necessary with the previous instrument. Besides discrimination of attributes withinjust the A. gambiae species, we also will show efforts to discriminate between different mosquito species. These developments
bring higher relevance to potential risk assessment strategies, and the further knowledge of specific cuticular hydrocarbons andlipids should help researchers develop more effective risk abatement and treatment strategies.

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 26: Lipidomics : Recent New Techniques and Applications

PWe-075
11:10 － 12:20

Liquid extraction surface analysis for local profile of lipid molecularspecies on mammalian tissue slices

Ryo Taguchi1, Akinori Mizuno1, Yoshiki Mizuno1, Miho Goto1, Kazutaka Ikeda2
1

Chubu University, Kasugai, Aichi, Japan, 2Institue for Advanced Biosciences, Keio University,Yamagata, Japan

Keywords:
phospholipid, surface-analysis, direct-extraction, tissues, localization.
Novel aspects:
Sensitive and practical analysis of localized position of tissue slices were effectively obtained by direct liquid extraction from tissue
slice surface.
Abstract:
Liquid extraction surface analysis（LESA）was applied to obtain local profiles of lipid molecular species on individual specific partsof mammalian tissue slices from brain, heart or kidney. Also LESA was applied for some transgenic and disease model animals.
Obtained mass spectometric data were automatically merged qualitatively and quantitatively by new version of our search engine“Lipid Search“（http：//lipidseach.hs.chubu.ac.jp）. Several characteristic differences in profiles of individual phospholipid
molecular species were further analyzed by principal component analysis（PCA）and discrimination analysis.
Method： Frozen mouse or rat tissues such as brain, heart or kidney were cut into slices at 15μm thickness by Leica CM1950
cryostat. Resulting slices were subjected to LESA-NanoMate（Advion）analysis with 4000Qtrap（ABSciex）or Orbitrap（Themo
Fisher）. Chloroform/methanol（ 1 ： 2 ）containing 5 mM ammonium formate was used as extracting and ionization solvent.
Solution volume of 1.6μL was used for each analysis area（ 1 - 2 mm diameter）. MS analysis was operated with flow rate at 200nL/
min and with using the analytical mode such as EMS, precursor ion scanning and neutral loss scanning both in positive andnegative ion mode by 4000Qtrap. Also more precise and high resolution MS data were obtained by Orbitrap.
Result： Detection efficiency by nanoESI by Triversa NanoMate combination with LESA system seems to be very sensitive, becauseof their high recovery by direct surface extraction. By LESA system most of major lipid molecular species were effectively extracted
and detected from specific localized parts at around 1 - 2 mm diameter of several mammalian tissue slice surface. We could
obtained characteristic mass profiles of most of major phospholipid molecular species from several organs, such as brain, kidneyand heart from mouse or rat.
As the results of LESA, it was revealed that molecular species such as 18： 0 /18： 1 of phosphatidylcholine （PC）,
phosphatidylethanolamine（PE）and phoshatidylserine（PS）and are rich in myelin layer of cerebellumn, and reticular formation,and relatively low in hypocampus, cerebral cortex, molecular and granular layer of cerebellum both in mouse and rat brain.
Detection efficiency of LESA with nano-ESI by NanoMate is revealed to be very sensitive. And the sensitivity is almost compatible
to MALDI-TOF, but detectable molecular species were more rich in nano-ESI by LESA than that of MALDI, especially for PE or PS
molecular species. Because of low space resolution efficiency of LESA, the narrow area less than 1 mm diameter such as callosum
was difficult to be analyzed as a specific limited area. Characteristic molecular profiles in localization of several major
phospholipid molecular species from mouse brain obtained by LESA were almost identical to those obtained by laser micro
dissection（LMD）combination with nanoESI-MS. The profiles in molecular species of glycosphingolipids such as gangliosides andsulfatides, and those of triglycerides were also effectively obtained with same extraction condition.
Local characterization of phospholipid molecular species in cortex and medulla from mouse and rat kidney was also effectively
obtained. Contents of arachidonic acid containing PC molecular species were richer in medulla than cortex. Concerning tophospholipids of kidney, content of docosahexsaenoic acid（DHA）containing molecular species were much higher in mouse thanrat.
Conclusion： Liquid extraction surface analysis（LESA）combination with NanoMate is revealed to be a practical and sensitive
method for the localization analysis of lipid molecular species in mammalian tissue slices at 1 - 2 mm resolution. We effectively
obtained 100-200molecular species of individual classes in phospholipids, triglycerides and glycosphongolipids such as gangliosides
and sulfatides. Characteristic features in each different domain in several mammalian tissues were effectively observed.

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 26: Lipidomics : Recent New Techniques and Applications

PWe-076
13:30 － 14:40

Comprehensive Standardized Methods of Lipid ProfilingToshiyuki Yamazaki, Noriko Fueki, Nembai Tomoko, Karasawa Kaoru
AB SCIEX, Tokyo, Japan

Keywords:
Lipidomics, Metabolomics, Q-TOF, Accurate Mass, Library
Novel aspects:
This presentation discribes two comprehensive standardized method of Lipid Profiling.These methods are aplicable forphospholipids and the other lipids.
Abstract:
Novel Aspect
We present comprehensive standardized methods of lipid profiling. These methods are applicable for phospholipids and the otherlipids.
Introduction
Phospholipids are very complex mixtures of closely related compounds. Shotgun lipidomics is a powerful approach for the analysis
of lipids from extracts of biological tissues by direct infusion. On the other hand, a reverse-phase high performance liquid
chromatography method is capable of separating isomers of lysophospholipids differing in the location of the aliphatic chain（sn- 1
orsn- 2 position）and the position（Δ 6 or Δ 9 ）.
AB SCIEX Triple TOFTM 5600 System delivering high-resolution for high-speed chromatography - up to 100spectra in a second or 50
spectra in a cycle while Information Dependent Acquisition（IDA）. In the shotgun lipidomics, the mass selective quadrupole, Q 1
is set to step 1 Da increments across the mass range, selecting lipid precursor ions at unit resolution. Isolated precursor ions aretransmitted through the LINAC (R) collision cell for complete CID fragmentation and all fragment ions are recorded as highresolution TOF spectra. Data processing was done with PeakView(R) Software. And lipid identification and quantitation was donewith LipidView™ Software.
Materials & Methods< Samples for methods development >Egg yolk extracted with Bligh-Dyer procedure using about 10mg of egg yolk and redissolved in IPA.
< MSMSALL（ infusion ）>Continuous-Infusion
Mass Spectrometry： AB SCIEX Triple TOFTM 5600 System
Accumulation time： TOFMS 1 or 10sec,Product Ion Scan 0.1 or 1 sec
Mass range： m/z 200 to 950 Da
Acquisition time： 1.3min or 13min
Collision Energy： 40V
Collision Energy Spread： 20V< Liquid Chromatography（MS screening and MS/MS Library Search）>
HPLC Column： ACQUITY UPLC HSS T 3 （2.1×50mm）
Solvent A： 5 mM ammonium formate in H2O, B： 5 mM ammonium formate in H2O IPA
Mass Spectrometry： AB SCIEX Triple TOFTM 5600 System
TOFMS 0.15sec, Product Ion Scan 0.05sec（20 times / cycle）
Mass range： TOFMS： m/z 200&#65374；950 Da, Product Ion ： m/z 150&#65374；950 Da
Acquisition time： 25 min
Collision Energy 40V, Collision Energy Spread20V
Preliminary Data
< MSMSALL（ infusion ）>MS/MS spectra were acquired from 200 to 950m/z accumulated for 100 ms each and the total cycle time for the experiment were
1.3 min per polarity. For minor component, 1 second accumulation time was used instead. MS/MS ALL workflow had shown thategg yolk has a combination of 7 major different acyl ions those were also detected providing molecular species quantification ina peak area response. Peaks of the acyl anion of FA 18： 0 （m/z 283.2643 ）and the product（ m/z 283.2431 ）of CO2 loss from theacyl anion of FA 22： 6 were distinguished with accurate mass in egg yolk.
< Liquid Chromatography >Isomeric lysoPCs were distinguished based on retention time and the peak intensity ratio of product ions, and 7 pairs of lysoPC
isomers were identified in egg yolk. XIC Manager, a function of PeakView(R)   Software, can be used for targeted and non-targeted
processing of high resolution MS and MS/MS data allowing for screening and identification with the highest confidence based on
retention time, accurate mass molecular ion, isotopic pattern, and automatic MS/MS library searching. We are going to establish
automatic identification and quantitative processing workflow using XIC Manager.

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 26: Lipidomics : Recent New Techniques and Applications

PWe-077
11:10 － 12:20

The utility of acetonitrile-related adducts formed in APCI sources for
localization of double bonds in lipids

Josef Cvacka1, Vladimir Vrkoslav1, Eva Hakova2, Petra Horka1,2Institute of Organic Chemistry and Biochemistry AS CR, v.v.i., Prague, Czech Republic, 2Charles University in Prague,
Prague, Czech Republic

1

Keywords:
lipids, structure elucidation, double bonds, HPLC/MS, gas phase reaction
Novel aspects:
Broadening the application area of recently discovered method for localization of double bonds for new lipid classes. Investigationof the gas-phase reaction mechanism.
Abstract:
Most lipids contain long chains derived from fatty acids, alcohols, or other long-chain compounds. The presence of double bonds,
their number, relative position and cis-/trans-isomerism strongly affects physical, chemical and biochemical properties of lipids,
which has important consequences on their functions in living organisms. Localization of double bonds in unsaturated long-chain
compounds is a classical analytical problem, especially challenging when the analytes are present in complex mixtures and low
quantities. There are numerous methods allowing for determination of double bond positions using mass spectrometry, but they
are mostly based on electron ionization（El）. The applications of EI are considerably limited for sufficiently volatile and thermallystable compounds. Most lipids are not directly amenable to GC/EI-MS and must by hydrolyzed prior to their analysis； animportant piece of information on their structures is lost. Therefore, methods making localization of double bonds possible in the
intact lipids are highly desirable.
We have recently shown that gas phase reactions of acetonitrile taking place in APCI sources can be utilized for localization of
double bonds in unsaturated esters. Wax esters and methyl esters provided［M+C 3 H 5 N］+. adducts. When fragmented by CID,
pairs of highly abundant ions allowing for unambiguous localization of double bonds were generated. In this work we investigatedunsaturated lipids from various classes to learn if they undergo similar type of reactions. Triacylglycerols with diverse FA chainswere prepared by a randomization reaction optimized for a small scale. It was found that all investigated unsaturated lipids
including long-chain compounds with various functional groups, diol diesters, acylglycerols and other lipids formed the above
mentioned adducts and provided expected diagnostic fragments suitable for localization of double bonds. Therefore, the method
can be generally used for the lipids amenable to APCI. As the reactant（acetonitrile）is a common solvent in HPLC, double bonds
can be conveniently localized in the molecular species separated by liquid chromatography, typically in non-aqueous reversed
phase systems. The influence of the arrangement of double bonds in polyunsaturated chains on the adduct formation and theappearance of the MS/MS spectra was studied using FAMEs with isolated, methylene-interrupted, conjugated and cumulated
double bonds. The mechanisms behind formation of the［M+C 3 H 5 N］+. adducts was investigated using stable isotope labeledacetonitriles and higher nitriles（propionitrile, butyronitrile）.
Financial support from the Czech Science Foundation GACR (Project No. 203/09/0139) and the Academy of Sciences of the
Czech Republic (Project RVO: 61388963) is acknowledged.

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 26: Lipidomics : Recent New Techniques and Applications

PWe-078
13:30 － 14:40

New mass spectrometric methods for structural characterization ofhydrocarbons

Vladimir Vrkoslav, Josef Cvacka

Institute of Organic Chemistry and Biochemistry AS CR, v.v.i., Prague, Czech Republic

Keywords:
Hydrocarbons, structural characterization, double bond position, branching position
Novel aspects:
New APCI and MALDI mass spectrometric methods for determination of double bond（s）and branching position（s）inhydrocarbons based on fragmentation of the molecular adduct.
Abstract:
Gas chromatography/electron ionization mass spectrometry（GC/EI-MS）is the most often used method for analysis ofhydrocarbons. EI spectra do not provide important information about position of double bonds. Characterization of branchingpositions is some times unclear. Double bond of hydrocarbons could be localized from fragments of acetonitrile related adductafter chemical ionization（CI）, or from fragments of EI ion source after suitable derivatization. However, derivatization is time
consuming. Moreover, themolecular weight of detected compounds is limited by the functional range of the method, i.e. thetechnique is limited to hydrocarbons with up to about 50 carbon atoms. Unsaturated hydrocarbons with even less carbons are
rapidly decomposed or rearranged due to high temperatures in the GC injector and/or column. Obviously, principally different
analytical methods are required to broaden the range of molecular weights and thus to allow analysis of hydrocarbons withconsiderably longer aliphatic chains. The main goal of this project is to develop new methods for structural characterization of
long-chain aliphatic hydrocarbons. The methods are based on MS/MS of molecular adducts obtained by MALDI and APCI
technique.
It is known that matrices containing lithium or silver cations work the best for MALDI of hydrocarbons. Hydrocarbons are detected
as lithium or silver molecular adducts（［M+Li］+,［M+Ag］+）. MALDI-MS allows determination of molecular weights, which is quite
important, but there are no structure-related fragments in the MS spectra. MS/MS spectra of long-chain hydrocarbons givefragments, which can by use for identification of position of branching point（s）of alkyl chain. We use LIFT technique（Ultraflextreme, Bruker, Bremen, Germany）to obtain MS/MS spectra of［M+Ag］+ and［M+Li］+ of various hydrocarbons like
Apolane-87 （22,24-diethyl-19,29-dioctadecylheptatetracontane） or squalene （2,6,10,15,19,23-Hexamethyl-2,6,10,14,18,22tetracosahexaene）. The technique provided fragments, which localized positions of branching.
The presence of double bonds and their number, position and cis-/trans- isomerism strongly affects physico-chemical properties
of hydrocarbons and their functions in biological systems. Acetonitrile-related adducts［M+55］+. formed in the APCI source for
localization of double bond position（s）in wax esters were described previously.［M+55］+. adducts in APCI ion source were also
observed, when unsaturated hydrocarbons were mixed with acetonitrile. Molecular composition of adduct was determined byaccurate mass measurement as［M+C 3 H 5 N］+. . The CID MS/MS of［M+C 3 H 5 N］+. yielded fragments allowing the localization of
double bond（s）in the hydrocarbon chain. Fragmentation corresponds to cleavage next to double bond.

Financial support from the Czech Science Foundation (Project No. P206/12/1093) and the Academy of Sciences of the Czech
Republic (RVO:61388963 ) is herewith acknowledged with appreciation.

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 26: Lipidomics : Recent New Techniques and Applications

PWe-079
11:10 － 12:20

Comparison of triacylglycerols in commercial DHA supplements utilizing
MALDI-TOF-TOF tandem MS with spiral ion trajectory

Ayumi Kubo, Yoshiyuki Itoh, Masahiro Hashimoto, Jun Tamura
JEOL Ltd., Tokyo, Japan

Keywords:
high energy CID, high precursor ion selectivity, MS/MS, MALDI,Triacylglycerol
Novel aspects:
Structural analysis of triacylglycerols in comercial DHA supplement using MALDI-TOF-TOF tandem MS without chromatographic
separation and derivatization.
Abstract:
Introduction
In recent years, docosahexaenoic acid（DHA）supplements are frequently promoted as functional foods because DHA is believed
to be helpful to people with heart disease, for premature infants, and to support healthy brain development. Main constituent ofmany of the DHA supplements is fish oil, or triacylglycerol derived from fish. It has been recognized that biological activities of
triacylglycerols depend not only on their overall fatty acids composition but also molecular species composition and positionspecific（or“regiospecific“）distribution of fatty acids. Such molecular and regiospecific analysis, however, has not been
performed very often because of the tedious analytical procedures required. We analyzed triacylglycerols of DHA supplements
using MALDI-TOF-TOF tandem MS without chromatographic separation and derivatization.
Methods
All of the DHA supplements were purchased at the local drugstore. They were almost in the form of soft gelatin capsule. The
contents of the capsules were dissolved into tetrahydrofuran（THF）at the concentration of 10 mg/mL. The 2,5-dihydroxybenzoic
acid DHB matrix solution was prepared at the concentration of 20 mg/mL in THF. The cationization agent solution was preparedby dissolving NaTFA at the concentration of 10 mg/mL in THF. For final sample preparation, one of the analyte solutions, matrix
solution, and cationzed agent solution were mixed at 1 ： 1 ： 2 volume ratio and 1
μL of the mixture was applied onto thestainless steel target plate. A JMS-S3000“SpiralTOF“ MALDI-TOF-TOF tandem MS（JEOL Ltd., Tokyo, Japan）was employed for all
the analyses.

Preliminary Data
On the mass spectra of all the supplements analyzed, peaks that can be attributed to sodium ion adduct of triacylglycerol
molecules containing polyunsaturated fatty acids（PUFA）, such as DHA and eicosapentaenoic acid（EPA）, are observed in mass
range over 900. The observed mass resolution of those peaks was between 50000 and 60000 at FWHM. The difference between
observed m/z value and theoretical m/z value for each of the triacylglycerol peak was less than 10 ppm by using sodiated poly
（propylene glycol）as the external mass calibration standard. By comparing mass spectra from various DHA supplements, it was
found that triacylglycerols contained in the supplements vary significantly. In addition, the chemical structures of several
triacylglycerols were investigated by acquiring the product ion mass spectra of monoisotopicaly selected precursor ions. As aresult, we found triacylglycerols containing DHA with the double bonds at w-3,6,9,12,15,18 positions. We also confirmed thepresence of several regioisomers of triacylglycerols.

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 26: Lipidomics : Recent New Techniques and Applications

PWe-080
13:30 － 14:40

Analysis of oxidized lipid derived volatiles by Gas Chromatography/Mass
Spectrometry

Shoji KAKUTA1, Yasuhiko BANDO2, Eiichiro FUKUSAKI1, Takeshi BAMBA1
1Osaka University, Osaka, Japan, 2AMR Inc., Tokyo, Japan

Keywords:
aldehyde, lipid peroxidation, In-tube extraction
Novel aspects:
Exhaustive volatile compounds analysis allowed us to understand oxidized lipid production mechanism and might lead to be able
to analyze diseases by malti-marker profiling.
Abstract:
Lipid, which is main component of cell membrane, might provide harmful peroxidation derivatives in the particular condition underoxidative stress. During the oxidative reaction, polyunsaturated fatty acid motifs are oxidized at a double bond carbon to produce
oxidized lipids. Volatile compounds including short-chain aldehydes are also released from oxidized lipids. However, theproduction of many kinds of volatile compounds depends on various carbon chain or different unsaturated degree fatty acids andthe production of these compounds is very trace. In addition, it is known that these compounds react with biological metabolites
and generate different compounds. Therefore, an advanced technique that enables exhaustive analysis of these compounds isrequired. In a previous study, it was reported that specific odors were released from individual diseases and these odors weregenerated differently in each diseases. Although there have been reports on the analysis of particular short chain aldehydes, an
exhaustive analysis for short chain aldehydes in blood or plasma samples has not been reported. In this study, Head-Space Solid
Phase Micro Extraction（HS-SPME）was selected as a method to extract volatile compounds. However, it was difficult to perform
detailed analysis due to the presence of some foreign substances in biological samples thus negatively affects the sensitivity.
Therefore, we attempt to develop the In-tube extraction（ITEX）method and gas chromatography/mass spectrometry to analyze
various volatile compounds produced by oxidizing lipids.
It is possible that there are many unreported volatile compounds exist in samples in addition to aldehydes. At first, oxidized
polyunsaturated lipid samples in vitro were prepared to construct the analytical system for volatile compounds derived fromoxidized lipids. We oxidized phosphatidylcholine（PC）samples by oxidant, 2,2’ - azobis（ 2 -aminopropane）dihydrochloride（AAPH）. As a result, aldehydes or hydroxyl compounds as well as cyclic volatile compounds； 2 -pentylfuran, and compoundswith a number of double bonds； 2,4-alkanedienal, were identified from these oxidized lipid samples. Furthermore, we applied themethod using mouse plasma as a biological sample. Aldehyde and hydroxyl compounds were identified in mouse plasma. In
addition, 2 -pentylfuran was also identified from these samples that are similar with previous volatile oxidized lipid samples.
For the future, we will continue to analyze various volatile oxidized lipid samples to construct volatile compounds library for real
biological sample analysis and apply the method for analysis of biological samples.

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 26: Lipidomics : Recent New Techniques and Applications

PWe-081
11:10 － 12:20

Structural analysis of triacylglycerols using desorption electrosprayionization mass spectrometry with precursor ion scanning and neutral loss
scanning

Mayuko Morita1,2, Toshie Takahashi3, Ryo Taguchi2,41
Kyoto Prefectural University of Medicine, Kyoto, Japan, 2Department of Metabolome, Graduate School of Medicine,
The University of Tokyo, Tokyo, Japan, 3Department of Biochemistry and Molecular Biology, Graduate School of
Medicine, The University of Tokyo, Tokyo, Japan, 4Department of Biomedical Sciences,Colledge of Life and Health
Sciences,Chubu University, Kasugai, Japan

Keywords:
Desorption electrospray ionization, triple quadrupole mass spectrometer, triacylglycerol, lipid
Novel aspects:
DESI, coupled with the triple quadrupole mass spectrometer, can provide detailed structural information and it is an effective
technique for the structural analysis for lipid.
Abstract:
Desorption electrospray ionization（DESI）is an ionization method that enables the direct and rapid analysis of biological samples
including whole organs. This ionization technique has been applied to lipidomics in combination with some mass spectrometers,
especially ion trap mass spectrometers. Nevertheless, few reports have described DESI-MS analysis using a triple quadrupole mass
spectrometer. This spectrometer, a powerful tool for structural analysis, can perform three kinds of MS/MS experiments, production scanning, precursor ion scanning, and neutral loss scanning.
This work demonstrates the availability of DESI-MS for structural analysis of lipid on surfaces using triple quadrupole mass
spectrometer without extraction. This analysis was also extended to the direct analysis of biological samples.
Commercially available edible meat, beef, pork, and salmon were used as samples directly without preparation. Samples wereanalyzed using positive or negative ionization mode. Product ion scanning was performed to confirm the structure. Neutral loss
scanning and precursor ion scanning were also performed for structural analysis. Neutral loss scanning of 273, 271, 301, 299, and 297
Da were used. To compare lipid profiles analyzed for ESI and DESI, the extracted lipids were analyzed using flow injection. Lipids
were extracted from meat according to solid-phase extraction and Bligh & Dyer method.
In this system, fatty acids（negative mode）, monoacylglycerol, diacylglycerol, and phosphatidylcholine（positive mode）were
detected. MG and DG ions were detected in higher sensitivity than ESI-MS was. These molecules were ionized more easily than
TG in this ionization method. Moreover, the intensity of TG molecular species consisted of numerous double bonds. Long acyl
chains were few in this system. These results suggest that the efficiency of ionization depended on the lipid class. The abundantfatty acids of beef were saturated fatty acid, palmitic acid, and stearic acid. Those of pork were mono-unsaturated fatty acids,
palmitoleic acid, and oleic acid. The results agree with those of previous reports. Product ion scanning and precursor ion scanninggave detailed information related to each TG structure. The TGs with a specific fatty acids were detected with neutral loss
scanning in positive mode. These results show the specific TG profiles for each edible meat. However, in this condition,
polyunsaturated and TG-containing polyunsaturated fatty acids, EPA and DHA, were not detected clearly. These results support
that the conditions for neutral loss scanning in DESI-MS analysis depended on the chemical structures such as the number ofcarbon and/or double bonds in the molecule. It is necessary for quantitative analysis and profiling analysis to optimize thedetailed analytical conditions for fatty-acid-containing long acyl chains.
DESI, coupled with the triple quadrupole mass spectrometer, can provide detailed structural information. It is an effective
technique for the structural analysis of lipid. These results show that DESI-MS is a useful approach for the lipid analysis on surface
providing detailed structural information. Moreover this simple and quick analysis system is expected to be valuable for the direct
structural analysis of lipids.

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 26: Lipidomics : Recent New Techniques and Applications

PWe-082
13:30 － 14:40

Multidimensional lipidomics for untargeted and targeted analyses

Giuseppe Astarita2, Marc V Gorenstein2, Andrew Baker2, Mark A Ritchie1, John P Shockcor2
1

Waters Pacific, Singapore, 2Waters, Milford, MA, USA

Keywords:
Lipids, HILIC UPLC, IMS ToF, MSE
Novel aspects:
Class based separation of lipids provides a diagnostic separation which is resolved and defined using IMS-ToF, allowing highly
complex samples to be screened with high descrimination
Abstract:
One of the main challenges for a global lipid analysis lipidomics is the separation of the wide array of lipid species present inbiological samples. The ability to perform multi-dimensional separations in one injection prior to MS analysis could improve ourability to map and measure complex lipid mixtures. Here, we present a complete workflow for the extraction, separation,
identification and quantification of lipid species in complex matrices.
We first used an off-line enrichment system for the extraction of lipid species from biological samples. Next, we analyzed lipid
extracts using Ultra Performance LC（UPLC）-MS giving class distinct separation of the lipids in a 10 minute gradient run. An ion
mobility-TOF system was used to discriminate lipids classes in their constituents based on the different cross collision sections.
Lipid ions with different degree of unsaturation and acyl length migrate with characteristic mobility times due to their uniqueinteractions with the nitrogen gas in the ion mobility cell. Using this novel technological approach, we applied a peak detection
algorithm to generate molecular maps of lipids present in various animal tissues and serum, which could facilitate comparative
lipidomic analysis. To gain more structural information, lipids were analyzed using parallel low and elevated collision energy（MSE）
to acquire both precursor and product ion information in a single chromatographic run, increasing the specificity of identification.
The database of mass transitions was used to program tandem quadrupole MRM method and automated processing methods forthe high throughput quantification of selected lipid species in biological samples.
In conclusion, the combination of UPLC with ion mobility-MSE delivers a multi-dimensional characterization of complex biological
mixtures, enhancing the lipidomic profiling and quantification.

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 26: Lipidomics : Recent New Techniques and Applications

PWe-083
11:10 － 12:20

Development of comprehensive identification and quantification ofpolyunsaturated fatty acids and eicosanoids in human plasma using LCMS/MS

Fumiyuki Nakagawa1, Katsutaro Morino2, Atsushi Ishikado2, Chiho Ikeda1, Yoko Hoshiya1,
Hiroshi Maekawa2
1

JCL Bioassay Corporation, Osaka, Japan, 2Shiga University of Medical Science, Shiga, Japan

Keywords:
Eicosanoids, PUFAs, Human plasma, Comprehensive identification and quantification, LC-MS/MS
Novel aspects:
Development of selectivity and sensitivity 3 several comprehensive quantification method（Free fatty acids, total fatty acids,
about 100 of eicosanoids）using LC-MS/MS to be able to apply in clinical study.
Abstract:
Polyunsaturated fatty acids（PUFAs）that are taken by human, these PUFAs are form to membrane phospholipids to composition
cell membranes. These membrane phospholipids are hydrolyzed by phospholipase A2（PLA2）then break down phospholipids into
fatty acids and cyclooxygenase（COX）or lipoxygenase（LOX）catalyzes converse the fatty acids to eicosanoids. Eicosanoids are
considering to be signaling compound act as autocrine or paracrine. Recently strong relationship with eicosanoids and various
diseases, such as arteriosclerosis, rheumatism and hyperalgesia are demonstrated by in vitro or using several model mice andhave revealed the mechanism of the diseases gradually in numerous studies. These researches about eicosanoids and relationshipwith various diseases are increase at an accelerated rate. However, there are few reports about eicosanoids analysis in clinicalstudies. These reasons are the body tissue possible to taken from alive human are confined like blood, urine or saliva. Especially,
the blood is one of the tissue of accurately reflect the physical health. However the blood is complex matrix and concentration of
the eicosanoids in blood is extremely low（most of the concentrations of eicosanoids are about pg/mL or fg/mL level）, therefore
high selectivity and sensitivity method is required. Furthermore, after blood is taken, PUFAs are metabolizing to eicosanoids ex
vivo because of the COX in platelet. Therefore, it’s difficult to measure the eicosanoids concentration stably in clinical study.
Therefore we validated the blood collection protocol from human and also developed the 3 several comprehensive quantification
method using liquid chromatography-tandem mass spectrometry（LC-MS/MS）to be able to apply in clinical study. First
methodology is quantitative total fatty acids（Arachidonic acid, Eicosapentaenoic acid, Docosahexaenoic acid）, second is freefatty acid and third is about 100 compounds of eicosanoids were targeted. Total fatty acids, free fatty acids were extracted from
20 uL of human plasma by liquid-liquid extraction respectively. And about 100 compounds of eicosanoids were extracted from 1
mL of human plasma by solid phase extraction. The extracted solution was injected to LC-MS/MS and all compounds were
performed in negative ion electrospray ionization scheduled SRM mode. For the calibration curve to quantitate the concentrationin plasma sample, we used PBS for the surrogate matrix. Finally, we developed the robust method for comprehensive identificationand quantification of polyunsaturated fatty acids and eicosanoids and we figure out the baseline level from the data of healthy
human subjects. Our developed method will be characterized the subjects andefficient platform in stage at evaluation of drug
efficacy in clinical study.

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 26: Lipidomics : Recent New Techniques and Applications

PWe-084
13:30 － 14:40

Chiral HPLC/MS Analysis of Triacyglycerol Enantiomers and Regioisomers

Miroslav Lisa, Michal Holcapek

Univerzity of Pardubice, Pardubice, Czech Republic

Keywords:
Lipidomics, HPLC/MS, chiral HPLC, triacylglycerol, enantiomer
Novel aspects:
Analysis of wide range of TG enantiomers and regioisomers in one analysis using chiral HPLC/MS method.
Abstract:
Triacylglycerols（TGs）are important components of human diet used as a source of energy, fatty acids（including essentialones）, fat soluble vitamins and other nonpolar compounds. The variety of TG species is enormous due to a high number of fatty
acids present in natural samples differing in acyl chain lengths, their position on the glycerol skeleton（region isomers,
enantiomers）and number, position（s）and cis-/trans- configuration of double bonds（DBs）, all having great differences in their
biological and nutritional properties. The detailed characterization of fatty acid composition including the determination ofindividual isomers is important from the nutrition point of view due to their different biological activity. The stereospecific analysis
of individual TGs represents a challenging task in the lipidomics due to different bioavailability of fatty acids on the glyceroleskeleton in the stereospecific environment of enzymes in the human body. We have optimized a chiral HPLC/MS analysis for the
separation of TG isomers using cellulose-based chiral HPLC column in the normal-phase mode. Under optimized chromatographic
conditions, both regioisomers and enantiomers are separated in one analysis. The retention behavior of TGs is governed mainlyby the number of DBs, i.e., retention times of TGs increase with increasing number of DBs. At least partial separation of TGs basedon acyl chain lengths or position and configuration of DBs is also achieved using optimized chiral HPLC method. TGs are identified
based on their positive-ion atmospheric pressure chemical ionization（APCI）mass spectra providing protonated molecules andhigh relative abundance of fragment ions formed by the neutral loss of fatty acid from the glycerol skeleton. The comparison ofrelative abundances of these fragment ions is used for the differentiation of individual regioisomers, because the loss of fatty acidfrom sn- 2 position is less preferred, which results in a fragment ion with lower relative abundance in comparison to sn- 1 andsn- 3 positions. No visible differences are observed in APCI mass spectra of enantiomers identified based on retention times ofidentical standards. We have developed a method for the synthesis of TG enantiomers with wide range of fatty acids commonly
present in natural samples（i.e., palmitic, stearic, oleic, linoleic, linolenic and arachidic acids）. The retention behavior of TGs
isomers is also studied using mixtures of TG isomers prepared by the randomization reaction of selected monoacyl TG standards
providing mixtures of all isomers in an equal amount. We have demonstrated the utilization of developed chiral HPLC/MS method
for the separation of complex natural TG mixtures of plant oils or human plasma samples.
This work was supported by the project 203/09/0139 sponsored by the Czech Science Foundation.

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 26: Lipidomics : Recent New Techniques and Applications

PWe-085
11:10 － 12:20

Structural analysis of oxidized triacylglycerols by using MALDI-SpiralTOFTOF tandem mass spectrometer with high precursor ion selectivity andhigh-energy CID

Akihiko Kusai2, Ayumi Kubo1, Yoshiyuki Itoh1, Masahiro Hashimoto1, Yoshihisa Ueda1
1

JEOL Ltd., Tokyo, Japan, 2JEOL (Europe) SAS, CROISSY-SUR-SEINE, France

Keywords:
MALDI, MS/MS, High-energy CID, High precursor ion selectivity, Oxidized triacylglycerols
Novel aspects:
Direct structural analysis of thermally oxidized triacylglycerols by using the MALDI-TOF-TOF tandem mass spectrometer
Abstract:
Triacylglycerols are lipids that consist of a glycerol moiety with each hydroxyl group esterified to a fatty acid. Almost all fats andoils produced by animals and plants contain these compounds as a main ingredient. Additionally, it is well known that
triacylglycerol, which has an unsaturated fatty acid moiety, is oxidized by air over time. Therefore, it is important to know thekinds of fatty acids and their position on the glycerol molecule in order for complete structural analysis of a particular oxidized/
non-oxidized triacylglycerol. A tandem mass spectrometer with high-energy collision induced dissociation（HE-CID）provides asuitable method for obtaining structural information of triacylglycerol such as the positions of double-bonds, branching, andhydroxylation by means of charge-remote fragmentation.［ 1 ］The MALDI-TOF/TOF tandem mass spectrometer（MS）used in thiswork combines a SpiralTOF for the first TOFMS with an offset parabolic reflectron for the second TOFMS.［ 2 ］This TOF/TOF
configuration offers several good performance characteristics for structural analysis of triacylglycerols in that it canmonoisotopically select precursor ions, eliminate post source decay（PSD）ions and perform HE-CID at 20keV.［ 3 ］
In this work, we report the structural analysis of thermally oxidized triacylglycerols by HE-CID using the MALDI-SpiralTOF-TOF
tandem MS.
As a starting point, triolein was used as a standard sample. Two triolein samples one as a pure sample, the other mixed with theantioxidant tocopherol（vitamin E）were oxidized in an oven set to 160 degrees Celsius in order to accelerate the oxidation
process. Afterwards, each sample was analyzed by MALDI-SpiralTOF-TOF tandem MS.
Accuracy of all observed ions was within 5 ppm with external calibration. The results showed that the presence of the antioxidant
was very effective in minimizing the oxidation of triolein. Additionally, the oxidized sample measured by HE-CID produced MS/MS
spectra that contained enough information to determine the position of not only oxidization but also the double-bonds in the fatty
acid moiety.
Next, commercial fats and oils like olive oil and margarine were also put into the oven at 160 degrees Celsius. These samples were
measured at various times during the heating process to observe the changes in the oxidization by MALDI-SpiralTOF MS. Similarly,
the structure of each oxidized triacylglycerol was determined using MALDI-SpiralTOF-TOF tandem MS.

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 26: Lipidomics : Recent New Techniques and Applications

PWe-086
13:30 － 14:40

Development of a high-throughput lipid profiling method by using aquadrupole orbitrap mass spectrometer and an automated lipid
identification software

Takayuki Yamada1, Takato Uchikata1, Shigeru Sakamoto2, Yasuto Yokoi3, Eiichiro Fukusaki1,
Takeshi Bamba1

Dept. Biotech., Grad. Sch. Eng., Osaka Univ., Suita, JAPAN, 2Thermo Fisher Scientific, Yokohama, JAPAN, 3Mitsui
Knowledge Industry, Tokyo, JAPAN1

Keywords:
lipidomics, high-throughput profiling, supercritical fluid chromatography, quadrupole orbitrap mass spectrometer, automatedidentification system for lipidome
Novel aspects:
We developed a high-throughput workflow for exhaustive lipid profiling by using Q Exactive and Lipid Search.
Abstract:
Lipids are the major components of biological membranes and energy storage substances, and they might function as signaltransducers in some occasion. To prove the universality of the important contribution of lipids related compounds, thecomprehensive analysis of lipid metabolism would be essential. Recently, the study of lipidomics, i.e., metabolomics for lipids, hasbeen encouraged. There are a large number of lipid molecular species because of the diversity of polar head groups andhydrophobic fatty acid chains. For example, phospholipids have a hydrophilic head and one or two fatty acid chains, andtriacylglycerols have three fatty acid chains. Thus, there are a variety of chemical structures and polarities in lipid molecularspecies. In mass spectrometry, certain molecular species show high sensitivity in the positive-ion mode, whereas others show highsensitivity in the negative-ion mode. Therefore, it is difficult to simultaneously analyze diverse lipid molecular species showing highsensitivity either in the positive-ion mode or in the negative-ion mode. In data processing, considerable time and effort is spent inmanually identifying and listing all the molecular species in a biological sample. This is because it is necessary to confirm themultiple fragment ions of the polar head group and the fatty acid chains in order to identify the chemical structures of lipid
molecular species.
In order to solve these problems, we developed a useful workflow using a quadrupole orbitrap mass spectrometer, which canperform high-resolution full scanning and product ion scanning in both the positive-ion mode and the negative-ion mode inpractical cycle time, and an automated lipid identification system. The target lipids in this study are phospholipids, triacylglycerols,
and free fatty acids. Supercritical fluid chromatography/mass spectrometry（SFC/MS）analysis was performed using the Analytical
SFC Method Station（Waters）with the Q Exactive mass spectrometer（Thermo Scientific）. Full MS/dd-MS 2 （data-dependent
MS 2 ）with the inclusion list, i.e., the parent mass list of the target lipids, was used. The Top 5 method was adopted, i.e., MS 2
was performed for 5 lipid ions in the inclusion list, starting with the ion with the highest intensity. Lipids were identified by the
product search mode in Lipid Search（Mitsui Knowledge Industry）, which is based on the accurate value of the parent mass andthe spectral pattern of MS2.
We investigated the SFC and MS conditions using a standard mixture of lipids. First, we optimized the MS detection conditions
such as the sheath gas flow rate, spray voltage, and heater temperature（temperature of the vaporizer）. Next, we optimized theMS 2 collision energy. Then, we investigated the separation conditions for lipids such as column type and gradient program.
Further, we optimized the identification conditions of Lipid Search. After the analytical method was developed, we analyzed thelipids present in mouse plasma. More than 200 lipid molecular species were detected and identified from each sample.
In conclusion, we demonstrated that the combination of the data-dependent MS 2 scan triggered by the positive or negative
parent mass list and Lipid Search software enables the efficient and feasible identification of lipids.

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 26: Lipidomics : Recent New Techniques and Applications

PWe-087
11:10 － 12:20

Simultaneous profiling of polar lipids by supercritical fluid
chromatography/tandem mass spectrometry with methylation

Jaewon Lee1, Shin Nishiumi2, Masaru Yoshida2, Eiichiro Fukusaki1, Takeshi Bamba1
1Osaka University, Osaka, Japan, 2Kobe University, Kobe, Japan

Keywords:
Methylation, Polar lipid, Supercritical fluid chromatography, Triple-quadrupole mass spectrometry
Novel aspects:
Advancement of peak tailing and detection sensitivity of polar lipids by applying TMSD methylation to analytical method based on
SFC/MS.
Abstract:
In lipidomics, the simultaneous analysis of diverse lipids with various structures and polarities is too difficult with regard to theirseparation, detection, and identification. In the previous paper, SFC/MS was applied successfully to analyze 14 types of lipids.
However, the analysis of polar lipids was not sufficient due to the peak tailing, low resolution, and low detection sensitivity. Next,
trimethylsilyl（TMS）derivatization was applied to advance the peak shape and detection sensitivity of polar lipids. However,
several polar lipids such as phosphatidylserine（PS）and lysophosphatidylserine（LPS）were not advanced. Finally, we attempt touse the methylation by TMS-diazomethane（TMSD）and to construct the analytical system for the simultaneous analysis of various
and low-abundance polar lipids in a biological sample. Several lipid standards that consist of 6 phospholipids, 6lysophospholipids, and 7 sphingolipids, dissolved in methanol were applied to simultaneous polar lipid profiling with TMSD
methylation. 2 mol/L TMSD in hexane was added to the standard lipids and reacted at 50 o C for 10 min. The reaction was
quenched with glacial acetic acid. The methylated lipids were subjected to SFC-QqQ/MS analysis that consists of SFC Method
Station and Xevo TQ（Waters, Milford, MA, USA）. In the analysis of biological sample, the TMSD methylation was applied to thelipid extracts with the same method. Simultaneous profiling of 19 polar lipids that consist of phosphatidylcholine（PC）,
phosphatidylethanolamine（PE）, phosphatidylserine（PS）, phosphatidylglycerol（PG）, phosphatidylinositol（PI）, phosphatidic
acid （PA）, lysophosphatidylcholine （LPC）, lysophosphatidylethanolamine （LPE）, lysophosphatidylserine （LPS）,
lysophosphatidylglycerol（LPG）, lysophosphatidylinositol（LPI）,lysophosphatidic acid（LPA）, sphingomyeline（SM）, ceramide（Cer）,
ceramide- 1 -phosphate（Cer 1 P）, sphingosine（So）, sphingosine- 1 -phosphate（So 1 P）, sphinganine（Sa）, and sphinganine- 1 phosphate（Sa 1 P）was carried out by using SFC/MS with TMSD methyltation. At first, MS/MS method for detecting 16 kinds ofmethylated polar lipids and 3 kinds of non-methylated polar lipids was optimized by performing the product ion scan to find theprecursor ion and fragment ion of each polar lipid. The values of cone voltage and collision energy were also optimized. For thebest efficiency of TMSD methylation, the reaction time and temperature were optimized as the reaction at 50 oC for 10 min. By thetest of repeatability, RSD % of TMSD methylation was under 8 % . And several columns were investigated for finding the bestcolumn for methylated polar lipid profiling. Finally, we successfully construct an effective polar lipid profiling system based on
SFC/MS with Inertsil ODS- 4 column by applying TMSD methylation. We also optimized the analysis of non-methylated polar lipids
for the comparison between methylated and non-methylated polar lipid profiling. As a result, by applying TMSD methylation, theimprovement of peak tailings was resulted in the analysis of 9 polar lipids that consist of PS, PI, PA, LPS,LPI, LPA, C 1 P, So 1 P and
Sa 1 P. Furthermore, the LODs of PS, PG, PA, LPS, LPG, LPI, LPA, Cer 1 P, So 1 P, and Sa 1 P were enhanced 7.5-, 5 -, 26.7-, 600-, 6.7-,
116.7-, 500-, 75-, 3000-, and 4500-fold, respectively. Validation study of the developed method represented high reproducibility andhigh coefficient of correlation. Simultaneous profiling of diverse polar lipids in a biological sample was also performed by thismethod. This result indicated that the application of TMSD methylation is effective for the analysis of polar lipids by SFC/MS. Thisdeveloped method is applicable to lipidomics study as a powerful tool, especially targeting polar lipids.

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 26: Lipidomics : Recent New Techniques and Applications

PWe-088
13:30 － 14:40

A screen using DART-MS to identify novel components of a pheromone
synthesis pathway in Drosophila melanogasterYin Ning Chiang1, Joanne Y Yew1,21National University of Singapore, Singapore, Singapore, 2Temasek Life Sciences Laboratory, Singapore, Singapore

Keywords:
DART-MS, lipids, pheromones, insects, hydrocarbons
Novel aspects:
DART-MS is used for the detection and semi-quantification of pheromones and other lipids from live insects.
Abstract:
Pheromones are molecules produced by an animal that can elicit complex social behaviours（eg, aggression and courtship）from
other individuals of the same species. Understanding the biochemical synthesis of pheromones and the role they play in behaviour
is useful for elucidating conserved lipid metabolism pathways and may provide novel, species-specific methods for controlling
reproduction of pests and disease-bearing insects. The fruit fly, Drosophila melanogaster, is a well-studied model organism forwhich many genetic tools are available. Pheromones of D.melanogaster are expressed on the cuticle and have been wellcharacterized. Analyses of insect cuticular hydrocarbons（CHCs）have conventionally been carried out with gas chromatography
coupled with mass spectrometry（GC-MS）. However, sample preparation for GC-MS is time consuming and involves placing
whole insects in organic solvent, a process that is lethal to the insects and provides no spatial information. Here, we use a form ofambient mass spectrometry, Direct Analysis in Real Time mass spectrometry（DART-MS）. This method requires little sample
preparation and allows rapid analysis of CHCs directly from whole flies. We combined DART-MS with genetic manipulation inorder to identify components of the pheromone synthesis and transport pathway in D. melanogaster. Specifically, we areinterested in genetic mutations that result in aberrant CHC profiles.
A library of 130 different transgenic fly lines was generated from 65 candidate genes. In each of these lines, the expression ofindividual lipid metabolism-related genes was genetically knocked down using RNA interference. The pheromone profiles of 5 - 7
individuals from each line were analyzed using DART-MS. CHC profiles of transgenic flies obtained by DART-MS were compared tothat of genetic controls to detect changes in CHC profiles. Individual peak intensities were normalized to the most abundant CHC
component in each profile. Relative intensity differences were compared across 65 different transgenic lines. Differences that weregreater than 2 standard deviations were selected for a secondary screen.
Approximately 10-15 different CHC species could be identified from each individual fly. Profiles between males and females aredifferent. Three positive controls（previously published genes known to affect CHC profile）were identified in the screen.
Additionally, in the primary screen, 11 transgenic lines were shown to produce significantly modified CHC profiles for females while
15 transgenic lines showed significant modifications for male CHC profiles. Six out of the 11 transgenic lines identified by the
primary screen for females and 10 out of 15 transgenic lines for males were found to contain significant modifications in CHC
profiles after a secondary screen with a larger sample size. The type of modifications identified from the secondary screen wasconsistent with that of the primary screen. In addition, 11 novel genes have been identified that contribute to pheromone synthesis
which includes genes involved in the elongation, long chain fatty acid transport, dehydrogenation, oxidation and reduction
processes. These results confirm that DART-MS is able to provide accurate and rapid analysis of chemical profiles and is effective
as a screening method.

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 26: Lipidomics : Recent New Techniques and Applications

PWe-089
11:10 － 12:20

Inter-strain comparative lipidomics of microalgae using an online multidimensional HILIC-RP-UPLC-QToF-MS

Jeremy Dietrich Netto1, Sophie Fon Sing2,3, William Ditcham4, Michael Borowitzka2, Mark Ritchie1

1
Waters Pacific Private Limited, Singapore, 2School of Biological Sciences and Biotechnology, Murdoch University,
Australia, 3Muradel Proprietary Limited, Australia, 4Separation Science and Metabolomics Laboratory, Murdoch
University, Australia

Keywords:
multi-dimensional separation, orthogonal chemistry, UPLC, lipidomics
Novel aspects:
We report for the first time the use of an online high throughput multi-dimensional separation UPLC-QToF-MS system utilizing
orthogonal HILIC-RP chemistries for the application of lipidomics and potentially other fields.
Abstract:
Introduction
With the increase of petroleum prices in recent years, alternative forms of biofuels are gaining significant interest as replacements
since they are both renewable and cleaner-burning as opposed to fossil fuels. One emerging form of biofuel production is fromthe cultivation of microalgae. Currently, adoption of commercial scale cultivation has been hindered by factors such as thegeography of the land, climate, costs of water replenishment due to evaporative losses, costs of nutrients etc. Many of thesefactors could be either eliminated or, the associated costs could be minimized by the suitable selection of the microalgal strain
from the beginning.
In this study, we compare the lipid profiles, particularly in the glycerolipid class which correlates to the potential biofuel yield, ofvarious strains of a chlorophyte using a novel orthogonal two-dimensional HILIC-RP UPLC coupled to quadrupole time of flightmass spectrometry.
Experimental
Lipids were extracted from the harvested algal biomass using the Folch method. The extracted sample was injected into a
2 D-UPLC system with a HILIC column as the first dimension and a charged surface C 18 column as the second dimension of
separation. An short isocratic run using acetonitrile and water buffered with ammonium acetate was utilized for the HILIC method.
A trapping column was placed in line to trap the lipids of interest eluting off the HILIC column. The trapped lipids were then backflushed into a charged surface C 18 column running a gradient of acetonitrile, water and isopropanol buffered with ammonium
acetate. Accurate mass data from both precursor and fragment ions of the lipids were collected simultaneously by a Q-ToF MS
running in MSe mode. Lipids were identified using SimLipid and comparative statistical analysis between the various strains was
performed using MarkerLynx™.
Preliminary Results and Discussion
Traditional direct infusion and reversed phase LC-MS methods suffer from poor sensitivity of low abundance but biologicallyimportant lipids due to the co-elution of interferences which could include lipids from other classes i.e. highly abundant
glycerophospholipids. Recently developed HILIC-UPLC methods show improved class separation but again, the individual lipids
within each class co-elute and thus interferences also form.
We see improved detection and identification of the glycerolipids as the starting high organic mobile phase composition flowing
through the HILIC column allowed only the hydrophobic classes to elute off the column while retaining the more polar
phospholipids. These classes of lipids were then further separated on the charged surface C 18 column according to theirhydrophobicity or alkyl chain length void of any other interferences. The orthogonal two dimensional approach utilized hereeffectively doubled the peak capacity of the separation method. This improved both the resolution and dynamic range and thus
the confidence level of lipid identification was higher when the data was analysed by SimLipid.
As more lipids were identified per sample due to the improved sensitivity of the method, there was a greater differentiation
between the various strains than previously reported from data obtained by direct infusion ion-trap MS.

Conclusion
The novel 2 D-UPLC-QToF-MS method described here eases the handling of complexity and dynamic range issues seen in
traditional lipid analysis methods, combining the benefits of orthogonal chemistries. Data obtained from the application of thismethod in algal lipidomics would be critical for future strain selection in commercial scale cultivation, potentially saving companies
resources that would have been wasted from the adoption of incorrect or low-yielding strains.

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 26: Lipidomics : Recent New Techniques and Applications

PWe-090
13:30 － 14:40

Lipid identification through MS and MS/MS analyses combined with new
database search software

Rainer Paape, Aiko Barsch, Jens Fuchser, Andrea Schneider, Ulrike Anders, Sebastian Goetz,
Detlev Suckau
Bruker Daltonik Gmbh, Bremen, Germany

Keywords:
Lipid identification, Lipid analysis software using MS and MS/MS data, MALDI-TOF/TOF and -FTICR, ESI-QTOF and ion trap
Novel aspects:
An automated lipid identification platform was validated with various mass spectrometry platforms.
Abstract:
Introduction:
Lipids are a large and diverse group of molecules that include fats, sterols, phospholipids, glycerides and many others. The main
biological functions of lipids include energy storage, as structural components of cell membranes, and as important signaling
molecules. These findings have led to an increasing interest into lipid analysis at large scale during the past years, also coined“Lipidomics“. The huge structural variety makes their analysis quite demanding. Today mass spectrometry and liquid
chromatography mass spectrometry represent versatile tools in the field of lipid analysis, also offering the possibility for molecular
structural identification. One of the most important aspects of mass spectrometric lipid data analysis is the structural identificationof lipids using precursor and MS/MS data.
Methods:
Glycerophospholipids standards（Avanti Polar Lipids）from different lipid classes were analyzed, such as phosphatidic acid（PA16： 0 -16： 0 ）, glycerophosphocholines（PC 18：0, PC 16： 0 -18： 1 （ 9 Z）, PC 18： 1 -18： 1 （ 6 Z）, PC 18： 1 -18： 1 （ 9 Z））,
glycerophosphoethanolamines（PE 16： 0 -18： 1 （ 9 Z））, glycerophosphoserines（PS 14： 0 -14： 0 ）, glycerophosphoglycerols（PG
16： 0 -16： 0 ）and sphingomyelins（SM 16： 0 ）. For each lipid mass spectra in positive and negative mode were acquired on
FTICR, ion trap, Q-TOF and MALDI-TOF/TOF instruments and raw data exported to the SimLipid software（Premier Biosoft）.
Individual lipids were identified by a structure database search using the SimLipid software.
Results:
In this study we analyzed a number of standard lipids using MALDI-TOF/TOF, MALDI-FTICR and ESI-QTOF and ESI-iontrap（infusion）techniques. For most analyses positive ionization mode was used, negative mode was successfully applied to theanalysis of lipids with an acidic characteristics such as PA, PG and PE.
The lipid identification software used MS and MS/MS spectra containing peak list data for lipid profiling and structural elucidation.
The program accepts experimental MS and MS/MS data obtained by mass spectrometry in text formats（e. g. MS Excel, mzDataand mzXML）and native file formats from all used mass spectrometers. Lipids were identified by searching precursor molecularweights and MS/MS fragments against known lipid structures available in a database provided by SimLipid and fragment masses
calculated based on the lipid structures.
All lipids were unequivocally identified by database searching（typically rank 1 to 3 in the result list）independent of the mass
spectrometer used and the ionization technique involved. The lipids were identified within MS/MS error tolerance settings that are
adequate for the various mass analyzers（0.005-0.2 Da）. The various tested lipid classes and their covalent gross structures, suchas the head group type and the fatty acyl chain composition were correctly attributed in the search results in all cases. However,the localization of double bonds by this approach was not possible due to lack of specific fragments； here chemical derivatization
remains to be required.
An instrument independent platform for lipid identification was established in this work, which can now be used in lipid
characterization or lipidomics by LC-MS/MS, infusion-ESI or TLC-MALDI（thin layer chromatography）workflows.

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 26: Lipidomics : Recent New Techniques and Applications

PWe-091
11:10 － 12:20

Lipidomic profiling of mammalian brain and biofluids by RP-LC/MS: Global
approaches and structural analysis using LTQ-Orbitrap.

Benoit COLSCH, Samia BOUDAH, Christophe JUNOT
CEA, Gif-sur-Yvette, France

Keywords:
Lipidomic, tissue, biofluids, LC-MS
Novel aspects:
We discribe an efficient methodology to profile lipid molecular species in biological matrices
Abstract:
Lipids are essential cellular constituents of all mammalian cells that have many critical roles in cellular functions. If many families oflipids such as glycerophospholipids, sphingolipids, glycerolipids and sterols are mainly present in outer layer of cell plasma
membranes, other lipids are also involved in energy storage or participate to cell signaling. Lipid metabolic or immunologicalalterations may have serious pathological consequences such as neurometabolic or neurodegenerative diseases. Due to the highstructural diversity of lipid families including polar or neutral molecules, a complete molecular profiling of biological matrices staya challenge in lipidomic for qualitative and quantitative method developments. Complex mixture analysis of lipids using mass
spectrometry technologies was reported since 1990s and actually recent developments in electrospray ionization mass
spectrometry（ESI/MS）using high resolution system instrumentation have rapidly expending the research on this field. Lipidomic
analysis can be performed using“shotgun“ approaches without any previous chromatographic separation, or using LC/MS toanalyze a larger number of molecules.
A comprehensive lipidomic methodology based on lipid fractionation using SPE cartridges（LC-NH 2 ）coupled or not with RP-LC/
MS was implemented to build a lipid database including exact mass, retention time and MSn spectra for further development of arobust lipid profiling method. Due to the high heterogeneity of lipid species in biological matrices, brain tissues, human plasmas
and cerebrospinal fluids（CSF）were selected in this study.
Brain lipid extracts were obtained using a Folch modified partition［ 1 ］from adult Sprague-Dawley rat and human biofluids wereobtained using modified Bligh and Dyer extraction［ 2 ］. The upper phases containing ganglioside species were desalted using
C18 columns and pooled with the lower phases which contain the other lipid families. An aliquot of total lipid extract（TLE）was
redissolved in chloroform and a lipid fractionation was performed using LC-NH 2 columns to separate crudely lipid classes［ 3 ］.
Six fractions were obtained and compared with TLE using a RP-LC/MS. TLE and fractions were separated, during 32 min per run, on
an Ascentis Express C18 2.1x150mm 2.7µm column connected to an Accela quaternary HPLC pump coupled with an LTQ-Orbitrap.
The tune methods were optimized using lipid standards.
All fractions obtained from LC-NH 2 columns were analyzed in postive and negative ion modes by direct infusion ESI-MS and LC/
MS and compared with the total lipid extract of all biological matrices. LC-NH 2 fractions were used to simplify lipid species
identification in order to build a homemade lipid database. In a single LC/MS run, numerous lipid species were identified in both
fractions and TLE. In presence of ammonium formate, in positive ion mode, salt adducts are drastically reduced（Na, K）and many
lipid classes such as phosphatidylcholines and their lyso-forms, ceramides, cerebrosides and sphingomyelins were detected as［M+H］+ ions. Other lipid species（mono- di- and tri-glycerides, and sterols）are also detected as［M+NH 4 ］+. However, to limit
the presence of phospholipids and neutral lipids in blank samples before and after sample analysis, a wash with organic solvents
must be used during the run. In the negative mode, numerous classes of lipids were also detected such as free fatty acids,
phosphatidylethanolamines and lyso-forms, sufatides, phosphatidylinositols, phosphatidylserines, phosphatidylglycerols, andgangliosides as［M-H］- ions. In this study, numerous lipid species was identified using high resolution mass spectrometry. As an
example, 250 phospholipid isoforms, 150 sphingolipid isoforms and 250 neutral lipid isoforms are listed in the database.
Interestingly, many lipid isoforms was found in only one investigated biological matrix.
［ 1 ］Folch J., et al. J.Biol.Chem. 1957, 226： 497-509.
［ 2 ］Bligh E.G and Dyer W.J. Can. J. Biochem. Physiol.（1959）37： 911-917.
［ 3 ］Bodenec J et al. J. Lipid Res.（2000）41： 1524-1531.
［ 4 ］Bird S and al. Anal. Chem. 2011, 83：6648-6657.

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 26: Lipidomics : Recent New Techniques and Applications

PWe-092
13:30 － 14:40

Development of ultra-high sensitive detection method of sphingosine-1-phosphate and its related sphingoids by LC/MS/MS

Daisuke Saigusa, Tomohiro Takahashi, Shiori Ootaku, Daisuke Jinno, Asuka Inoue, Michiyo Okutani,
Takaaki Abe, Naoto Suzuki, Junken Aoki, Yoshihisa Tomioka
Tohoku University, Miyagi, Japan

Keywords:
Sphingoids, Sphingoid- 1 -phosphates, Laser micro dissection, Brain, LC/MS/MS
Novel aspects:
We have developed a method for analyzing intracellular kinetics and disposition of sphingoids and sphingoid- 1 -phosphates at acellular level, combining a LMD with an ultra-high sensitive method using LC/MS/MS.
Abstract:
Introduction
Sphingoids and sphingoid- 1 -phosphates are endogenous compounds, and their function of lipid mediator has been noticed inthese days. However, the biological kinetics and intracellular disposition at a cellular level have not been demonstrated. A micro
dissection is useful tool for analyzing the distribution of intercellular endogenous compounds with cytological staining. However, it
is difficult for determining sphingoid- 1 -phosphates because phosphate groups are easily removed and unstable in biologicalsamples. Therefore, it has been required ultra-high sensitive detection and stable sample preparation method to determine
sphingoids and sphingoid- 1 -phosphates with a micro dissection sample.
PurposeIn this study, we have developed a method for analyzing biological kinetics and intracellular disposition of sphingosine- 1 phosphate and its related sphingoids at a micro level, combining a laser micro dissection（LMD）system with an ultra-highsensitive detection method using LC/MS/MS.
Methods
An ultra-high sensitive determination method of sphingosine- 1 -phosphate（S 1 P）and 5 sphingoids has been developed by LC/
MS/MS. LC separation was optimized using a NANOSPACE SI- 2 HPLC system（Shiseido）. MS/MS was conducted on a TSQ
Quantum Ultra（Thermo Fisher Scientific）triple quadrupole mass spectrometer equipped with a heated ESI source. The detection
was selected ion monitoring mode. The symmetry factor, theoretical plate number and signal to noise（S/N）were evaluated toestablish an ultra-high sensitive method. To indicate the effect of the presented method, brain samples were prepared from male
C57BL 6 mouse and dissected the brain section by LMD system（LMD 6000, Lica）. After perfusion with PBS, brain was quickly
removed and buried into optimal cutting temperature compound. A 10 µm slice of mouse brain has been prepared by cryostat（Microm HM550, Thermo fisher scientific）, and put on a slide glass. Then slice sample was dissected several brain regions by
LMD system. The dissected samples were prepared by simple methanol deproteinization.
Results and discussion
We have improved the sample preparation method using on-line column switching, and particularly focused on changing the HPLC
flow passages. The optimized online column-switching system consisted of a trap column, a CAPCELL PAK ACR（1.0 mm i.d. × 35
mm, 3 µm, Shiseido）and an analytical column, a CAPCELL PAK ACR（1.0 mm i.d. × 150 mm, 3 µm）. The elution mobile phase（A）, 5 mmol/L HCOONH4-H2O/acetonitrile（ 5 /95, v/v； pH 4 ）（B）,
were 5 mmol/L ammonium formate（HCOONH4）-H2O（pH 4 ）and loading was A/B（70/30, v/v）, and the flow rate was 60 µL/min. These conditions had the best performance of symmetry
factor（0.99）, theoretical plate number（213912）and S/N（14581）of 100 nmol/L std. solution, and no peak tailing was observed on
the chromatograms. A sensitive and selective assay was developed with a 50 pmol/L lower limit of detection for S 1 P and its
related sphingoids. All of dissected samples of mouse brain were detected S 1 P and some of sphingoids, and samples wererequired 5,000 - 20,000 cells to detect by LC/MS/MS. The concentration of S 1 P in cerebellum, cortex and nigra were 1.22±0.03, 0.40
±0.13 and 1.53±0.16 nmol/L, respectively.
Conclusion
We have developed an ultra-high sensitive method for S 1 P and its related sphingoids. The dissected brain samples were stablefor S 1 P on LMD system. The distribution of S 1 P in the brain regions was demonstrated. These results are indicated that thepresent method is powerful tool for analyzing the intracellular distribution of S 1 P and its related sphingoids in tissue dissection
samples.

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 26: Lipidomics : Recent New Techniques and Applications

PWe-093
11:10 － 12:20

Mass spectrometry based assessment chimeric mouse liver metabolite
profiles following oral dosing of troglitazone

Alan J Barnes1, Neil J Loftus1, David R Baker1, Ian Wilson2, Yoshio Morikawa3

1
Shimadzu Corporation, Manchester, United Kingdom, 2Astra Zeneca, Alderley Park, UK, 3PhoenixBio Co. Ltd, HigashiHiroshima, Japan

Keywords:
LC/MS, metabolomics, accurate mass, profiling, MetID
Novel aspects:
Lipid metabolite profiling used to characterise animal groups as a tool to gauge sample integrity for drug metabolism experiments.
Abstract:
The use of chimeric（PXB）mice, with up to 85% humanized livers, have become well established in the study of drug metabolism
and disposition in which the majority of the hepatocyte population of the mouse liver has been replaced by human hepatocytes.
Expression of human Phase I and II metabolic enzymes and hepatic transporter proteins enable identification of drug metabolites
that may not be detected by other means. Histological based methods to assess the percentage human tissue can offer a visualgauge of the amount of human tissue. For known compounds with metabolism characteristic to human, drug metabolites can alsooffer a guide as percentage humanised tissue in chimeric livers. Endogenous lipid differences, also occurring between PXB andwild type（SCID）mice may also offer a guide as percentage humanised.
To assess the viability of the chimeric PXB mouse troglitazone（TGZ）was dosed orally over 7 days. In pre-clinical studies TGZ
showed inter-species differences in metabolism particularly in sulfation and glucuronidation pathways. The present study
evaluated the metabolic profile of troglitazone and endogenous metabolites in the PXB compared to SCID（severe combined
immunodeficiency）mouse model by high mass accuracy MS/MS analysis.
Liver extracts from SCID and PXB mice were analysed using a high resolution LC/MSn system（Nexera LC coupled with a LCMS-ITTOF； Shimadzu Corporation）. Aqueous extracts were analysed using a Phenomenex Kinetex XB C18 1.7um（2.1x100mm）held at
30C； components were separated with a 40 minute gradient（mobile phase was A - 0.1% formic acid solution + 10mM ammonium
acetate and B - acetonitrile + 0.1% formic acid）at a flow rate of 0.6mL/min. The LCMS-IT-TOF acquired negative MS and MS 2 data
（m/z 150-800）.
In preclinical studies, TGZ human hepatic metabolism is broadly similar to rat, dog, marmoset and cynomolgus monkey with thesulfate conjugate being the most abundant metabolite detected while glucuronidation was predominant in mouse. Consistent topublished results, in this study the sulphate metabolite was measured at higher levels in humanised whereas the glucuroniode
was reduced. Marked differences in endogenous metabolic profiles were also observed between the humanized and wild typemice with several glycerophosphocholine compounds both elevated and decreased up to twenty fold difference in peak area. In
addition, several fatty acid compounds were decreased only in troglitazone treated animals. These results may in future studies
offer the possibility to provide a non-histological based estimation of percentage humanised liver whilst also providing an
indication of endogenous metabolite changes occurring from drug administration.

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 26: Lipidomics : Recent New Techniques and Applications

PWe-094
13:30 － 14:40

High-throughput lipid profiling system for dried plasma spots by using
online-supercritical fluid extraction-supercritical fluid chromatography/
mass spectrometry

Takato Uchikata, Atsuki Matsubara, Eiichiro Fukusaki, Takeshi Bamba
Osaka University, Osaka, Japan

Keywords:
supercritical fluid extraction, supercritical fluid chromatography, Dried plasma spot, lipidomics, phospholipid
Novel aspects:
Phospholipids in dried plasma spot could be analyzed without any pretreatment in 15 min using online-SFE-SFC/MS/MS. 172
phospholipids from only 3 µl plasma could be detected.
Abstract:
Although biological analysis is usually performed using plasma obtained by venipuncture, the use of dried blood spots（DBS）has
grown in popularity in the clinical and pharmaceutical communities over the past decade as an alternative sampling procedure.
DBS and dried plasma spots（DPS）analysis is available as a diagnostic tool for genetic diseases and is useful for the screening of
biomarkers.
In this study, a high-throughput analytical system based on online-supercritical fluid extraction-supercritical fluid chromatography
with tandem mass spectrometry（online-SFE-SFC/MS/MS）was developed for phospholipids profiling of DPS. This system is able tosimultaneously perform extraction and separation, allowing phospholipids that have common polar head groups to be analyzed.
We investigated conditions of the online-SFE-SFC/MS/MS. First we optimized SFC and MS conditions to separate and detect thephospholipids such as column type and collision energy. Next we optimized online-SFE-SFC conditions using mouse plasma to
extract and separate these phospholipids such as modifier concentration and extraction time.
In the result phospholipids in only 3 µl of plasma can be extracted in 5 min and analyzed within 15 min using this system. A totalof 172 phospholipids, including phosphatidylcholine（PC）, lysoPC, sphingomyelin（SM）, phosphatidylethanolamine（PE）andlysoPE, were annotated, and 78 phospholipids were analyzed with good repeatability. The online-SFE-SFC/MS/MS, which is able toperform high-throughput lipid profiling analysis for clinical diagnosis and drug discovery, may also be suitable for the screening of
biomarkers.

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 26: Lipidomics : Recent New Techniques and Applications

PWe-095
11:10 － 12:20

Systems based LC-MS metabolite profiling of mice treated with ethanol
enriched liquid diets

Ashton Simon1, Neil J Loftus1, Alan J Barnes1, Filippos Michopoulos2, Ian Wilson2, Ji Cheng3

1
Shimadzu Corporation, Manchester, United Kingdom, 2Astra Zeneca, Alderley Park, UK, 3University of Southern
California, Los Angeles, USA

Keywords:
LC/MS, metabolomics, profiling, alcohol, ethyl glucuronoide
Novel aspects:
Using a systems based approach to metabolite profiling, significant alcohol marker compounds were detected by accurate mass
MetID software.
Abstract:
Through measurement of liver, plasma and urine, a systems based approach to metabolite profiling can provide a perspective ofchange in endogenous compounds through modified dietary factors. Previous studies into the harmful effects of alcohol have
utilised the intragastric feeding models in which animals have surgically inserted feeding cannulas. In this study a less invasive fourweek ethanol enriched liquid diet was evaluated as an alternative vehicle to modified diet. To gauge the impact of ethanol
exposure, known ethanol metabolites were monitored by high resolution mass spectrometry to monitor metabolic change.
Animal liquid diets（Dyets, Inc., Bethlehem, PA）were administered to control mice（Dyets #710079 and ethanol treated mice（Dyet#710301）. The percentage alcohol derived calorie in the alcohol diet was 10% for week 1, 20% - week 2, 35% - week 3 and 4. When
35% of the calorie was derived from alcohol, alcohol concentration was ~6.5%（v/v）. Urine, plasma and liver tissue samples wereseparated by Nexera UHPLC（Shimadzu）using a Phenomenex Kinetex column（XB C18 1.7um 2.1x100mm）over 12 minutes（A - 0.1%
formic acid solution + 20mM ammonium formate and B methanol + 0.1% formic acid + 20mM ammonium formate）at a flow rate of
0.7mL/min. LCMS-IT-TOF was used to acquire high mass accuracy MSn data.
In this current study ethylated lipid marker compounds were detected, consistent to previous work published from intragastricfeeding models. The profound lipid differences observed from previous work were markedly reduced in the current liquid dietstudy with the emergence of ethylated lipid markers at week 4 but not at week 2. These included ethyl arachidonate, ethyl
linolate and ethyl DHA which were not detected in control or 2 week samples. In broad agreement to previously published work,
retinol palmitate and retinol were also significantly reduced in ethanol treated animals. Comparing urine, plasma and liver tissue,
elevated levels of two phosphatidylcholine were measured at significantly elevated levels in urine and liver and to a lesser extent
in plasma also in 4 week ethanol treated animals. Urine samples were found to contain ethanol metabolites including ethyl
sulphate and ethyl glucuronide, metabolites commonly used to determine recent ethanol consumption. The implications of thistargeted metabolomics study suggest that the level of alcohol consumption in this work was lower than in the previous
experiments. It may also provide marker compounds that highlight prolonged low level continuous alcohol consumption andpossible indicators of alcohol addiction in humans.

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 26: Lipidomics : Recent New Techniques and Applications

PWe-096
13:30 － 14:40

Identification of Insect Species by Direct UV-Laser Desorption/ Ionization
Mass Spectrometry of Cuticular Lipids and Pheromones

Jacqueline Chin1,2, Qi Ling Koh3, Joanne Yew1,2

Temasek Life Sciences Laboratory, Singapore, 2National University of Singapore, Singapore, 3Nanyang Technological
University, Singapore

1

Keywords:
LDI, pheromone, lipid, hydrocarbon, identification
Novel aspects:
Direct UV-LDI MS of cuticular lipids from related species of insects reveals distinct chemical profiles that could be used for species
identification and evolutionary mapping
Abstract:
Introduction
Insect cuticles are covered with a lipid layer consisting of hydrocarbons, fatty acids, and triglycerides. Many of these compounds
serve as pheromones, signaling molecules that influence social behaviors such as mate choice and aggression. Because of theirunique behavioral properties, many pheromones are species-specific and sex-specific. As such, pheromone lipid markers can beused for biotyping of insects and could be particularly helpful for identifying cryptic species. The standard method for cuticularhydrocarbon analysis is gas chromatography MS. We recently showed that UV-laser desorption/ ionization（LDI）MS could be
used as a complementary analytical method（ 1 ）. UV-LDI MS detects larger, polar compounds and allows spatially-resolved
profiling of single insects. We show here that UV-LDI MS can be used for rapid biotyping of different drosophilid（fruit fly）species
based on cuticular lipid profiles.
Methods
Intact, individual adult virgin males and females from 14 different drosophilid species and populations were analyzed. The UV-LDI
MS measurements were performed using a modified ion source of an orthogonal TOF mass spectrometer equipped with a N 2
laser. 6 - 8 flies from each species and of each sex were mounted on a custom-built sample plate and analyzed under elevatedsource pressure conditions（ 2 - 4 mbar）. No matrix was applied. Profiles of individual body parts were obtained with a spatial
resolution of ~200um.
Preliminary data
The lipid profiles of both closely- and distantly-related Drosophila species and populations show clear quantitative and qualitative
differences. Among the different kinds of lipids identified from the cuticular surface are C20-C37 length unsaturated hydrocarbons（including alkenes, dienes, trienes）, oxygen-containing hydrocarbons, and triglycerides. Closely-related species and populations
show quantitative rather than qualitative differences. In contrast, distantly-related species exhibited striking differences in lipid
profiles, particularly in the pheromone components of male sex glands. Males and females of all species tested exhibited distinct
sexually dimorphic lipid compositions. The cuticular profiles of recently-mated females also showed changes, indicating that somemale-specific compounds were transferred from males to females during copulation. These results indicate that cuticular lipids ofinsects can be used as biomarkers specifying species, sex, and mating status. Such information is important for species
identification and may be used to trace evolutionary relationships.
1. Yew JY, Dreisewerd K, Luftmann H, Müthing J, Pohlentz G, Kravitz EA. 2009. A new male sex-pheromone and novel cuticularcues
for chemical communication in Drosophila. Curr Biol. 19（15）： 1245 1254.

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 26: Lipidomics : Recent New Techniques and Applications

PWe-097
11:10 － 12:20

Systems-Based Approaches to Elucidating Gender-Specific Mechanisms in
the Etiology of Atherosclerosis

F L D’Alexandri1, Dmitry Grapov2, Peddiniti Gopalacharyulu3, T L Pedersen2, A Razuvaev1, S Arvidson1,
H Hedin1, K Lund1, T Gustavsson1, K Caidahl1, Jesper Z Haeggstrom1, R Laaksonen4, M Janis4,
T Hyotylainen3, M Oresic3, J W Newman2, Craig E Wheelock1
1
Karolinska Institute, 2United States Department of Agriculture ARS Western Human Nutrition Research Center, Davis,
CA, USA, 3VTT Technical Research Centre of Finland, Espoo, Finland, 4Zora Biosciences, Oy, Espoo, Finland

Keywords:
eicosanoid, lipidomics, metabolomics, atherosclerosis, cardiovascular disease
Novel aspects:
A systems-based approach integrated transcriptomics, lipidomics, metabolomics and lipid mediator data from carotid plaques.
Multivariate-statistical analyses highlighted lipid mediator pathways that evidenced gender-specific shifts at the gene and lipid
level.
Abstract:
Cardiovascular disease is the major cause of premature death in Europe, resulting in > 4 million deaths annually. Cardiovasculardisease is often regarded as a“men’s“ disease； however, it is the leading cause of mortality among women. In particular, since
1984, more US women than men have died annually from cardiovascular disease. Studies have suggested a key role for oxidized
fatty acids（oxylipins）in inflammatory reactions of atherosclerosis； however, it is still unclear to what extent pro- and antiinflammatory factors determine whether an atherosclerotic lesion develops into a stable plaque orruptures, leading to stroke or
myocardial infarction. To further probe the etiology of plaque development and subsequent rupture, we performed a combinationof lipidomics and metabolomics as well as targeted oxylipin, freefatty acid and endocannabinoid lipid mediator profiling in human
carotidatherosclerotic plaques and matching circulating plasma. These data were combined with sonographic gray-scale medianplaque imaging and AffymetrixGeneChip® data as well as patient clinical parameters to develop a multivariate model of plaquegender-specificity. PCA analysis showed that plaque and plasma have unique composition（R^ 2 =0.54, Q^ 2 =0.41）, which was
mainly driven by oxylipin levels, despite the fact that endocannabinoids represent >90 % of the lipids measured. Orthogonal
projections to latent structures（OPLS）analysis based on gender resulted in a robust oxylipin-driven model with high predictive
power, which was specific for plaque（R^ 2 =0.87, Q^ 2 =0.43）. No model could be generated using data from circulating plasma.
The most important variables driving the separation between genders were primarily products of the12/15 lipoxy genase pathway（12/15-LOX）, suggesting gender-specific differences in this key pathway. These trends were supported by gene set enrichment
analyses（GSEA）showing enrichment in linoleic- and eicosanoid-specific pathways in plaque, but not plasma. Accordingly, theobserved changes in lipid and gene data agreed in the vector and magnitude of the alterations, suggesting that gender-specificpathways do exist in carotid plaques. Imaging data suggested that plaques in women had distinct morphological differences, withwomen having overall more calcified plaques. Collectively, results point to gender-specific shifts in inflammatory lipid species aswell as morphological differences that could potentially explain the higher incidence of cardiovascular disease in women.

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 26: Lipidomics : Recent New Techniques and Applications

PWe-098
13:30 － 14:40

Molecular lipidomic workflow solution identifies a single lipid marker forthe diagnosis of statin myalgia

Kim Ekroos1, Dimple Kauhanen1, Tuulia Sylvänne1, Kirill Tarasov1, Reini Hurme1, Giulianna Mombelli2,
Cesare R Sirtori3, Michael Phillips4, Marie-Pierre Dube4, Nathalie Laplante4, Jean-Claude Tardif4,
Reijo Laaksonen1
Zora Biosciences, Espoo, Finland, 2Niguarda Hospital, Milan, Italy, 3University of Milan, Milan, Italy, 4Montreal Heart
Institute, Montreal, Canada

1

Keywords:
Molecular lipidomics, statin, myalgia, high-throughput, biomarker
Novel aspects:
Molecular lipidomics identifies diagnostic lipid markers
Abstract:
Statins, globally most prescribed drugs, can cause a wide range of muscular side effects, from non-specific myalgias to the fullblown rhabdomyolysis. Statin intolerance may substantially reduce treatment compliance and statin efficacy in preventing
cardiovascular end points. The prevalence of myotoxicity is clearly higher in clinical practice than has been shown in clinical trials.
Statin myotoxicity is underdiagnosed partially because the currently used biomarkers such as creatine kinase（CK）are notsensitive enough to detect most patients who develop muscle weakness or pain. Here, we assessed proprietary state-of-the art
molecular lipidomics high-throughput workflows for the discovery of more sensitive biomarkers for statin induced muscle toxicity.
Plasma samples were obtained from statin users with or without muscle complaints（n=334）. The main muscle symptoms were
myalgia and weakness. The lipidomic assessment was carried out using an in-house Zora lipidomics solution composed ofmultiple tailor-made lipidomics platforms supported by Vostok, bioinformatic processing tools, providing stringently quality
controlled outputs of molecular lipids in absolute amounts. Plasma samples were carefully prepared using a lipidomics customized
robotic sample preparation and extraction workflow, followed by high throughput quantitative shotgun and targeted lipidomic
workflows assessed on QTRAP 5500 technology equipped with a robotic nanoflow ion source and UHPLC systems, respectively.
The molecular lipidomic assessment revealed that the concentration of 12-hydroxyeicosatetraenoic acid（12-HETE）was
significantly higher in statin intolerant patients compared to patients with no clinical signs ofmuscular side effects（mediandifference +125%, p = 1.0x10-13）. 12-HETE discriminated cases and controls equally well both in men and women. Taken together,
the proprietary in-house molecular lipidomics solution identified 12-HETE as a sensitive and specific biomarker for statin-induced
myalgia. This single lipid marker may serve as a new tool for physicians, and will help them to rationally select treatments forpatients sensitive to statin induced adverse muscle side-effects. This study successfully demonstrates the value of stringently
executed quantitative molecular lipidomics in biomarker discovery.

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 26: Lipidomics : Recent New Techniques and Applications

PWe-099
11:10 － 12:20

Development of comprehensive glycerophospholipid profiling methods
using liquid chromatography / high-speed triple quadrupole mass
spectrometry

Suzumi M Tokuoka1, Yoshihiro Kita1, Masaki Yamada1,2, Takao Shimizu1
1

The University of Tokyo, Tokyo, Japan, 2Shimadzu Corporation, Kyoto, Japan

Keywords:
Lipidomics, glycerophospholipids, LC/MS, SRM
Novel aspects:
High-speed triple quadrupole mass spectrometry enabled high-throughput and comprehensive profiling of glycerophospholipids.
Abstract:
Glycerophospholipids are important as structural and functional components of cellular membranes. Based on the polar
headgroup at the sn- 3 position of the glycerol backbone, glycerophospholipids are divided into distinct classes such as
phosphatidylcholine （PC）, phosphatidylethanolamine （PE）, phosphatidylinositol （PI）, phosphatidylserine （PS）,
phosphatidylglycerol（PG）, phosphatidic acid（PA）, and cardiolipin. Each glycerophospholipid class consists of numerous
molecular species due to a variety of the linkage between the fatty acid（FA）chains and the sn- 1 position of the glycerol
backbone（i.e. acyl-, alkyl-, and alkenyl-linked fatty chains）, and the complexity of the combinations of fatty acids at the sn- 1 andsn- 2 positions which may differ in carbon number and degree of unsaturation. The major fatty acids in glycerophospholipids canvary in length from 14 to 22 carbons and may have 0 to 6 double bonds. In theory, over a thousand different glycerophospholipids
molecular species are possible. Compositions of glycerophospholipids differ among tissues as well as cell types. Such diversity
may be critical for various cellular functions, although the roles of the various molecular species of each class ofglycerophospholipids are still unclear.
Recent advances in mass spectrometry have provided the means for rapid and sensitive analysis of cellular lipids. We developed
methods to perform lipid profiling utilizing the enhanced capabilities of a recently developed“high-speed“ triple quadrupole
mass spectrometer with high-speed scanning, fast polarity switching, and short dwell time. Two liquid chromatography /electrospray ionization/mass spectrometry（LC/ESI/MS）methods were compared for measuring cellular glycerophospholipids.
One was a multiple scanning method in which several precursor ion scans and neutral loss scans in both positive and negative ion
modes were performed in one analytical run.. The other method was based on SRM（selected reaction monitoring）in which
hundreds of glycerophospholipid species are measured simultaneously.
Precursor ion scans and neutral loss scans are commonly used to identify specific polar head groups of glycerophospholipids.
SRM-based approaches are routinely used to analyze many small molecules such as drug metabolites, hormones and pesticides
with high sensitivity and throughput and are also gaining popularity in proteomics because of the higher sensitivity than scanning
methodologies. Here, we compare sensitivities and coverages of glycerophospholipids detected using both scan-based and SRMbased methodologies with various scan speeds and dwell times and discuss advantages and disadvantages of each approach.

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 26: Lipidomics : Recent New Techniques and Applications

PWe-100
13:30 － 14:40

Development of a comprehensive detection method for quantifying
eicosanoids using high-speed liquid chromatography / mass spectrometry

Masaki Yamada1,2, Yoshihiro Kita1, Suzumi Tokuoka1, Takao Shimizu1
1

The University of Tokyo, Tokyo, Japan, 2Shimadzu Co., Kyoto, Japan

Keywords:
Lipidomics, eicosanoid, LC/MS, SRM
Novel aspects:
A comprehensive and high sensitive detection method of eicosanoids, their metabolites and isomers using a novel LC/MS system
was developed.
Abstract:
Eicosanoids are generated from arachidonic acid（AA）as lipid mediator in various pathological and physiological events ofmammalian organisms. Enzymatic oxidation of AA by cyclooxygenase（COX）, lipoxygenase（LOX）and cytochrome P450 including
non-enzymatic oxidation induce synthesis of various eicosanoids such as prostaglandins （PGs）, leukotrienes （LTs） orhydroxyeicosatetraenoic acids（HETEs）. Isoprostanes（iPs）known as oxidative stress biomarkers can be generated via chemical
reaction of reactive oxygen or nitrogen species. Comprehensive analysis of eicosanoids and their metabolites or isomers is
expected to serve as a key technology for disease studies. Selected reaction monitoring（SRM）using liquid chromatography/mass
spectrometry（LC/MS）has been widely used for detecting eicosanoids(1-3). Although SRM is a selective and sensitive detection
method, performing a large number of SRM channels can often cause poor sensitivity. To analyze eicosanoids comprehensively
without compromising sensitivity, we developed a new quantitative method using a high-speed LC/MS system.
The analytical system consisted of Nexera UHPLC system and triple quadrupole（TQ）mass spectrometer LCMS 8040（Shimadzu
corporation）. The TQ-MS has a new type of collision cell（UFsweeper TM ）which emits product ions without decreasing theirvelocity that enable to detect product ions with keeping sensitivity even if a few milliseconds（ms）were set for one SRM transition
such as 1 ms dwell time and 1 ms pause time. Over 50 eicosanoids and their metabolites including not only PGs, LTs, HETEs or
iPs such as 8 -iso PGE2, but also platelet activating factor PAF were purchased from Cayman Chemical. Samples were dissolved inmethanol. We evaluated several HPLC columns such as Shim-Pack ODS XR-III（2.0 × 150 mm, Shimadzu Co.）, CAPCELL PAK ODS
IF-II（2.1 × 100 mm, Shiseido Co.）or Kinetex C 8 （2.1 × 150 mm, Phenomenex）. A typical flow rate using Kinetex column andgradient of B concentration were 0.4 mL/min and 15%（ 0 min）35%（10 min）85%（20 min）, respectively.
A mixture of 50 eicosanoids was separated and detected using the LC/MS system after SRM optimization for each compound. Allcompounds resolved in 20 min. Most of compounds detected in negative mode. Several lactone or ethanolamide species wereappeared in positive mode. Enantiomers such as PGF 2a and ent-PGF 2a were not resolved. Both LTC 4 and PAF peaks werebroadened when Shim-Pack ODS XR-III column was used. The broadenings were improved using CAPCELL ODS PAK IF-II column.
Further improvement of peak shape was observed using Kinetex C 8 column. Therefore we used Kinetex column for following
quantitative analysis. Setting 10 ms for each transition to over 50 SRMs including twice of polarity switching（15 ms × 2 ）, linear
calibration range spanning from 0.5 to 1000 pg was obtained（R2> 0.999）. Limit of quantifications（LOQ）were below 1 pg for anumber of eicosanoids and isomers including 8 -iso PGE2, PGD2, LTB4 and several HETEs. To examine the sensitivity under ultimate
short transition, we set 2 ms transition including 1 ms dwell and 1 ms pause time to 100 SRM transitions. Consequently, LOQ of
PGE2 was 1 pg even when such a high-speed SRM set was applied. These results suggest that several hundreds of eicosanoids
can be simultaneously detected with up to 1 pg sensitivity. We will extend the method to cover a large number of fatty acidmetabolites and other mediators without compromising throughput and sensitivity.
References
Y. Kita, T. Takahashi, N. Uozumi, T. Shimizu, Anal. Biochem. 342（2005）134-143.
M. Masoodi, M. Eiden, A. Koulman, D. Spnaer, D.A. Volmer, Anal. Chem. 82（2010）8176-8185.
S.A. Brose, B.T. Thuen, M.Y. Golovko, J. Lipid Res. 52（2011）850-859.

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 26: Lipidomics : Recent New Techniques and Applications

PWe-101
11:10 － 12:20

Formation and collision-induced dissociation (CID) of unsaturated fatty
acid-alkali metal anionic complexes: A new approach for determining
double bond position

Michael C Thomas, Thomas Gallagher, Geoffrey W Nette
Independent Marine Biochemical Research, Dunwich, Australia

Keywords:
Fatty acid analysis, collision-induced dissociation, double bond position
Novel aspects:
Presented for the first time is the collision-induced dissociation of fatty acid-alkali metal anionic complexes and two methods for
producing these anionic complexes.
Abstract:
Fatty acids are arguably the most important lipid class and are the building blocks for the majority of lipid classes. Isomeric fatty
acids differing only in double bond position are common in nature and include； 18： 1 n- 9 /n-7, 18： 3 n- 3 /n-6, 20： 3 n- 3 /n- 6 /n- 9 and 22： 5 n- 3 /n- 6 （where ’n’ defines the location of the terminal double bond from the methyl end）. Fatty acids are
essentially long-chain carboxylic acids and therefore readily produce［M-H］- ions upon electrospray ionisation（ESI）. The collisioninduced dissociation（CID）of fatty acid［M-H］- ions results mainly in the neutral losses of water and carbon dioxide while isomerspecific product ions are not observed using ion-trap mass spectrometry. This precludes the“de novo“ determination of doublebond position. Consequently, the CID of cationic fatty acid-metal ion complexes has been extensively studied and has, in general,
proved more successful in providing rich structural information.
We present here, for the first time, the CID of anionic fatty acid-alkali metal complexes. We have found that fatty acid［M- 2 H+Alk］- anions（where Alk = Li+, Na+, K+）may be generated efficiently by two distinct methods, these are； 1 ）Electrosprayionisation of a solution containing a fatty acid and alkali metal fluoride to form the［M-H+AlkF］- anion. CID of the［M-H+AlkF］anion results in the formation of the［M- 2 H+Alk］- anion via the neutral loss of HF. Or 2 ）direct formation of the［M- 2 H+Alk］anion by electrospray ionisation of a solution containing a fatty acid and alkali metal hydroxide, bicarbonate or carbonate. The
CID of［M- 2 H+Alk］- anions（in either an MS3 or MS2 experiment）results in extensive fragmentation of the fatty acid acyl chain
providing a series of ions which allow the assignment of double bond position（s）.
Typically, fatty acids are deprotonated at the carboxylic acid functional group during, or prior to, negative ion electrosprayionisation to form［M-H］- anions. The formation of［M- 2 H+Alk］- anions requires the removal of an extra proton which can only
occur from the fatty acid acyl chain. We propose that this deprotonation occurs at the bis-allylic position（s）of polyunsaturated
fatty acids（or the allylic position of monounsaturated fatty acids）resulting in the formation of a resonance-stabilised carbanion.
Strong interactions between the alkali metal cation and resonance-stabilised carbanion likely prohibits the migration of the charge
site and resulting randomisation of double bond position（s）. Upon CID, charge-driven fragmentations at the site of theresonance-stabilised carbanion then provides product ions indicative of the original double bond position（s）.
These results essentially lay the foundation to previously inaccessible negative ion chemistry. Furthermore, the negative ion
approach to determining double bond position by low-energy CID presented here may be thought of as complementary to the
CID of dilithiated fatty acid cations； an established positive ion-ESI method for determining double bond position

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 26: Lipidomics : Recent New Techniques and Applications

PWe-102
13:30 － 14:40

Phosphoinositides analysis by liquid chromatography and mass
spectrometry

Federico Torta, Pradeep Narayanaswamy, Michael P Sheetz, Markus R Wenk
National University of Singapore, Singapore, Singapore

Keywords:
Phosphoinositides, lipidomics, mass spectrometry
Novel aspects:
The analysis of phosphoinositides is still a challenging field in the lipidomics world. In this work we developed new methods tomake this kind of measurements faster and more sensitive.
Abstract:
Phosphoinositides are important lipid mediators of signalling events in all cellular compartments. They play a key role in theattachment of the cytoskeleton to the plasma membrane, exocytosis, endocytosis, membrane trafficking and enzymes activation.
The analysis and quantification of biologically important lipids became a routine process in lipidomics, however thephosphoinositides analysis has not progressed at the same rate and it is still a challenging research field. This is due to their low
cellular concentration, their highly charged head groups that affects extraction and detection and their presence as a complex
with phosphoinositide binding proteins. In this work, new approaches to examine native and derivatized phosphoinositides inbiological samples, by HPLC and nanoLC-ESI-MSMS, are presented.

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 27: Progress in Microbiology

PWe-103
11:10 － 12:20

Secondary metabolite profile of Fusarium oxysporum by LCMS using
QTOF,different sources, ion modes and metal solution post-column
addition

Adriana M Cirigliano1, Brenda V Bertinetti1, Maria A Rodriguez2, Alicia M Godeas2, Gabriela M Cabrera1
Depto Quimica Organica y UMYMFOR-CONICET Buenos Aires University, Buenos Aires, Argentina, 2Depto
Biodiversidad y biología experimental Buenos Aires University, Buenos Aires, Argentina

1

Keywords:
Fungal metabolite profile； metal complexation
Novel aspects:
The post-column addition of metal solutions, together with the use of different sources and ion modes for metabolic profiling,enabled the differentiation of isomers such as cyclosporine and isocyclosporine A
Abstract:
Metabolite profiling is crucial for many aspects of fundamental research and industrial applications of filamentous fungi and othermicroorganisms. The use of a quadrupole - time-of-flight mass spectrometer has proved to be very suitable for this purpose due to
its mass high resolution capability, and ESI is the most widely used ionization method in both positive and negative ion modes.
In this work, we report the metabolite profiling using HPLC-MS in a QTOF, of a strain of Fusarium oxysporum that has significant
antifungal activity against pythopathogens. After optimization of LC conditions such as different solvent systems and phasecolumns（C18, C8, PFP,HILIC）and in order to explore the whole universe of chemical structures that may be present in the extract,different sources were employed like ESI, APCI and APPI in both positive and negative ion modes. C18 chromatographic phase wasselected for the analysis of the mycelium extract, PFP for medium analysis and HCOOH 0.1 % -MeOH as solvent system in bothcases.
A complex metabolite pattern was observed in all LCMS runs. When ESI was employed, a greater number of metabolites wereobserved compared to APCI and APPI runs, while APCI and APPI gave similar results.
Using the obtained molecular formulas and MS/MS data, a search in different libraries and literature1 was performed allowing theidentification of seven cyclosporines, sansalvamide, N-methyl sansalvamide, a related cyclodepsipeptide not previously described,
fusarubin and related compounds.
APCI and APPI resulted more sensitive for cyclic peptides, and MS/MS analysis of［M+H］+ was especially useful for thesecompounds, which give predominantly sodiated forms in the ESI LCMS runs.
The use of negative ion mode allowed the identification of some compounds such as the antibiotic F244, where MS/MS fragments
were very informative for its identification.
Many of the identified structures were confirmed by NMR spectroscopy after the isolation of the compounds.
Based on our previous results in the application of ESI-LCMS with post-column addition of metal solutions for the differentiation ofisomers, as in the case of hydroxypyridine N-oxides 2 and dihydroxyarenes 3 , we used this methodology to obtain further
information about the metabolites produced by this fungus. The use of metal complexation by post-column addition of a solutionof Ca（II）, Cu（II）or Zn（II）allowed the differentiation of isomeric Cyclosporine A from Isocyclosporine A, which are known to beindistinguishable by MS/MS 4 . In the MS spectra, complexes of cyclosporine A with the corresponding divalent metal wereobserved, whereas complexes were not observed for isocyclosporine A.
In conclusion, the use of different sources, ion modes and post-column addition of metal solutions allowed the complete analysisof the extracts of this fungal strain, providing complementary information.
1-Havlicek, V.; Jegorov, A.; Sedmera, P.; Ryska, M. Sequencing of cyclosporins by Fast Atom Bombardment and linked-scan mass spectrometry.Org. Mass Spectrom. 1993, 28, 1440-1447.
2- Butler M.; Arroyo Mañez P.; Cabrera G. M. Differentiation of Isomeric Hydroxypyridine N-Oxides Using Metal Complexation and Electrospray Ionization Mass Spectrometry.
J. Am. Soc. Mass Spectrom. 2011, 22, 545-556.
3- Butler M.; Arroyo Mañez P.; Cabrera G. M. Differentiation of dihydroxyarene isomers using metal complexation and electrospray ionization mass spectrometry.
59º Congress American Society for Mass Spectrometry, Denver, Colorado, USA 2011.
4- Jegorov, A.; Havliek, V. Spontaneous N-O acyl shift in the [M+H]+ ions of [MeBmt1]-cyclosporins in an ion trapJ. Mass Spectrom. 2001 36, 633-640.

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 27: Progress in Microbiology

PWe-104
13:30 － 14:40

Pyruvate Phosphate Dikinase Phosphorylation Increases Lipid Production
in Rhodopseudomonas palustris: a Phosphoproteomic Analysis

Chia-Wei Hu2, Miao-Hsia Lin1,3, Tsun-Hsuan Yi2, Hsuan-Cheng Huang4, Wei-Chi Ku1, Chia-Feng Tsai5,
Yu-Ju Chen5, Naoyuki Sugiyama6, Yasushi Ishihama1,6, Hsueh-Fen Juan2,7, Shih-Hsiung Wu2

Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto, Japan, 2Institute of Molecular and Cellular
Biology, National Taiwan University, Taipei, Taiwan, 3Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan,
4
Institute of Biomedical Informatics and Center for Systems and Synthetic Biology, National Yang-Ming University, Taipei,
Taiwan, 5Institute of Chemistry, Academia Sinica, Taipei, Taiwan, 6Institute for Advanced Biosciences, Keio University,
Daihoji, Tsuruoka, Japan, 7Department of Life Sciences, National Taiwan University, Taipei, Taiwan
1

Keywords:
Phosphoproteome, Rhodopseudomonas palustris, lipid production
Novel aspects:
Lipid production is regulated by protein phosphorylation which is identified by phosphoproteomic analysis
Abstract:
Rhodopseudomonas palustris（R. palustris）is a purple nonsulfur anoxygenic phototrophic bacterium with metabolic versatility. It
is able to grow under photoheterotrophic and chemoheterotrophic states and has the potential for bioenergy production andbiodegradation. This study is the first to identify the phosphoproteome of R. palustris including 100 phosphopeptides from 54
phosphoproteins and 74 phosphopeptides from 42 phosphoproteins in chemoheterotrophic and photoheterotrophic growth
conditions, respectively. Among the phosphopeptides, phosphorylation at the threonine residue, Thr 487, of pyruvate phosphate
dikinase（PPDK, RPA1051）was found to be involved in the regulation of carbon metabolism. Here, we show that PPDK enzyme
activity is higher in photoheterotrophic growth, with Thr 487 phosphorylation as a possible mediator. Under the same
photoheterotrophic condition, bacteria with wild-type PPDKs showed enhanced accumulation of total lipids than those with the
mutant（T487V）forms. This study reveals the role of the PPDK in the production of biodiesel material, lipid content, with threonylphosphorylation as one of the possible regulatory events during photoheterotrophic growth in R. palustris.

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 27: Progress in Microbiology

PWe-105
11:10 － 12:20

Rapid and Simple Characterization of Mycolic Acids in the Suborder
Corynebacterineae Using MALDI Spiral-TOFMS

Kanae Teramoto1, Takafumi Sato1, Shinichi Mukosaka1, Tomohiko Tamura2, Moriyuki Hamada2,
Ken-ichiro Suzuki2
1

JEOL Ltd., Tokyo, Japan, 2NITE Biological Resource Center, Kisarazu, Japan

Keywords:
Mycolic acids, Rapid and simple characterization, high mass resolving power, MALDI Spiral-TOFMS, Corynebacterineae
Novel aspects:
Crude mycolic acids in the suborder Corynebacterineae were successfully characterized using MALDI Spiral-TOFMS rapidly andeasily.
Abstract:
Introduction
Mycolic acids are 2 -alkyl 3 -hydroxy fatty acids which are present in the cell envelope of the suborder Corynebacterineae, andused for their chemotaxonomy. Since compositions and contents of mycolic acids are related to virulence of Mycobacterium
tuberculosis, their synthetic pathway is one of the targets for antitubercular drugs. As a result, the determination of thecompositions and contents of mycolic acids is important. Mycolic acids in bacterial cells are classified based on the functionalgroups and the number of unsaturated bonds, and each mycolic acid has a distribution in the number of total carbon atoms.
Though thin layer chromatography（TLC）methods have been used widely, it is impossible to analyze such complex mycolic acids
without the help of other methods. Accordingly, to examine exact distribution of carbon chain length, each mycolic acid separated
by TLC is further analyzed using MALDI-TOFMS. However, TLC methods are cumbersome so that it is not suitable for analysis of alarge number of samples.
We expected that mycolic acids are analyzed using MALDI Spiral-TOFMS［ 1 ］without prior separation by TLC, because eachmycolic acid with slight mass differences can be distinguished owing to high mass resolving power. In this study, rapid and simple
compositional analysis of crude mycolic acids using MALDI Spiral-TOFMS was demonstrated.
Experimental
Mycolic acids were extracted from the genera Corynebacterium, Dietzia, Rhodococcus, Gordonia, and Nocardia by saponification
with KOH followed by methyl esterification with methanol-toluene-sulfur acid. As the matrix for sample ionization,
2,5-dihydroxybenzoic acid was used. Samples were measured by the SpiralTOF（JMS- S3000； JEOL Ltd., Tokyo, Japan）, of whichflight length was ca. 17 m along with eight cycles of the spiral trajectory to achieve high mass resolving power. The mass spectral
data were analyzed using the Polymerix software（Sierra Analytics, Modesto, CA）.
Results
Many peaks of mycolic acid methyl esters were observed. Especially, in the MALDI mass spectra of the genus Nocardia, adjacentpeaks originated from CH2-CH2 and C=O, whose mass difference is only Δ0.036 Da, were clearly separated thanks to the highmass resolving power of the MALDI Spiral-TOFMS. As a result, the number of total carbon and oxygen atoms, and unsaturated
bonds were successfully obtained. Furthermore, relative peak intensity of each mycolic acid could be calculated. In this study, 16
categories based on 3 - 5 of oxygen atoms and 0 - 5 of unsaturated bonds were used for analysis of mycolic acids. These
categories are related to the functional groups such as cyclopropane ring, methoxy group, and keto group. By showing the relative
peak intensities of each mycolic acid in the contour map, in which horizontal axis indicates the number of oxygen atoms andvertical axis indicates the categories of mycolic acids, different distribution of mycolic acids in each strain were clearly visualized.
This would be a new rapid and easy technique for the characterization of mycolic acids. This method could be applied not only
for the chemotaxonomy, but also for the development of antitubercular drugs and vaccines.
Acknowledgment
This study was partly funded by a grant from the Institute for Fermentation, Osaka（IFO）.
［ 1 ］Satoh T.et al., J. Am. Soc. Mass Spectrom., 18, 1318（2007）.
［ 2 ］Barry C. E. et al., Prog. Lipid Res., 37, 143（1998）.

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 27: Progress in Microbiology

PWe-106
13:30 － 14:40

Evaluation of Proteomic Profile with Principal Component Analysis inGroup A Streptococci

Akira Okamoto1, Taguchi Yoshihiro2
1

Aichi University of Education, Aichi, Japan, 2Chuo University, Tokyo, Japan

Keywords:
Group A Streptococci, Shotgun proteome, Principal Component analysis, data-mining
Novel aspects:
We applied Principal Component Analysis（PCA）for shotgun-proteomic analysis of Group A Streptococci. The results suggested
that the PCA is useful for data-mining of proteomic analysis.
Abstract:
Microbes modify its proteome to adapt the environment. It is well known that many pathogens produce exotoxins, cell surface
proteins, or effectors to attach, survive, and spread in living host organisms. Therefore, the shotgun proteomic analysis is aneffective approach to evaluate the phenotype of harmful infectious bacteria. The next challenge is to handling big data thatderived from mass-spectrometer, and data mining to discover what proteins characterize the pathogenic phenotype. To harvest
the fruitful information from proteomic profile, we applied the principal component analysis（PCA）to shotgun proteomic analysisof Streptococcus pyogenes. S.pyogenes is well known human pathogen that causing many infections including pharyngitis,
impetigo, and toxic-shock like syndrome. This pathogen produces large amount of exotoxins such as proteases and superantigens.
The proteome, including exotoxins, are influenced by culture conditions, however, the overall picture of changing proteome withgrowth phase have not clearly evaluated in S. pyogenes. Herein, we analyzed the proteomic profile with growth phase of S.
pyogenes, and evaluated its profile with PCA.
In this study, the proteome of S. pyogenes SF370 was evaluated. The four point of cell culture were harvested during middle
logarithmic to stationary phase under oxidatively（with shaking）or statc culture condition. The cell culture was divided into two
subcellular fractions, supernatant（secreted proteins）, and whole cell fractions. The protein mixture was digested with trypsin,and the resulted peptides were served to the shotgun proteomic analysis. Detection of fragmented proteins are performed by
LTQ- Orbitrap XL（ThermoFisher SceintificInc.）combined with Paradigm MS 4 LC system（MichromBioResources Inc.）.Obtained
spectrum by LTQ is identified by MASCOT program based upon in-house amino acid database. To be identified, at least two
unique amino acid sequences for each protein is required. Each of samples was measured in triplet for each of two subcellular
fractions, four time points under two distinct incubation conditions. Analyzed quantity by PCA is percent of experimentally
modified protein abundant index（%emPAI）.
As a result, 810 proteins were identified in two subcellular fraction, four time point and two（wish or without shaking）culture
conditions. With the PCA analysis that 48 experiments（three subcellular fractions with two growth phases in triplet）wereembedded into plane（D= 2 ）, each experiments formed separate clusters. In first-round PCA, twenty-three proteins were pickedup as the factors that characterized each experiment. After remove them, additional thirty proteins were picked up in secondround PCA. These picked up proteins were classified with functional category： translation（24 proteins； including 20 ribosomal
proteins）, carbohydrate metabolism（ 9 ）, chaperone（ 4 ）, amino acid metabolism（ 2 ）, replication（ 1 ）, energy production（ 1 ）,cell division（ 1 ）, signal transduction（ 1 ）, function unknown or prediction only（ 4 ）, and virulence associated proteins（ 6 ）.
Furthermore, we tried to assign the biological meaning for these proteins. For example, peroxiredoxin reductase（SPy2079：
AhpC）, which is estimated to be involved in oxygen metabolism and hydrogen peroxide decomposition, was found in shaking
culture condition rather than static condition. It seems reasonable that the increasing amount of AhpCin shaking condition
because the shaking condition induces the higher oxygen stress. Another examples, many virulence associated proteins, pyogenic
exotoxin B（SpeB； SPy2039）, pyogenic exotoxin C（SpeC； SPy0711）, mitogenic factors（Mf； SPy2043, Mf 2 ； SPy0712, and Mf 3 ；SPy1436）, and M protein（Emm； SPy2018）, are picked up by PCA analysis. SpeB increases with time-dependent, in both shaking
and static culture condition. Both Mf 2 and SpeC increase under the shaking condition, but decrease under the static condition.
The amount of both M protein and Mf increased, but that of Mf 3 decreased in shaking condition, although their amount keep
constant value under the static incubation condition.
These results indicated that the PCA is useful for the data mining with proteomic analysis.

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 27: Progress in Microbiology

PWe-107
11:10 － 12:20

Glycopeptidolipids with novel peptide composition from Mycobacterium
avium-intracellulare complex

Naoya Ichimura, Takeshi Kasama

Tokyo Medical and Dental University, Tokyo, Japan

Keywords:
Mycobacterium, MAC, Glycopeptidolipid, GPL, Lipopeptide
Novel aspects:
We found novel glycopeptidolipids containing valine or leucine in the peptide core from Mycobacterium avium intracellulare
complex. These structure were confirmed with MS, MS/MS and amino acid LC/MS analysis.
Abstract:
Introduction
Mycobacterium avium-intracellulare complex（MAC）is a widely distributed opportunistic pathogen that causes lung infections
such as tuberculosis in immune-compromised hosts, and disseminated infections in AIDS patients. MAC specifically expresses
glycopeptidolipids（GPLs）on their cell walls. GPL expression is strongly associated with colony morphology, sliding motility andbiofilm formation.
In MAC, GPLs are commonly composed of a lipopeptide core structure, consisting of a peptide moiety（D-Phe-D-alloThr-D-Ala-Lalaninol）, and a fatty acid. The lipopeptide is glycosylated with a 6 -deoxy-talose to the D-alloThr and a rhamnose to theL-alaninol, respectively. This is called nonspecific GPL which is commonly expressed in all serotypes of MAC. This is further
glycosylated with specific oligosaccharides at the 6 -deoxy-talose. These are called sero-specific GPLs（ssGPLs）, and have been
serologically classified into ~30 species to date. Molecular diversity can occur on parts of the acyl moiety（chain length andmethylation of hydroxy group）and sugar moieties（methylation and acetylation）. These modifications influence host immune
responses. Elucidation of the intact structure of GPL, including its molecular diversity is essential to understand MAC virulence.
We have studied the structure of ssGPLs using MALDI TOF-MS. When we applied total lipids of MAC serovar 4 to MS, we found
two atypical ions of ssGPLs, which were 34 or 48 Da less than the dominant ion of ssGPLs. These ions could not be explained as
resulting from conventional molecular diversity. Additionally, these atypical ions were also found in previous reports by otherresearchers using MAC. However, there have been no detailed studies performed on these ions to date. Therefore, weinvestigated the reason as to why these atypical ions appear.
Material and method
The reference strain of MAC was ATCC 700737 for serovar 4, and was cultured in Middlebrook 7 H 9 broth with oleic acid albumin
dextrose complex enrichment（Becton Dickinson Co.）for 3 weeks. Whole lipids of autoclaved MAC were extracted using Folch’s
procedure. After alkaline methanolysis for eliminating acetyl groups from GPLs and other alkali-labile lipids, the alkali-stable lipids,
including the GPLs, were recovered by Folch’s procedure, followed by purification of deacetylated ssGPLs with the one-stepcolumn method. For the preparation of the lipopeptide component of GPLs, the purified deacetylated GPLs were subjected toacidic methanolysis. After that, the lipopeptide component was extracted by Folch’s procedure.
MALDI TOF-MS and MS/MS spectra were obtained using an ultrafleXtreme（Bruker）.
For preparation of amino acids, the lipopeptide component was subjected to acidic hydrolysis. The recovered amino acids were
derivatized with 3 -aminopyridyl-N-hydroxysuccinimidyl carbamate（APDS）. The amino acids were analyzed by ESI-LC/MS using
HPLC1100（Agilent）and Esquire 3000plus（Bruker）.
Results and Discussion
We observed ions at m/z 1671.9 and 1685.9, which were 48 and 34 Da less than m/z 1719.9, dominant ssGPLs ions of serovar 4,
respectively. MS/MS spectra of these ions showed product ions such as rhamnosyl lipopeptide portion which kept the massdifference. Furthermore, MS/MS analysis of purified lipopeptide components showed that Phe was replaced by other amino acid.
LC/MS analysis of APDS derivatives of amino acids showed presence of Phe, allo-Thr, Ala, Val, Leu. However, we did not observe
other amino acids. Considering these results, there were at least three lipopeptide species in the amino acid sequence, including
Val-alloThr-Ala, Leu-alloThr-Ala and Phe-alloThr-Ala. Because Val, Leu and Phe closely resemble each other in hydrophobic
property, mycobacterial peptide synthetase may incorporate these amino acids into GPLs.
We confirmed that mass spectra showing atypical mass differences were also observed in MAC serovar 2 （ATCC25291）and
serovar 16（ATCC13950）in our experiment. Therefore, Val- or Leu- containing GPLs are commonly expressed in MAC. However, it isunknown whether other Mycobacterium species that contain GPLs also express these atypical GPLs.

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 27: Progress in Microbiology

PWe-108
13:30 － 14:40

Identification of Medically Important Candida Species by LC-Selective
Tandem Mass Spectrometry

Po-Chih Chang1, Muralidhar Reddy1, Chien-Liang Chen1, Tsung C Chang2, Hsien-Chang Chang3,
Yen-Peng Ho1

Department of Chemistry, National Dong Hwa University, Hualien, Taiwan, 2Department of Medical Laboratory Scienceand Biotechnology, National Cheng-Kung University, Taiwan, 3Department of Biomedical Engineering, National ChengKung University, Taiwan
1

Keywords:
candida infections clinical LC-MS/MS markers
Novel aspects:
Detection of Candida species in a microbial mixture using a new approach - selective proteotypic-peptide analysis.
Abstract:
Candida infections are life threatening in neutropenic and AIDS patients. In particular, C. albicans, C. glabrata, C. lusitaniae, C.
krusei, C. parapsilosis and C. tropicalis, are the most frequently occurring species in the clinical practice. Therefore, accurate
detection and species identification is of utmost importance to initiate appropriate therapy. Here we focused on reliable detection
and identification of candida species present in a mixture by LC- selective proteotypic-peptide analysis method. The LC-MS/MS
analyses of species-specific peptides and database search were used to accurately identify the fungal/microbial mixture. The
differentiation/characterization of many candida species can be achieved by performing a series of MS/MS analyses of selectedmarker peptide ions. Furthermore, we managed to identify different candida species mixed in various abundances simultaneously
during a single LC-MS/MS analysis.
Further, the complete genome of C. krusei is currently not available in NCBI protein databases（only 116 proteins are presented）.
Therefore, we cannot use the database search algorithm to accurately identify proteins associated with C. krusei. To address thisproblem, we used an overlapping approach to find C. krusei biomarkers that are not unique to this species but are simultaneously
present only in this species. C. krusei is considered present in the sample if these markers are all present in a sample andidentified.
The method presented here（LC-selective MS/MS）provides the advantages of excellent selectivity and high accuracy forcharacterizing complex microbial mixtures. Also our findings indicate that the proposed approach could be an alternate in thenear future of the traditional pathogenic identification methods.

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 27: Progress in Microbiology

PWe-109
11:10 － 12:20

Metabolome of Aspergillus: Towards the Early Diagnosis of Invasive
Aspergillosis

Havlicek Vladimir1, Strohalm Martin1, Kavan Daniel1, Lemr Karel1, Schug Kevin2, Sulc Miroslav1
1

Institute of Microbiology, Prague, Czech Republic, 2University of Texas at Arlington, TX, USA

Keywords:
Aspergillus, Siderophore, Biomarker, Diagnosis, Aspergillosis
Novel aspects:
Focusing to metabolome in fungal diagnostics represents a similar benefit to what we have learned from proteomics： going frompeptide mapping to peptide sequencing could give better identification rates.
Abstract:
Classification of microorganisms by MALDI profiling has already become a standard. With the growth of fungal databases, theremaining obstacles are represented by strain mix analysis, the longer cultivation period mandatory for sufficient fungal material
production and presence of melanine-like structures interfering with MALDI analysis. At present, no data are available on thesensitivity of MALDI protein typing approaches of filamentous fungi including Aspergillus.
In this presentation we will report on development of highly specific molecular approaches for fast and early identification ofaspergillosis, one of the most prevailing fungal infections occurring in immunocompromised humans. Targeted and untargeted
metabolomic1 and proteomic2 approaches, utilizing combinations of separation and enrichment strategies, allowed us to define
new small molecule and/or proteinaceous fungal biomarkers useful in early diagnostics, the analysis of mixed strains and fungal
phenotype discrimination. In metabolomic approaches a special focus was dedicated to siderophores, mycotoxins, melaninstructures, non-ribosomal peptides and lipids. For Fe-containing compound classification the negative mass defect andthe 54Fe/56Fe isotope ratio data filtering was used.
Strain-specific low molecular weight biomarkers were identified in eight reference Aspergillus strains（including A. fumigatus, A.
flavus, A. niger）using our in-house developed and publicly available software mMass 3 . The updated Aspergillus metabolite
database was used for MALDI and NALDI-FTMS profiling data interpretation, as well as for fungal strain classification by HPLC-ESI/FTMS. In addition, a new de novo sequencing tool dedicated to non-ribosomal cyclic microbial products was also developed. In
this software a large database of non-ribosomal peptide fungal building blocks was incorporated. The software did not only
annotate and characterize the tandem mass spectrum of a known cyclic peptide, but also provided a sequence tag in an unknown
natural product. We used the strategy of fragment ion“pre-characterization“ based on its characteristic neutral losses andidentification of the neighboring residues according to characteristic（pre-calculated）exact mass differences of one building block［or its di-（tri-）mers］. One unknown building block in the sequence did not necessarily hamper the sequence determination.
The final sequences were scored by using comparison of the calculated theoretical peaklists（a- and b-ions, water and ammonia
losses）with the acquired data.
We conclude that metabolomic approach conducted by mass spectral techniques provides not only the disease biomarkers but
also may define new antifungal drugs. Siderophores being secreted in 8 -12 hour time frame upon inoculation can serve as early
diagnosis features also useful in 68Ga-enhanced high-resolution computer tomography 4. Biomarker enrichment strategies combinedwith advanced mass spectral software tools can help mass spectrometry to be soon translated from bench to bedside in small
molecule area.
Acknowledgement：
The authors acknowledge support from the Ministry of Education, Youth and Sports of the Czech Republic（ME10013）, IMIC
institutional research concept RVO61388971 and Czech Science Foundation（P206/12/1150）.
References：
1. Pavlaskova, K.； Nedved, J.； Kuzma, M.； Zabka, M.； Sulc, M.； Sklenar, J.； Novak, P.； Benada, O.； Kofronova, O.； Hajduch, M.；
Derrick, P. J.； Lemr, K.； Jegorov, A.； Havlicek, V., Characterization of Pseudacyclins A-E, a Suite of Cyclic Peptides Produced by
Pseudallescheria boydii. Journal of Natural Products 2010, 73,（ 6 ）, 1027-1032.
2. Sulc, M.； Peslova, K.； Zabka, M.； Hajduch, M.； Havlicek, V., Biomarkers of Aspergillus spores： Strain typing and protein
identification. International Journal of Mass Spectrometry 2009, 280,（ 1 - 3 ）, 162-168.
3. Strohalm, M.； Kavan, D.； Novak, P.； Volny, M.； Havlicek, V., mMass 3 ： A Cross-Platform Software Environment for Precise
Analysis of Mass Spectrometric Data. Analytical Chemistry 2010, 82,（11）, 4648-4651.
4. Petrik, M.； Haas, H.； Schrettl, M.； Helbok, A.； Blatzer, M.； Decristoforo, C., In vitro and in vivo evaluation of selected Ga-68siderophores for infection imaging. Nuclear Medicine and Biology 2012, 39,（ 3 ）, 361-369.

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 27: Progress in Microbiology

PWe-110
13:30 － 14:40

Design Of Specific methodology For The Identification Of Beta-Lactamase
Producing Enterobacteriaceae Using MALDI Mass Spectrometry

Emmanuel Q wey1, Matthew Openshaw2, Philippa Hart2, Omar Belgacem2, Pranav Somaiya3,
Shervanthi Homer-Vanniasinkam3, Indran Balakrishnan1

Royal Free Hospital Foundation Trust London United Kingdom, 2Shimadzu, Manchester, United Kingdom, 3NPIMR-UCL,
London, United Kingdom

1

Keywords:
Extended spectrum beta-lactamase, MALDI, sample preparation
Novel aspects:
The design of an algorithm and optimised sample preparation for the detection of beta-lactamase production in Escherichia coli.
Abstract:
The emergence of multi-drug resistant（MDR）enterobacteriaceae that can produce Extended Spectrum β-lactamases（ESBLs）iscurrently a major worldwide concern. They confer resistance to a wide spectrum of beta-lactam antibiotics currently used as 1 stline empirical therapy in the management of gram negative bacterial infections. Escherichia coli (E. coli) is the most frequent gramnegative bacillus isolated from blood cultures in clinical settings and CTX-M-15bla gene the most common coding for an ESBL
worldwide. The use of MALDI mass spectrometry for microbial speciation is generally performed by MS only fingerprinting
methods and is now widely applied. The technique is now standardized, but the commonly accepted methods do not allow
differentiation between an ESBL producing E. coli and antibiotic sensitive E. coli. The aim of this study is to explore the variety ofsample preparation methods inherent to MALDI and apply them directly to clinical/laboratory isolates in order to create a
valuable diagnostic tool that can be used on an everyday basis in microbiology laboratories.
The isolates of E. coli usedcarry fully characterised plasmids with a variety of beta-lactamase genes under an inducible andcontrollable expression system. These isolateswere grown overnight on LB agar and a variety of extraction protocols and layering
methods were employed. The utilisation of different layering methods led to changes in the crystallisation process. These were
ultimately used in the optimisation of the resolving power of the generated superspectra. Several matrices were applied to thesecolonies as per standard protocol using layering. MS spectra were obtained on organisms using linear mode MALDI-TOF-MS（AximaTM Assurance, Shimadzu, Manchester, UK）.
An MS-only pattern recognition method using CHCA, sinapinic acid and DHB matrices was employed alongside machine learning
using the SARAMIS™ software platform to create individual“superspectra“ for fully phenotypically and genotypically characterised
isolates.“Superspectra“ created were then used in an attempt to identify both single and mixed populations of fully characterised
E. coli isolates. Subsequently further E. coli isolates with full phenotypic and genotypic characterisations were submitted forcomparison to the previously generated“superspectra“.

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 27: Progress in Microbiology

PWe-111
11:10 － 12:20

Differentiation of Single Attachment versus Double Attachment of
Phycoerythrobilin Chromophores to Phycobiliproteins of Fremyelladiplosiphon Cyanobacteria using MALDI Mass Spectrometry

M Nazim Boutaghou2, Christina M Kronfel2, Leanora S Hernandez2, Avijit Biswas2, Wendy M Schluchter2,
Richard B Cole1,2
1

Université Pierre et Marie Curie (Paris 6), Paris, France, 2University of New Orleans, New Orleans, LA, USA

Keywords:
Fremyella, phycobiliproteins, phycoerythrobilins, MALDI, bilin-lyase
Novel aspects:
MS methods allow characterization of the site-specificity of cyanobacterial bilin lyases. A novel MALDI approach to distinguishbetween singly- and doubly-attached PEB chromophore-peptide complexes overcomes current MS limitations.
Abstract:
Introduction
Cyanobacteria use chromophorylated proteins called phycobiliproteins as light-harvesting structures. Bilin chromophore
attachment to phycobiliproteins, an enzyme-catalyzed post-translational modification process, serves to improve the lightabsorption characteristics. Bilin-lyases attach a bilin chromophore to the phycobiliprotein through a thioether bond that covalently
links the chromophore to a cysteine moiety of the phycobiliprotein. The tetrapyrollic bilin chromophores attach to the proteinsthrough the 3 1 carbon of the bilin. Double attachment of the same bilin can occur as well, and in this case, carbons 3 1 and 181 of
the bilin are each forming covalent linkages to distinct cysteine moieties on the phycobiliprotein.
State-Of-The-Art
Mass spectrometry has become a method of choice for investigating the site-specificity of cyanobacteria bilin-lyases. Mass
spectrometric analysis of peptides generated by enzyme proteolysis enables the detection of proteolytic peptides containing bilin
chromophores. Further analysis using tandem mass spectrometry allows localization of the specific site（s）of attachment bydetection and identification of the peptide-linked chromophore. We have used this approach to characterize the site-specificity of
newly developed bilin lyases CpeY & CpeZ, as well as CpeS. For the CpeY & CpeZ heterodimer, we were able to localize the siteof PEB attachment to Cys-82 of the C-phycoerythrin alpha subunit, whereas CpeS attaches PEB specifically to Cys-80 of theC-phycoerythrin beta subunit. However, there is a mass spectrometric limitation when examining tryptic peptides containing two（or more）cysteines if one seeks to ascertain whether chromopeptides are singly or doubly attached. The problem is that singly
and double attached chromopeptides appear at the same m/z value, thus, up until the present, only NMR analysis has beensuccessful at determining the number of sites of chromophore attachment. Moreover, if the molecular weight of the peptidechromophore complex is large, tandem mass spectrometry experiments relying on collision induced decompositions may beineffective at generating fragments in high yields.
Novel Advance
We report in this work a new, fast and accurate method for discriminating singly from doubly attached chromophores using
MALDI TOF/TOF mass spectrometry. This method was developed from mass spectral analysis of chromopeptides that had
undergone in vitro and in vivo attachment of bilin chromophores to phycobiliproteins. Distinction is based on a difference
corresponding to a characteristic neutral loss that appears in the MALDI-TOF mass spectrum only when the bilin is singly
attached. When a tryptic peptide bearing a singly-attached PEB（covalently bound at the A-ring）is subjected to laser photons（λ= 355 nm）during a MALDI experiment, the C 15 -C 16 bond of PEB was observed to undergo a heterolytic cleavage which
systematically results in the loss of the D-ring moiety of PEB. This cleavage reproducibly produces a fragment peak that is 121 Da
below the mass of the intact protonated chromopeptide complex in the MALDI-ToF mass spectrum. In cases where the PEB
moiety is doubly attached to the phycobiliprotein through the A- and D-rings, no such decomposition is observed. The appearanceof a peak at 121 Da below that of the protonated tryptic peptide is deemed to be diagnostic of attachment of the A-ring of PEB ata single site on the phycobiliprotein. Conversely, failure to detect a peak at 121 Da below that of the protonated tryptic peptide isstrong evidence of double attachment at the A- and D-rings of PEB. The validity of using the above-described photophysicalproperty to identify the number of linkages is shown for singly attached PEB chromopeptides resulting from proteolysis ofrecombinant C-phycoerythrin alpha and beta subunits, singly attached PEB chromopeptides and a doubly-attached PEB
chromopeptide resulting from the proteolysis of holo-phycoerythrin isolated from Fremyella diplosiphon.

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 27: Progress in Microbiology

PWe-112
13:30 － 14:40

The study and characterization of growth factors produced by murine
herpes virus using multistage MS techniques

Andrea Stanova, Jozef Marak, Miroslava Supolikova, Livia Cikova, Frantisek Golais, Pavol Kois
Comenius University, Bratislava, Slovak Republic

Keywords:
mass spectrometry, murine herpes virus, growth factor, cellular transformation, separation methods
Novel aspects:
Detailed study of growth factor, its purification and physico-chemical characteristics along with its structure, using multistage MS
techniques might open a new direction in research of herpes viruses.
Abstract:
At the present, the trends in modern analytical chemistry and biosciences are closely connected with the necessity to solve thecomplex analytical problems associated with multi-component qualitative and quantitative analyses of substances present incomplex biological matrices at too different concentration levels（ 3 -10 decimal orders）. For successful solution of analyticalproblems associated with the analysis of substances present in complex matrices, one is always looking for new analytical
approaches, often based on already known high-performance separation and detection techniques. Mass spectrometry（MS）is
able to detect and identify analytes with high selectivity without a lost of sensitivity, dynamic range and/or speed of analysis.
However, if the analyte is part of a complex mixture of substances, obtained mass spectra contain ions from all substances which
are present at the same time in the ion source and, consequently, if the analyte is a minor component of the mixture, its
identification with a certain degree of certainty is very difficult and in some cases mostly impossible. Taking the above mentioned
facts in mind high performance liquid chromatography with mass spectrometry（HPLC-MS）took a significant and important
position in bioanalytical chemistry.
There exist at least 8 strains of murine herpes virus（ MHV）, 5 strains（76, 78, Šumava. 4556, 5682）were isolated from Apodemus
flavicolis, and 3 strains（60, 68, 72）were isolated from Myodes glareolus. A complete sequence and genome analysis is known
only with MHV-68. It was shown later, that MHV-76 represents a naturally occurring mutant that lacks 9, 538 bp of the left end of
the unique portion of the genome encoding nonessential pathogenesis-related genes. As concerns other isolates of MHV, they are
only poorly characterized, if at all. Recently, some monoclonal antibodies（moabs）directed against gB glycoprotein of MHV-68
have been characterized, some of them, especially those recognizing N-terminal domain neutralize the virus, and some other give
good reaction in immunoblotting or ELISA method. The use of these moabs might contribute to further characterization of all MHV
isolates. Furthermore, there is known that herpes viruses are able to induce several types of persistent or latent infections andmay be under certain conditions oncogenic. In studies of interaction between the various types of host cells and various herpes
viruses a new class of substances resembling growth factors probably encoded by virus genome have been obtained. These
factors are produced in certain cells in conditions non-permissive for virus replication, e. g. at supraoptimal temperature or in thepresence of inhibitors of DNA replication, or in the cells which are naturally non-permissive for virus replication. Although thesefactors are only partially purified at present, their fundamental characteristics already exist. The cultivation of non-transformed
cells in the presence of these factors leads to the appearance of the transformed phenotype, while the phenotype of transformed
cells becomes changed towards the normal ones, As shown with the factor associated with human herpes simplex virus（HSV）,
the gB glycoprotein of this virus plays an important role in its synthesis and some moabs against gB of HSV neutralized thebiological activity of this factor and the same has been shown with the factor associated with pseudorabies virus. Similar factor
has been also demonstrated in cells infected with MHV-68.
This work was focud on detailed study of growth factor with the use of moabs against gB of MHV-68, its purification and physicochemical characteristics along with its structure, using multistage MS techniques in combination with different separation
techniques.
Acknowledgement： This work was supported by a grant from the Slovak Research and Development Agency（No.VVCE-0070-07）
and the grants of Slovak Grant Agency, No`s.1/0265/11 and 1 /1305/12.

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 27: Progress in Microbiology

PWe-113
11:10 － 12:20

MALDI Mass Spectrometry and MALDI-MS/MS Investigation into
Chemically Induced Peptides of Beta-Lactamase Producing
Enterobacteriaceae

Emmanuel Q Wey2, Philippa J Hart1, Matthew Openshaw1, Omar Belgacem1, Pranav Somiya3,
Shervanthi Homer-Vanniasinkam3, Indran Balakrishnan2
1

Shimadzu, UK, 2Royal Free Hospital NHS Foundation Trust, London , UK, 3NPIMR-UCL, London, UK

Keywords:
MALDI, Beta-lactamase production, sample preparation
Novel aspects:
The use of specific chemical cleavages prior to bio typing methods
Abstract:
The emergence of multi-drug resistant（MDR）enterobacteriaceae that can produce Extended Spectrum β-lactamases（ESBLs）iscurrently a major worldwide concern. ESBLs are a group of enzymes that are transferred via plasmids in gram negative bacteria.
They confer resistance to a wide spectrum of β -lactam antibiotics currently used as 1 st line empirical therapy in the
management of gram negative bacterial infections. Escherichia coli (E. coli) is the most frequent gram-negative bacillus isolated
from blood cultures in clinical settings and CTX-M-15 bla the most prevalent gene coding for an ESBL worldwide. The use of
MALDI mass spectrometry for microbial speciation is generally performed by MS only fingerprinting methods and is now widely
applied. The aim of this investigation was to use a bottom-up proteomics approach to highlight peptides/proteins associated with
ESBLs, and consequently antibiotic resistance.
The methodology applied in this study deviates from MS-only pattern recognition methods. Here, isolate extracts underwent
chemical cleavage, under optimised conditions using a variety of endopeptidases. MALDI-MS was used to generate polypeptide
profiles thought to be related to the presence of β-lactamases in phenotypically and genotypically characterised isolates of E. coli
carrying fully characterised plasmids with a variety of beta-lactamase genes under an inducible and controllable expression
system. Tandem mass spectrometry was then used in an attempt to confirm the identity, and thus the relevance, of these
peptides.
E. coli strains were grown overnight on LB agar prior to extraction. E. coli carrying fully characterised plasmids with a variety ofbeta-lactamase genes were identified using linear mode MALDI-TOF-MS（AximaTM Assurance, Shimadzu, Manchester, UK）and
SARAMIS TM software. Once identity was confirmed, the β-lactamase containing E. coli and antibiotic sensitive controlswere
subjected to chemical cleavage, and co-crystallised with matrices under varying conditions. In-silico digests and blasts of the β-lactamase enzyme were performed via the Expasy Bioinformatics Resource Portal. The peptides generated in-silico were thensearched for in the peptide mass fingerprints generated as above. Subsequently, any m/z species that were found to be present inboth the theoretical and experimental data were subject to MALDI-MS/MS. All MS/MS data were submitted to MASCOTTM for
peptide/protein identification. Antibiotic sensitive E. coli was analysed and processed alongside ESBL containing E. coli to confirm
the specificity and relevance of the possible peptides/proteins detected. It is hoped that this workflow will allow for the
reproducible identification of peptides/proteins that are truly representative of ESBLs and as such, may be applied as markers forthe purpose of rapidly determining the B-lactamase profile of E. coli.

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 27: Progress in Microbiology

PWe-114
13:30 － 14:40

Exploring the Patellamide Biosynthetic Pathway

Shirran L Sally1, Fuszard Matthew1, Koehne Jesko1, Bent Andrew1, Houssen Wael2, Jaspars Marcel2,
Botting H Catherine1, Naismith H James1
1

University of St Andrews, 2University of Aberdeen

Keywords:
patellamide, cyclic peptide, MRM, biosynthetic pathway, MALDI
Novel aspects:
Biosynthetic pathway dissection, novel substrate utilization to produce products of unknown biological activity, tailoring enzymes
to produce novel cyclic peptides containing heterocycles
Abstract:
The patellamide cyclic peptides have been shown to have biological activities which include cytotoxicity and the ability to reverse
multiple drug resistance in human leukaemia cells by antagonizing the p-glycoprotein export of vinblastine 1. They are synthesizedribosomally as a pre-propeptide（PatE）, which is then tailored by a series of enzymatic（and possibly non-enzymatic）chemicaltransformations to produce a modified cyclic octapeptide, containing oxazoline and thiazole heterocycles derived from serine andcysteine2. We are using a mixture of mass spectrometric, protein X-ray crystallographic and biochemical methods to elucidate thebiosynthetic pathway by characterising the individual enzyme products and, hence, tease out the mechanistic details of thetransformations. We aim to exploit any flexibility in the enzymes’ substrate tolerance to produce novel cyclic peptides, which may
have interesting biological activities. We have developed a range of mass spectrometric methods, utilizing both MALDI（ABSciex
4800 TOF/TOF Mass Analyser）and ESI（Waters LCT, ABSciex QTrap 4000 and ABSciex TripleTOF 5600）instruments, to follow theindividual steps in the biosynthetic pathway. MRM analysis delivers sensitive detection of signature MS/MS transitions in acomplex background and hence allows us to follow the fate of specific functional groups in order to determine the products ofthe enzyme reactions and monitor enzyme turnover. Also presented are the MSMS fragmentation patterns observed for our cyclic
peptides.
1. Houssen and Jaspars, 2010, Chembiochem, 11, 1803-15.
2. Schmidt et al., 2005, Proc Natl Acad Sci U S A, 102, 7315-20.

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 27: Progress in Microbiology

PWe-115
11:10 － 12:20

Liquid chromatography-electrospray ionization mass spectrometry formetabolism studies of mogroside V by Ganoderma lucidum mycelium

Chun-Hui Chiu, Ting-Jang Lu

National Taiwan University, Taipei, Taiwan

Keywords:
Siraitia grosvenorii, Ganoderma lucidum , LC-MS, mogroside, metabolism
Novel aspects:
The enzymatic system has a potential for mogrosides transformation, owing to its high specificity, yield, and productivity, and thismethod could be apply to the structural modification and metabolism studies.
Abstract:
The sweetness of Siraitia grosvenorii（Lo Han Kuo ）fruit comes from a mixture of triterpenoid glycosides, mogrosides I-V, withmogroside V as the major component. Mogrosides have demonstrated blood glucose regulating activity. The purpose of thisstudy was to investigate the structural conversion of mogroside V by Ganoderma lucidum mycelium. The metablolites ofmogroside V were analyzed by high-performance liquid chromatography coupled with electrospray ionization tandem mass
spectrometry and NMR spectrometer. The mogroside V molecule sequentially lost its glucosyl unit as the mass spectrometry
being operated at collision-induced dissociation mode. Adding 1 or 2 % of water extract of the fruit did not impact the growth ofmycelia in a malt extract medium. During the growth of the G. lucidum, the mycelium showed significant activity of betaglucosidase and able to hydrolyze the glucosyl residues of mogroside V. The major metabolite of mogroside was tritepene
triglucoside, Mogroside III E. The mycelia of G. lucidum were able to further utilize one glucosyl residue on Mogroside IIIE andconverted it to triterpene diglycoside, Morgroside II A when the carbon source was limited. According the results of highperformance liquid chromatography coupled with electrospray ionization tandem mass spectrometry, the beta-（1,2）linkage of
glucosyl were more resistant to hydrolysis of beta-glucosidase of G. lucidum.

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 27: Progress in Microbiology

PWe-116
13:30 － 14:40

Identification of Bacteria and Fungi by Rapid Evaporative Ionization Mass
SpectrometryÁkos Szekeres1, Imola Körtvélyessyné Györi2, Júlia Balogh2, Tamás Szaniszló3, Katalin Dr Kristóf3,
Judit M Dr Molnár3, Zoltán Dr Takáts4
1
4

Masprom Ltd., Budapest, Hungary, 2Medimass Ltd., Budapest, Hungary, 3Semmelweis University, Budapest, Hungary,
Imperial College London, London, UK

Keywords:
REIMS, Indentification, Bacteria,
Novel aspects:
Novel mass spectrometric method for rapid identification of microorganisms without sample preparation direct from the agar
plate.
Abstract:
The rapid identification of microorganisms has a great importance in routine clinical investigations, by aiding the fast commence ofadequate antimicrobial treatments. Using mass spectrometric（MS）techniques, characteristic spectral fingerprint of bacterial cells
is obtained in a short time frame, which provides chemotaxonomic information as a basis for identification. Matrix Assisted Laser
Desorption（MALDI）ionization is already implemented at the routine level for the rapid screening of unknowns, replacing classicalapproaches and carbon source utilization tests. Intact cell MALDI allows a relatively fast microbial identification of isolatedcolonies, however its application also requires some level of culturing, which dramatically increases the overall time demand of
identification. A fast and cost effective technique for exact identification of bacteria and/or fungi would avail rapid introduction ofadequate therapies.
In our study we assessed the possibility of mass spectrometric identification of different microorganisms by the use of the newly
developed Rapid Evaporative Ionization Mass Spectrometry（REIMS）using a LTQ linear ion trap mass spectrometer. REIMS spectraof bacterial cells similarly to that of mammalian tissue samples features predominantly cellular membrane forming polar lipid
components. These molecules are present in all examined organisms and possess a proper heterogeneity for species- or strainlevel identification and differentiation. During the examinations nearly 2000 well defined clinical isolates belonging to 20 different
genera were tested, which were precultured for 24 hours on three types of diagnostically used agar media； blood, Mueller Hintonand chocolateagar. REIMS analysis of the fingerprinted molecules was carried out by evaporating bacterial biomass using highfrequency electric current. Modified electrosurgical bipolar forceps were employed as an atmospheric pressure ion source
producing gaseous ions of certain bacterial cell components. Aerosol formed on the rapid evaporation of bacterial cells wastransferred to mass spectrometer using a Venturi air jet pump. It is important to note, that the whole experimental setup was
microbiologically separated from the working environment with an isolation box design arrangement. The mass spectra werecollected in single stage MS, negative ion mode, in the mass range of 600-900 m/z at unit mass resolution. The obtained spectra of
replicate analyses of strains on different media were compared and found highly specific and characteristic for the species
analyzed. Clinical isolates with similar taxonomical classification grouped well using principal component analysis, while individual
groups showed complete separation using the first 3 principal components.
Identification method for unknown isolates was developed, based on the combination of principal component analysis and linear
discriminant analysis. Authentics trains obtained from ATCC were analyzed by REIMS, and resulting data was used as a learning
set. Learning set data was subjected to dimension reduction by PCA, and first 60 PC was subjected to LDA. Data points
corresponding to unknowns were localized in the 60 dimensional LDA space and were classified into closest strain-specific datagroup. The method was proven to be insensitive for any examined perturbation, including colony age or substrate type.
The accuracy of identification procedure was higher than 99 % achieved using the PCA/LDA algorithm and aspectral databasecollected from ATCC strains.

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 27: Progress in Microbiology

PWe-117
11:10 － 12:20

Lysosomal storage diseases diagnostics in dry blood spots by fluorimetry
and multiple reaction monitoring mass spectrometry

Claudia Cozma1, Marius-Ionut Iurascu1, Laura Ion1, Stefan Maeser2, Michael Przybylski1
1

University of Konstanz, Germany, 2Centogen GmbH, Germany

Keywords:
Lysosomal storage disease, diagnostic, multiple reaction monitoring
Novel aspects:
Novel working aproach for the deternimation of lysosomal ezymes by fluorimetry mass spectrometry
Abstract:
The loss of lysosomal enzyme activity is a characteristic of lysosomal storage diseases（LSDs）, a group of mostly genetic
metabolism （e.g.,α-galactosidase Fabry’s Disease； β-glucocerebrosidase Gaucher’s Disease； α-L- iduronase
mucoplysaccharidose I ）. The enzyme substrates can no longer be processed and are accumulating in the lysosome causing
severe disease symptoms leading to multiple organ failure and finally death. For Fabry’s Disease（FD）enzyme replacement
therapy is available from 2001 with high success, thus rendering rapid and efficient diagnosis of key importance. In the present
study we have developed and established specific and highly sensitive diagnostics tools using mass spectrometry multiple
reaction monitoring approach（MS-MRM）. A fluorometric determination for monitoring of α-galactosidase, β-galactosidase andβ-glucuronidase activity was developed as control assay. A clinical diagnostic study was performed using the dry blood spot
（DBS）method with samples from ca. 100 healthy controls and Fabry patients. Tandem mass spectrometry offers a qualitative
dimension by identification of the product, but also a quantitative dimension by introducing in the reaction an internal standard.
The novelty of the present assay is the adaptation of the working conditions for enzymatic determination for the mass
spectrometers used, reducing the time for sample preparation and also the introduction of MS/MS diagnostic spectra. The MSMRM diagnostics of FD was carried out using a special mixture of substrate and internal standard. Substrate used used for
enzymatic determination of alpha-galactosidase is（ 6 -benzoylamino-hexyl）-{ 2 -［ 4 -（3,4,5-trihydroxy- 6 -hydroxymethyl-tetrahydropyran- 2 -xyloxy）-phenylcarbamoyl ］-ethyl}-carbamic acid tert-butyl ester（SGLA）（C33H47N3O10； MW 645.7Da）and the expected
product is（ 6 -benzoylamino-hexyl）-［ 2 -（ 4 -phenylcarbamoyl）-ethyl］-carbamic acidtert-butyl ester（PGLA）（C 27H37N5O5；MW483.6 Da）. The internal standard for qualitative monitoring of the reaction was deuterated PGLA（C27H32N5O5D5；MW 488.6 Da）.
Diagnostic spectrum was established for the product and the internal standard for identification, and the concentration of bothwere monitored by MRM（multiple reaction monitoring）. Using the relative intensity of the peaks and knowing the concentrationof the internal standard, the concentration of the product was evaluated using an equation characteristic for each mass
spectrometer. Atypical healthy control sample in the MRM- MS assay gives an average substrate conversion of 6.6 mM pro liter per
48h（or 2,13nmols/spot/48h）. A DBS sample collected from male FD patient presents an activity 7 to 18x less（under 0,28 nmols/
spot/48h）.

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 28: IR Spectroscopy of Gas-phase Ions

PWe-118
13:30 － 14:40

UV action spectroscopy of peptides and proteins via photodissociation ofiodine labelled tyrosine residues.

Benjamin B Kirk1, Adam T Trevitt1, Haibo B Yu1, Yuanqi Tao2, Benjamin N Moore2, Ryan R Julian2,
Stephen J Blanksby1
1

University of Wollongong, Wollongong, Australia, 2University of California, Riverside, USA

Keywords:
Action Spectroscopy, Peptides
Novel aspects:
A method of measuring ultraviolet action spectra of peptides and proteins using selectively iodinated tyrosine residues.
Abstract:
The advent of soft ionisation techniques such as electrospray ionisation, coupled with its unprecedented speed and sensitivity,
has imparted on mass spectrometry an important role in the structural characterisation of peptides and proteins. The threedimensional structure of proteins directly affects their biological activity. The capability to ionise and transfer both small peptides
and large proteins intact into the gas phase provides a versatile tool with which to interrogate these molecules. While ion
spectroscopy is an attractive means of directly probing the structure of these molecules, the low concentrations employed, anddifficulty in gaining unrestricted access to the trapped ions, means structural characterisation is typically carried out by action
spectroscopy. Instead of measuring absorbance and fluorescence directly, action spectroscopy is a measure of ion fragmentation
yield relative to excitation wavelength.
Unfortunately, as molecular weight increases so does the energy required to fragment an ion. Intramolecular vibrational
redistribution（IVR）results instead of direct bond cleavage, as any energy imparted on the ion is delocalised from the absorbing
chromophore and redistributed into vibrational degrees of freedom. To overcome this limitation, one may instead measure fastdissociation phenomena, such as electron photodetachment, as recently employed by Dugourd and co-workers.［ 1 ］While aneffective technique for measuring multiply charged anions, this method cannot always be used to measure singly charged anions
or ions that are positively charged. An alternative is to introduce a functional group that dissociates prior to IVR. Recent work has
demonstrated that peptides and proteins containing selectively iodinated tyrosine residues dissociate promptly on irradiation witha 266 nm laser pulse, generating an iodine atom and a carbon centred radical. Evaluating this dissociation as a function of
wavelength affords a powerful, extensive method of measuring structural changes in a peptide or protein using action
spectroscopy.
In these experiments, a Thermo Fisher LTQ linear quadrupole ion trap mass spectrometer has been modified to allow irradiationof trapped ions with a tunable laser pulse. The laser is a frequency-doubled OPO pumped by the third harmonic（355 nm）of a
Nd：YAG laser, providing a tunable wavelength range between 220 - 350 nm. Coupling the Thermo Fisher Xcalibur analysis
software with custom LabView scripts enables a simple, semi-automated process to measure action spectra at nanometre
resolution.
In this presentation, we introduce iodinated tyrosine residues as target chromophores for action spectroscopy and outline aninvestigation of the charge-state modifications of small iodine labelled peptides and proteins. Action spectra of several chargestates of 3 -iodotyrosine, iodinated KGYDAKA and ubiqituin are reported and a comparison made to the reported threedimensional structures of these molecules measured by UV absorption and electron photodetachment yields in solution and thegas phase, respectively.
［ 1 ］Bellina et al.； Int. J. Mass. Spectrom.（2010）,297,36.

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 29: The Advances in Biological Mass Spectrometry in Drug Discovery andDevelopment: Current State of the Art and Challenges

PWe-120
13:30 － 14:40

Influence of end-capping reagent of LC column on sensitive determination
using LC/MS

Chiaki Aoyama, Yuko Yui, Kensuke Okusa, Kosuke Osaka, Takao Tamura, Masakazu Takahashi
GL Sciences Inc., Iruma, Japan

Keywords:
LC/MS, quantification, sensitivity, peak shape
Novel aspects:
Reagent used for end-capping often influences on sensitivity, and electrically neutral column is most recommended for highly
sensitive analysis.
Abstract:
Introduction
LC/MS is a powerful and absolutely necessary technique for analysis of organic compounds. LC columns, which are used toseparate target compounds from interfering material, play an important role as ion source, mass analyzer, and detector.
The most widely used columns for LC/MS are ODS columns. End-capping is exceptionally important for ODS columns because it
greatly influences on peak shape. If tailing or leading occurs, peak height is diminished and sensitivity is lowered. Therefore, mostof commercially available LC columns are end-capped with a variety of reagents.
In this study, LC columns end-capped with several reagents are compared. Peaks shape and sensitivity of drug compounds werecompared in our poster.
Material and method
LC/MS system used in this study was consisted of an LC800 liquid chromatograph（GL Sciences）and a 4000QTRAP mass
spectrometer（AB Sciex）. Isocratic or gradient elution was carried out using 0.02% formic acid aqueous solution as mobile phase
A and acetonitrile as mobile phase B.
Trimethylchlorosilane（TMCS）, hexamethyldisilazane（HMDS）, and trimethylethoxysilane（TMES）were used as end-capping
reagent and compared. Same silica gel to which octadecylsilyl group was chemically bonded was reacted with each reagent.
Procainamide, ranitidine, acetaminophen, caffeine, chlorpheniramine, propranolol, ketoprofen, salicylic acid, and carbazochrome
sulfonate were chosen as analyte. Salicylic acid and carbazochrome sulfonate were detected in negative mode, while the others
were in positive mode.
Preliminary results and discussion
TMCS and HMDS have frequently been used as end-capping reagent. However, it is known that residual hydrochloric acid andammonia, respectively, exist on the surface of silica gel after the end-capping reaction. TMES was also selected in this study
because we considered that electrostatically charged by-product should not be yielded by end-capping with this reagent.
Firstly, peaks of chlorpheniramine, which is a basic compound, obtained with each column were compared. The compound waseluted with good peak shape from HMDS column and TMES column. However, peak tailing occurred and S/N ratio was decreased
when TMCS column was used. It was thought to be caused by partial adsorption owing to electrostatic interaction between
positive charge of the analyte and negative charge of residual chloride ion on the surface of the silica gel.
Secondly, retention times of procainamide, which is a basic and highly hydrophilic compound, were compared. Although thecompound was retained on TMES column to some extent, almost no retention was observed on HMDS column. We consideredthat its retention on HMDS column was prevented by electrostatic repulsion between positive charge of the analyte and that ofby-product of HDMS end-capping. HMDS column is not desirable for determination of procainamide because its peak should besusceptible to ion suppression caused by unretained material.
As for carbazochrome sulfonate, significant adsorption occurred only when HMDS column was used. It can also be explained byinteraction between negative charge of carbazochrome sulfonate and positive charge of HMDS.
In conclusion, only TMES column provided good peak shape and retention for all compounds examined in this study. LC columns
which do not bear any charge on the surface of silica gel, such as InertSustain C18, are most recommended as first choice column
for highly sensitive determination using LC/MS.

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 29: The Advances in Biological Mass Spectrometry in Drug Discovery andDevelopment: Current State of the Art and Challenges

PWe-121
11:10 － 12:20

Simultaneous, Fast Analysis of Melamine and Analogues inPharmaceutical Components Using Q Exactive - Benchtop Orbitrap LCMS/MS

Kate J Comstock, Hongxia (Jessica) Wang, Tim Stratton, Yingying Huang
Thermofisher Scientific

Keywords:
Benchtop-Orbitrap. Identification, Quantification. Melamine. Pharmaceutical
Novel aspects:
Novel approach - simultaneous, fast analysis of melamine and analogues in pharmaceutical components by high resolution
benchtop Orbitrap LC-MS/MS
Abstract:
Potential drug contamination by melamine and its analogues remain a major concern by FDA. We present a workflow for analysisof melamine and its analogues in at-risk pharmaceutical components using high resolution benchtop Orbitrap LC-MS/MS： Q
Exactive. Simultaneous, fast screening and quantitation for melamine and its analogues in complex matrices were achieved by
HRAM full scan, ms/ms in a data-dependent fashion with polarity switching. Comparing with commonly used Triple Quadrupole
MS method, this workflow allows for added post analysis flexibility, it avoids the upfront selection of specific compound masses
pertaining to SRM methods. Confident identification is achieved by accurate mass measurement of both precursor and fragment
ions, as well as the fine isotope pattern of 13C and 15N.
Methods
A calibration curve, containing melamine, ammeline, ammelide, and cyanuric acid, was prepared by serial dilution of a 1 mg/ml
stock solution to give a final concentration range from 25ppb to 10ppm. Sample preparation recovery was determined by spiking
neat solutions into the commercial pharmaceutical excipients tested in this study. Q Exactive coupled to Ultimate 3000 UHPLC
system（Thermo Scientific）was employed for the studies. LC base line separation of all four compounds was achieved within 5
minutes using Thermo Accucore HILIC column. Full scan MS at resolving power 70,000 FWHM and MS/MS at resolving power 17.500
FWHM were collected in a data-dependent fashion with polarity switching. The US FDA method for melamine and cyanuric acidwas referenced with modifications.
Preliminary Data
Initial data showed that full scan analysis at 70,000 resolution（FWHM）provides spectra of high quality that have sub- 2 ppm massaccuracy with external calibration and well-separated isotopic pattern of A+ 2 isotope ion on 13C and 15N. The high resolution
accurate mass measurement, fine isotope pattern and signature product ions provide a multi-dimensional confirmation formelamine and its analogues analysis. Product ions from MS/MS spectra were utilized for quantitation. The results have good
linearity on five-point calibration curves over the range from 5 ppb to 50ppm with excellent linear regression coefficients（r 2 >0.99）
and above 85% recovery rate. The preliminary data suggested that this robust UHPLC- HR MS/MS method provides simultaneous,
simple, fast determination and quantitation of melamine and its analogues at a high confident level in a variety of pharmaceuticalcomponents.

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 29: The Advances in Biological Mass Spectrometry in Drug Discovery andDevelopment: Current State of the Art and Challenges

PWe-122
13:30 － 14:40

Measurement and Optimization of Organic Chemical Reaction Yields by
GC-MS with Supersonic Molecular Beams

Aviv Amirav, Alexander Gordin, Youlia Hagooly, Shlomo Rozen, Bogdan Belgorodsky, Boaz Seemann,
Hanit Marom, Michael Gozin, Alexander B Fialkov
Tel Aviv University

Keywords:
GC-MS, Cold EI, Chemical Reaction,
Novel aspects:
New method and GC-MS instrument for the measurement and optimization of organic chemical reaction yields. GC-MS withenhanced molecular ion, extended range of analysis and uniform response
Abstract:
Chemical reactions are typically performed via mixing reactants together in an appropriate solvent, often with a catalyst, andallowing them to undergo a reaction for a few hours. At the perceived end of the reaction, the products are separated andpurified, a process which could take several days. Subsequently, the purified products are analyzed by NMR and high resolution
ESI-MS（typically with HR-QTOF）. However, no information is obtained about the synthesis yield, products purity, availability ofisomers and on the reaction mechanism. GC-MS with its standard electron ionization, can provide such information but only forvolatile compounds that exhibit molecular ions. Thus, a new MS system is needed to enable semi-on-line, monitoring of thereacting compounds and products in organic reactions.
Measurement and optimization of chemical reaction yields was performed by our Aviv Analytical 5975-SMB GC-MS with supersonicmolecular beams（SMB）. It is based on the coupling of SMB interface and its fly-through electron ionization ion source with an
Agilent 7890 GC + 5975 MSD. The GC eluting molecules are mixed with helium make up gas, expand from a supersonic nozzle intoa vacuum chamber, vibrationally cooled, skimmed, collimated into a SMB, pass a fly-through electron ionization ion source where
they are ionized with uniform response and mass analyzed. The vibrational cooling enhances the molecular ions and the
combination of short columns and high column flow rates about doubles the range of thermally labile and large molecules
amenable for analysis.
We used our 5975-SMB GC-MS with SMB for semi-on-line monitoring of synthetic organic chemical reactions for their reaction yield
optimization, mechanism elucidation and for obtaining information on the reaction products identity, purity and availability ofisomers. Several different organic synthetic reactions were studied and will be described and demonstrated. These reactions
included the synthesis of a C14H18N2O3S2 potential anti fungal drug compound（MW=362）, a C25H25NO4S compound（MW=435）
which is targeted for chemical sensing of explosives, a C50H96N2O8 compound（MW=852）which is aimed at environmental metal
remediation and the oxidation reaction of few thioesters.
The key features of the 5975-SMB GC-MS with Cold EI （EI of vibrationally cold molecules） that uniquely enabled suchmeasurements are：
A）The fly-through EI ion source provides degradation and tail-free close to uniform compound independent response, thereby
enabling the measurement of reaction yields from total ion count peak areas while using the standard Chemstation percent area
report.
B）The molecular ions are enhanced and are practically always observed.
C）Isotope abundance analysis automatically inverts the molecular ions into elemental formulas.
D）The use of short columns and high column flow rates significantly extends（~doubles）the range of thermally labile and low
volatility compounds amenable for analysis.
E）The high total ion count signal to noise ratio obtained enables the identification and quantification of both major and minor byproducts and impurities
F）The chromatography measurement time is much faster than with standard GC-MS, from one minute to several minutes analysis
cycle time.
Based on the collected data, we were able to better understand how the reaction conditions should be optimized in order tomaximize the yields and purity of target products. Consequently, we propose that GC-MS with SMB can serve as a novel tool forthe optimization of chemical reactions in organic chemistry departments, drug synthesis and in the chemical industry.
Our goal is to advance towards a change in the way in which organic chemistry is being performed and eliminate the lengthy
steps of post reaction sample separation and purification while enabling reaction yield optimization and product purity
improvement.

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 29: The Advances in Biological Mass Spectrometry in Drug Discovery andDevelopment: Current State of the Art and Challenges

PWe-123
11:10 － 12:20

The Utility of a Novel High Resolution Quadrupole Time-of-Flight Mass
Spectrometer for Improved Characterization of Antibodies and ImmunoConjugates

Asish Chakraborty1, St John Skilton1, Steve Pringle2, Nick Tomczyk2, Weibin Chen1
1

Waters, Milford, USA, 2Water MS Technologies, Manchester UK

Keywords:
Antibody, ADC, Antibody-drug conjugate, SEC-MS
Novel aspects:
Sensitive and high resolution MS characterization of immuno-conjugates using high-throughput on-line size exclusion
chromatography coupled directly to electrospray ionization mass spectrometry
Abstract:
Introduction: Monoclonal antibodies（mAbs）and derivatives for the treatment of cancers and inflammatory diseases are a rapidly
growing class of biotherapeutic. mAbs are often combined with cytotoxic drugs to enhance their therapeutic efficacy, andalternatively, small anti-neoplastic molecules can be chemically conjugated to mAbs. Typically, several molecules of a potent
cytotoxic-agent are covalently linked to a MAb, and therefore structural characterization is challenging due to the heterogeneous
nature of the conjugate, the large molecular-size of antibodies and low abundances of the modified residues. Additionallyimmunoconjugates might differ in number of drugs linked and the sites of linkage. Here, we present a high-sensitivity quadrupole/time-of-flight system with novel ion-guide source for characterization of mAbs and conjugates that helps unravel some of thecomplexity.
Methods: Intact and deglycosylated-conjugates, and naked antibodies were introduced to the ESI mass spectrometer using two
different chromatographic conditions： either a generic size-exclusion chromatography（SEC）with high-throughput MS-friendly
conditions, or high-resolution reversed-phase chromatography（RP）method. The naked antibody, conjugate, and their
deglycosylated forms were also reduced with DTT to the light chain and heavy chain fragments and were separated on a SEC
column with sub- 2 micron particles, and analyzed using a prototype quadrupole/ time-of-flight system equipped with an off-axis
conjoined stacked ring ion guide. RP separation was also used to separate the naked and conjugated-antibody light and heavy
chain fragments and to automatically determine the conjugation sites by peptide mapping.
Preliminary Data: Antibody drug-conjugate analysis is more complex than the naked antibodies. The use of MS-friendly SEC with
sub- 2 micron column packing materials, directly coupled to a quadrupole time-of-flight mass spectrometer for the mass analysisof antibody drugs and conjugates provides improved detail and productivity than previous generations of instrument. Light andHeavy chains were baseline separated for all antibody and conjugate samples. SEC, using aqueous 30% acetronitrile with acidic
modifiers as mobile phase, directly coupled to ESI-MS provided a routine analysis method for profiling of antibodies andconjugates, degree of conjugations, sequence confirmation（isotope resolution）, and molecular-weight measurement.
Intact subunits of mAb and conjugates from DTT reduction were separated by high-resolution RP chromatography which resolved
different isoforms coexisting in the sample. High-resolution MS detection was able to istopically resolve light chain（25 kDa）.
Identities of either intact mAbs or conjugates, or the subunits were automatically confirmed by deconvoluting the mass spectrausing both maximum entropy and BayesSpray deconvolution algorithms. The deconvoluted mass spectrum of deglycosylatedconjugate shows several peaks with approximately same mass differences and with varying intensities. The masses of these peaks
are assigned to naked mAb and to mAb conjugates with varying number of covalently linked drug-molecules. The deconvoluted
MS spectra of deglycosylated reduced mAb-conjugate show both light and heavy chains are modified and conjugated with drugs.
The total number of linkers attached to the light and heavy chains agrees with the number of linkers attached to thedeglycosylated intact conjugate. The LC-TOF MS approach presented here facilitates rapid characterization of antibody
conjugates. We are currently working on detecting the conjugation sites using trypsin/AspN peptide mappingPoster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 29: The Advances in Biological Mass Spectrometry in Drug Discovery andDevelopment: Current State of the Art and Challenges

PWe-124
13:30 － 14:40

Method Development for Quality Control of Traditional Chinese Herbal
Medicine- Guizhi Tang by HPLC and Electrospray Ion Trap Mass
SpectrometryMing-Yu Chao, Ying-Yu Kuo, Jui-Ching Chen, Che-Yi Lin, Yuan-Ling Ku, Leah Lo

Medical and Pharmaceutical Industry Technology and Development Center, Taipei, Taiwan (R.O.C.)

Keywords:
Guizhi Tang； Ion Trap； Mass.
Novel aspects:
New LC/MS analysis method of the Chinese Medicine prescription- Guizhi Tang.
Abstract:
Introduction:
Traditional Chinese Herbal Medicine（TCM）is utilized regularly in the multi-herb prescription for treating people’s disease. The
quality control of the prescription and their derived products is more difficult than the single herb. In this study we have used LC/
ion trap MS analysis to develop a simple and highly efficient analysis method for the Chinese Medicine prescription- Guizhi Tang.
Methods：
An extract PDC-1689 as Guizhi Tang was prepared from traditional Chinese herbal medicines containing Ziziphus jujuba Mill,
Zingiber officinale Rosc, Paeonia lactiflora Pall., Glycyrrhiza glabra L., and Cinnamomum cassia Presl（material ratio as follows, 5 ：6 ： 6 ： 4 ： 6 ）. PDC-1689 was analyzed by Agilent1100 high performance liquid chromatography（Agilent Technologies, CA,USA）, Brukeresquire HCTion trap mass spectrometry（Bruker, Bremen, Germany）equipped with Agilent 1100 HPLC, and Nuclear
Magnetic Resonance Spectroscopy（Bruker, Bremen, Germany）.
Results and conclusion:
Different elution conditions of reversed-phase HPLC for PDC-1689 assay were developed and optimized. The optimal conditions
were found of C-18 reversed phase column with a mobile phase of 0.05% Formic acid and Acetonitrile at a flow rate of 0.8mL/min.
UV detection was achieved at 254 nm. Under the optimal conditions, seven compounds with retention time of 21.2, 23.2, 24.1, 26.1,
32.9, 64.9, 72.6 min were analyzed by electrospray ion trap mass spectrometry with positive-ion mode at m/z 563.1, 551.1, 431.0, 839.4,
823.4, and negative-ion mode at m/z 833.5, 510.8. The structures were elucidated by LC-SPE-NMR and led to the identification ofglycyroside, isoliquiritin apioside, formononetin 7 -O-glucoside, isoliquiritin, liquiritigenin, licorice-saponin G 2 and glycyrrhizic acid.

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 29: The Advances in Biological Mass Spectrometry in Drug Discovery andDevelopment: Current State of the Art and Challenges

PWe-125
11:10 － 12:20

High-throughput sequence determination of screened 90 micron single
beads on which a cyclic octa-peptide is immobilized

Kiyoshi NOKIHARA1, Takafumi OHYAMA1, Noriko ONO1, Takeshi KASAMA2
1

HiPep Laboratories, Kyoto, Japan, 2Tokyo Medical and Dental University, Tokyo, Japan

Keywords:
HTP-sequencing, cyclic-peptide, PEG-based resin, OPOB
Novel aspects:
High throughput sequencing for cyclic peptides immobilized on PEG-based beads, screened by assays in aqueous media, hadbeen developed. The method involves partial hydrolysis followed by nano-LCMS without encoding technologies.
Abstract:
Peptide library constructed using the concept of“one peptide immobilized on a single bead（OPOB）“ is useful for the discovery
of interacting molecules［Lam KS, Salmon SE, Hersh EM（1991）Nature 354： 8284］. In particular preliminary information regarding
ligands can not be obtained. While gel-type polymer beads are often used as supports, peptide assembly is carried out in organic
solvents, although assays are performed in aqueous media. Hence library construction is often performed on polyethylene glycol（PEG）based resins. Sophisticated encoding technologies such as DNA or small functional groups followed by deconvolution
using DNA-sequencer or GC-MS have been carried out［Still WC（1996）Acc. Chem. Res. 29： 155-163］, although the simplest
deconvolution method is the Edman degradation using a peptide-sequencer［Liu R, Lam KS（2001）Analytical Biochemistry 295：
9 -16］. However the Edman method has restrictions as relatively larger amounts（tens of picomoles）of sample and N-terminal
free amino groups are required. Therefore N-terminal amino acid should not be blocked during screening/assays. Additionally the
Edman cycle is time consuming since the determination of amino acids is performed on a one-residue basis and thus costly. To
overcome these restrictions we have attempted top down sequencing of one bead by mass spectrometer. However, the large
diversity of PEG derived signals were encountered and peptide peaks could not be identified. The present library has been
constructed by the“one peptide on one bead（OPOB）basis“ applied by the split and combine method on TentaGel® S NH 2（90µm）, which is a graft co-polymer of polystyrene and polyethylene glycol. The library contains ca. 200 million octa-peptide beads
constructed from 19 natural and 5 non-proteinogenic amino acids which include two D-cysteines that were used for enzymatic
resistance during bioassays. Completion of acylation was monitored by the Kaiser test and double coupling was employed forincomplete acylation. Intra-molecular disulfides were formed by on-resin cyclization in 10% DMSO（pH6.0）at room temperature for4 days. Since one bead carries ca 100 pmole peptides（calculated from resin substitution）, complicated sample pretreatment is
not practical due to loss of sample. To overcome these restrictions and problems we have attempted to develop partial hydrolysis
followed by characterization using LCMS. In the present study the peptide-bead was screened using an assay with human cancer
cells. The whole procedure has been carried out using microscopic observation to find particular beads which attached cells. The
peptide on the bead was liberated by partial acid hydrolysis under optimized conditions. The partial hydrolysate, which contained
not only target peptide but also PEG-derived materials, was subjected to nano-scale LCMS to give the primary structure of thetarget peptide. Based on the MS analyses the target peptides have been individually synthesized and confirmed the target
sequence using the same assay. In the present study we have screened 24 beads carrying individual peptides whose structures
have been elucidated.

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 29: The Advances in Biological Mass Spectrometry in Drug Discovery andDevelopment: Current State of the Art and Challenges

PWe-127
11:10 － 12:20

MsXelerator: A Software Platform for (Reactive) Drug Metabolite
Detection and Identification using High-Resolution Mass Spectrometry andPost-Acquisition Data Mining

Marco Ruijken

MsMetrix, Maarssen, the Netherlands

Keywords:
Reactive Drug Metabolites, Identification, DataMining
Novel aspects:
A novel software platform combining multiple data mining algorithms has been developed for the detection and identification of
drug metabolites.
Abstract:
Introduction：
Recent post-acquisition data mining tools applied to the detection of drug metabolites in complex matrices using high resolution
mass spectrometry include： Mass Defect Filtering, Precursor Ion Filtering, Neutral Loss Filtering, Background Subtraction and High
Resolution Isotope Pattern Filtering. Starting point for any of the above data mining tools can be either a targeted or non-targeted
approach. Most of the above techniques have distinct advantages and disadvantages. Here, we report on a new software platform
combining the above mentioned algorithms and results into one automatic procedure. Combining the results from different filters
significantly decreases the chance of missing important metabolites.
Methods：
After high resolution peak picking using the full scan MS data, the following algorithms are available： Mass Defect Filtering,
Neutral loss and Product Ion Filtering, High Resolution Background Correction and Isotopic Pattern Matching for detection of
metabolites containing Cl, Br or a mixture of a drug and its stable isotope labeled drug（reactive metabolite detection）. Filters
can be used individually or sequentially in any selected order. In each step, peaks that conform to the applied filter can bedeleted or marked. Most software tools will report a“filtered total ion current“ after each step. The result of the MsXelerator
software implementation is a table of ions that pass or fail each test. Identification is done by searching Metabolite Prediction lists.
Preliminary Results：
Several commercial test compounds were incubated with human liver microsomes for 60 min. Some other test compounds,
Nefazodone, Verapamil, Diclofenac, Imipramine, Ticlopidine and Clozapine were incubated in rat liver microsomes with a mixtureof GSH/stable isotope labeled GSH at a 1 ： 1 ratio for 30 minutes or using KCN/K13C15N.
In order to assess the formation and detection of（reactive）metabolites in a discovery stage, and to test the MsXelerator
performance, several ultra-high resolution mass spectrometer platforms were used and compared： the LTQ-Orbitrap and Velos
and the Sciex Triple TOF 5600.
Incubation samples were injected and full-scan MS and MS/MS datasets were recorded. For stable isotope labeled samples, data
dependent MS/MS was performed on the top two most intense peaks that have a mass difference of 3 Da（both high and low
masses of mass tag）with a relative ratio of 80-100%.
Isotope Pattern filtering in combination with the post-processing algorithms almost doubled the number of detected GSH adducts
compared to more traditional detection methods that are normally applied in this application field. In all cases IPF outperformed
direct Neutral Loss analysis, especially for low abundant metabolites. Probably the most important step is the removal of common
peaks, present in both sample and control by using the background correction algorithm. It is preferred to use this filter as a firststep in the analysis. In general it can be concluded that product ion and neutral loss filtering are very useful as a confirmatory
information source. It is not advised to apply a“hard filter“ on these tools. The same is true for mass defect filtering. In manycases it was observed that mass defect filtering removes some of the unexpected metabolites.
A typical analysis, combining all post acquisition tools takes about one 1 - 2 minutes.

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 29: The Advances in Biological Mass Spectrometry in Drug Discovery andDevelopment: Current State of the Art and Challenges

PWe-128
13:30 － 14:40

Non-targeted identification of novel Buspirone metabolites using a
Spectral Similarity score derived from common fragment ion and neutral
loss species

Kirsten Hobby, Alan Barnes, Neil Loftus
Shimadzu MSO, Manchester, UK

Keywords:
drug metabolism IT-TOF buspirone
Novel aspects:
Identification of novel pyrimidine ring cleavage metabolites of Buspirone via spectral similarity correlation score
Abstract:
Buspirone is an anxiolytic psychoactive drug of the azapirone chemical class, and is primarily used to treat generalized anxiety
disorder（GAD）. Buspirone is a common model compound for xenobiotic metabolism. Here we describe the application of a
Spectral Similarity Correlation methodology（’S Score’）to the analysis of data generated from a human microsomal incubation of
Buspirone. Non-targeted data-dependent MSMS data was collected from a human microsomal incubate using a Shimadzu LC ITTOF system. The ’S scores’ were derived from the correlation of fragment ion and neutral loss species of unknown
chromatographic peaks to those of Buspirone. This approach was unbiased and success was not influenced any user interaction.
Buspirone（30µM）was incubated with human liver microsomes and NADPH at 37°C for 45 minutes. 5 µl of incubate was injectedusing a Shimadzu LC IT-TOF system onto a 2.1 x 50 mm C18 BEH 1.7um； A - Water + 0.1 % formic acid； B - Acetonitrile + 0.1 %formic acid. Flow 0.6ml/min @ 65°C. Gradient： 2 %B 0 min, 40%B 8 min, 90%B 9 -10min, 2 %B 10.5min.
The Spectral Similarity scoring was performed by Shimadzu Met ID solutions software（v1.2）. Two data files were used in theanalysis. The t 0 sample was used to supply reference fragment ion and neutral loss data for Buspirone, and a t45 sample wastreated as the unknown.
Initial data analysis by Met ID Solutions software was performed without a search for ’expected’ metabolites and 37 peaks werereturned with an S Score >0. Re-analysis by the software with a list of expected metabolites containing all previously describedPhase 1 metabolites reduced the number of candidate ’unknown’ peaks to 12. Manual scrutiny of the data indicated that some of
the unknown peaks could be attributed to combinations of dealkylation and oxidative metabolic reactions but two peaks（m/z350.2531 & m/z 438.2707）could not be assigned to previously described metabolites of Buspirone. Further analysis of the MSMS
data supported the hypothesis that m/z 350.2531 was a product of cleavage of the pyrimidine ring in Buspirone. The m/z 438.2707
ion was tentatively identified as an intermediate in the metabolic cleavage of the pyrimidine ring as MSMS data showed the m/z
350 ion as a fragment.
Analysis of the literature showed that pyrimidine ring cleavage has been previously described for a structurally similar drug. The
metabolic cleavage of the pyrimidine ring results in a loss of C3 relative to Buspirone. In the absence of prior knowledge of thismetabolic step it is unlikely that a metabolism scientist would search for metabolites containing the C3 mass difference.

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 29: The Advances in Biological Mass Spectrometry in Drug Discovery andDevelopment: Current State of the Art and Challenges

PWe-129
11:10 － 12:20

Analysis of interaction between TNF-alpha and anti-TNF-alpha agents byhydrogen deuterium exchange/mass spectrometry

Shiori Nakazawa1,2, Noritaka Hashii2, Nana Kawasaki1,2
1

Hokkaido University, Sapporo, Japan, 2National Institute of Health Sciences, Tokyo, Japan

Keywords:
TNF-alpha, HDX/MS, anti-TNF-alphaNovel aspects:
We found that a common region in TNF-alpha is shielded from the solvent when it is bound to four of anti-TNF-alpha therapeutic
agentsAbstract:
Anti-TNF-alpha agents are used in treatment for some autoimmune diseases or inflammatory diseases like rheumatoid arthritis.
There are several biological anti-TNF-alpha agents approved, one is a decoy receptor, and others are monoclonal antibodies.
Although they are directed against identical antigen, they were established independently, thus their complementarity-determining
regions（or ligand-binding regions）differ from each other. Then, what kind of structural differences are there when different antiTNF-alpha agents capture TNF-alpha? For the purpose of elucidating this, we applied hydrogen deuterium exchange/mass
spectrometry（HDX/MS）to TNF-alpha in the presence and absence of anti-TNF-alpha agents. HDX/MS is a known method which
has its advantage in analyzing the dynamic aspects of protein higher order structures.
We examined four anti-TNF-alpha agents； infliximab, a chimeric antibody, adalimumab and golimumab, human antibodies, andetanercept, a fusion protein of TNF receptor and IgG Fc domain. We immobilized anti-TNF-alpha agents on beads, deuterated
TNF-alpha trimers on the anti-TNF-alpha beads or without anti-TNF-alpha agents, then quenched and eluted TNF-alpha with acid,and determined the regions which have differences in deuteration levels between in the presence and absence of the anti-TNFalpha agents. Interestingly, the same region of TNF-alpha had exhibited decreased deuteration levels when TNFalpha was boundto anti-TNF-alpha agents, whichever anti-TNF-alpha agents it was bound to. The region we identified is supposed to be tucked
between two TNF-alpha monomers in the trimer, and it also disagrees with the reported epitopes. These results and facts suggestthat it possibly is a region which has been shielded from the solvent by the conformational changes induced by anti-TNF-alphaagents.

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 29: The Advances in Biological Mass Spectrometry in Drug Discovery andDevelopment: Current State of the Art and Challenges

PWe-130
13:30 － 14:40

Validation and application of UHPLC-ESI-MS/MS method for thequantification of probe-drugs and metabolites of CYP3A4 and UGT1A1 inpharmacokinetics study

Daryl Kim Hor Hee, Zhangyan Yao, Lawrence Soon-U Lee
National University of Singapore, Singapore

Keywords:
Pharmacokinetics, probe drugs, metabolites, CYP 3 A4, UGT 1 A 1
Novel aspects:
Simultaneous quantification of probe-drugs, metabolites, inhibitors and inducer of metabolic enzymes CYP 3 A 4 and UGT 1 A 1
and its application in clinical trial pharmacokinetics study
Abstract:
An UHPLC-ESI-MS/MS based method for the quantification of metabolic enzymes CYP 3 A 4 and UGT 1 A 1 probe-drugs（midazolam and raltegravir, respectively）and metabolites（ 1 ’-hydroxy midazolam, 1 ’-hydroxy midazolam glucuronide andraltegravir glucuronide）simultaneously with the inhibitors（ketoconazole and ritonavir）and inducer（rifampicin）was developed.
Analytes were extracted from 100 µl of plasma using CaptivaTM NDLipids. Chromatographic separation was performed on a reverse
phase C18 column（50 mm × 2.1 mm × particle size 1.8 µm）with a stepwise gradient using water and acetonitrile containing 0.1 %formic acid, at a flow rate of 0.5 - 0.6 ml/min. Separation of the 8 analytes was completed within the analytical run time of 4 min.
The triple quadrupole mass spectrometer was operated in the positive ionization mode and drug quantification was performed
using multiple reaction monitoring with deuterated internal standards. The method has been validated according to the FDA
guidelines. The assay was linear over the concentration ranges that covered various analytes in the plasma samples. The lower
limit of quantification for midazolam, 1 ’-hydroxy midazolam, 1 ’-hydroxy midazolam glucuronide, raltegravir, raltegravir
glucuronide, ketoconazole, ritonavir and rifampicin were 0.1, 0.05, 0.2, 1, 1, 50, 50 and 50 ng/ml, respectively, in plasma. The method
was proven to be specific, sensitive and robust. Accuracies and precisions of intra- and inter-assay at all concentration levels forvarious analytes ranged from 88.1 to 111.7 % and 0.4 to 13 %, respectively. The validated method has been successfully applied tothe clinical trial pharmacokinetics study of inter-ethnic differences in the degree of induction and inhibition of CYP 3 A 4 and
UGT 1 A 1 in a multi-ethnic Asian population and the results will be discussed.

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 29: The Advances in Biological Mass Spectrometry in Drug Discovery andDevelopment: Current State of the Art and Challenges

PWe-131
11:10 － 12:20

MALDI High Resolution Mass Spectrometry in the Identification of
Atorvastatin and Its Oxidative Metabolites

Robert Jirasko1, Veronika Jedlickova1, Tomas Mikysek1, Michal Holcapek1, Ales Svatos2, Martin Kunes3,
Milan Nobilis3
Department of Analytical Chemistry, Faculty of Chemical Technology, University of Pardubice, Studentska 573, 53210
Pardubice, Czech Republic, 2Max Planck Inst. Chem. Ecol,. Res. Grp. Mass Spectrometry, D-07745 Jena, Germany,3
Faculty of Pharmacy, Charles University, Heyrovskeho 1203, CZ-50005 Hradec Kralove, Czech Republic
1

Keywords:
drug metabolites, Orbitrap, MALDI imaging, UHPLC/MS, electrochemical generation
Novel aspects:
Electrochemical generation of atorvastatin oxidative products and choice of suitable MALDI matrix followed by MALDI imaging
high resolution mass spectrometry of atorvastatin in rat tissues.
Abstract:
The choice of suitable analytical techniques for the sample characterization in the area of drug metabolite analysis is an importanttask. Common analytical tools - combination of separation techniques and mass spectrometry（MS）are usually used for the
analysis of body fluids, but this technique is unable to localize the distribution of particular compounds in the studied tissues andorgans. The most widespread MS imaging scanning method is matrix-assisted laser desorption/ionization（MALDI）with aprominent role in the analysis of synthetic polymers and biopolymers. Nowadays, the MALDI is also considered in the area ofsmall molecule analysis. The successful application of this technique is, however, hampered by low molecular weight matrixderived interference signals and poor reproducibility of signal intensity arising mainly from inhomogenity of matrix deposition. The
sublimation of suitable matrix followed by MALDI high resolution mass spectrometry can be used to avoid these complications.
In our study, various matrices typical for the small molecule analysis, such as 2,5-dihydroxybenzoic acid（DHB）, its sodium salt
（NaDHB）, 2,4,6-trihydroxyacetophenone（THA）and 9 -aminoacridine are tested on the mixture of atorvastatin and its
electrochemically generated oxidative products previously identified by UHPLC/MS/MS. Experiments are performed using highresolution MALDI mass spectrometer LTQ Orbitrap XL equipped with nitrogen UV laser（337 nm, 60Hz）. Tandem mass spectra arealso measured to obtain supplementary structural information. The most suitable matrix with regard to atorvastatin signal
enhancement is further studied in the atorvastatin distribution in rat tissues. The matrix coating is done by the sublimation to
obtain small crystal size and avoid local variations in the ionization efficiency.
The financial support of this project was provided by the Czech Science Foundation（No. P206/12/P065）.

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 29: The Advances in Biological Mass Spectrometry in Drug Discovery andDevelopment: Current State of the Art and Challenges

PWe-132
13:30 － 14:40

Metabolic Stability Screening Workflow using a Second Generation High
Resolution Accurate Mass Benchtop Instrument

Shigeru Sakamoto1, Sun Hai-Ling3, Soohoo Daniel3, Comstock Kate2, Stratton Tim2

1
ThermoFisher Scientific, Yokohama, Japan, 2Thermofisher Scientific, San Jose, CA, USA, 3Gilead Sciences, Foster City,
CA, USA

Keywords:
Benchtop-Orbitrap MetQuest Quan/Qual metabolic-stability-screening
Novel aspects:
Ultra high resolution mass spectroscopy, automated software acquisition and processing for simultaneous relative quantitativeand qualitative（Quan/Qual）analysis.
Abstract:
Introduction
In vitro metabolic stability screening performs a very important role in the drug discovery stage for compound selection inpharmaceutical companies. The screening is primarily supported by LCMS, which involves the monitoring of the disappearance ofparent compounds, using selected reaction monitoring（SRM）on triple-quadrupole instruments. If moderate to high turnover isobserved, separate metabolite identification experiments are then conducted to characterize the biotransformation products. In
this study, we present a novel workflow using a high resolution accurate-mass benchtop Mass spectrometer, Exactive. This
workflow combines relative metabolic stability and initial metabolite information from the same analysis. The high mass resolution
with high scan speed data acquisition is compatible with UHPLC for high throughput screening.
Methods
Six model compounds were selected for this study. Compounds（ 3 µM）were incubated using human, dog and rat hepatic
microsomes at nominal 0.5 mg protein/mL at 37°C for up to 60 minutes in the presence of NADPH. At 0, 5, 15, 30, 45 and 60 minutes,
aliquots of the reaction mixture were transferred and mixed with quench solution. The samples were centrifuged, and supernatant
was injected for analysis. The time-concentration profiles of parent compounds and identified metabolites were estimated fromthe acquired data by using an Exactive II benchtop HR mass spectrometer in positive full scan / all ion fragmentation（AIF）scans
at 70,000 FWHM resolution. Simultaneous acquisition and processing were performed using the relative Quan/Qual software
package, MetQuest 1.1.
Preliminary Data
The metabolic stabilities of six diverse compounds- trifluoperazine, carbenoxolone, raloxifene, diclofenac, paclitaxel, and piroxicam
were analyzed by UPLCMS on a benchtop Orbitrap MS with higher energy collision dissociation（HCD）. Data were acquired as afull scan followed by an all-ion-fragmentation（AIF）scan. The data acquisition was conducted by using MetQuest 1.1 metabolic
screening software, which performed automatic relative Quan/Qual data analysis. Further metabolite structure determination was
possible using the same data files, without reinjection, by using data mining software. Initial processing of the time-course samples
was performed automatically, using a list of 50 mass changes representing commonly found biotransformations as well as
screening for unexpected metabolites. To be considered an unexpected metabolite, the extracted ion chromatogram of anobserved m/z value had to follow a peak shape, and not appear in the control sample. Expected metabolites were included only
if the area, after comparison to the control, differed by more than 5 X and whose exact mass value was within 5 ppm of the
expected value. This workflow reduces data turnaround time, increases compound throughput, and maximizes mass spectrometer
usage.

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 29: The Advances in Biological Mass Spectrometry in Drug Discovery andDevelopment: Current State of the Art and Challenges

PWe-133
11:10 － 12:20

Development of High Performance Liquid Chromatography Tandem Mass
Spectrometry Method for Analysis of Bacopaside-I in Rat Urine and Feces
Samples

Sontaya Sookying2, Kornkanok Ingkaninan*2, Dumrongsak Pekthong3, Sarawut Oo-puthinan3, Jie Xing1,
Zhaoqi Zhan1
Shimadzu (Asia Pacific) Pte Ltd, 79 Science Park Drive, #02-01/08, CINTECH IV, Singapore, 2Department ofPharmaceutical Chemistry and Pharmacognosy, Faculty of Pharmaceutical Sciences, Naresuan University, Phitsanulok
65000, Thailand, 3Department of Pharmacy Practice, Faculty of Pharmaceutical Sciences, Naresuan University,
Phitsanulok 65000, Thailand
1

Keywords:
Pharmacokinetics, Quantification, LC-MS/MS, Validation
Novel aspects:
A LC-MS/MS quantitative analysis method of Bacopaside I in rat urine and feces samples method has been developed andvalidated
Abstract:* Corresponding author
Introduction
Bacopaside I（C 46H 74O 20S）is one of active components isolated from Bacopa monnieri (L.) Wettst.（Brahmi）, which has beenused as an Ayurvedic medicine plant for centuries［ 1 ］. It has been reported that Brahmi has potential therapeutic effect intreatment and prevention of neurological diseases and improvement of cognitive processes［2,3］. Memory enhancing effect ofBrahmi has been established through animal experiments and in healthy volunteers. The damarane triterpenoid saponin,
Bacopaside I is a major active compounds in Brahmi. HPLC method［ 4 ］has been established and used to determine thebacopaside I and other active components in the plant extracts as well as biological samples［ 3 ］. However, so far LC/MS
method for quantitative analysis of Bacopaside I in biological samples has not been reported. We report for the first time thedevelopment and validation of a LC-MS/MS method, aiming for pharmacokinetic study of bacopasides I.
Experimental
A LCMS-8030（Shimadzu Corporation, Japan）tandem triple quadrupole LC-MS/MS was used for this method development study. A
fast gradient elution separation program on a Kinetex C18 HUPLC column（1.7 um, 50 mmL x 2.1mmID）was developed andoptimized. The MRM transitions in ESI positive mode used are 979.4 > 473.4 for bacopaside I and 609.3 > 195.0 for reserpine asinternal standard. Rat urine and feces were used as the biological matrix for method development and validation of bacopaside I
quantitative analysis. Liquid-liquid extraction method was employed in sample extraction and purification.
Results and Discussion
The performance of the method has been evaluated systemically using extract samples prepared from rat urine and feces. The
linearity（r 2 ）was 0.9999 for a range from 8.6 ng/mL to 900 ng/mL. The LOQ of the method was lower than 8.6 ng/mL. The peakarea reproducibility at this concentration level（spiked into urine）was 17.2%（RSD, n= 6 ）. The peak area reproducibility were 6.3&
and 2.9% for concentration levels of 25.7 ng/mL and 85.5 ng/mL, respectively. The recoveries of bacopaside I spiked into urine were
between 100% and 137.1% for three concentration levels（50, 80 and 100 ng/mL）. However, the recovery of feces extract samples
was found lower than that of urine samples at 30~60%. Further study to improve the recovery of bacopaside I in feces samples isundergoing. The method has been applied to in vitro hydrolysis samples.
Conclusions
For the first time, a high sensitivity MRM method on LC-MS/MS was developed and validated using rat urine and feces samples.
The method with further improvement in feces sample pretreatment to improve the recovery is ready for use in pharmacokinetics
study.

References
1. H.K. Singh, B.N., Dhawan, Indian J. Pharmacol. 29（1977）S359
2. A. Russo, F. Borrelli, Phytomedicine 12（2005）305
3. D. Vohora, S.N Pal, K.K. Pillai, J. Ethnopharmacol. 71（2000）383
4. M. Deepak, G.K Sanggli, P.C. Arun, A Amit, Phytochem Anal 16（2005）24

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 29: The Advances in Biological Mass Spectrometry in Drug Discovery andDevelopment: Current State of the Art and Challenges

PWe-134
13:30 － 14:40

Novel Mass Spectrometry Based Method for Quantitation of Antibody
Drug Conjugates

Kim Alving1, James Stefano1, Richard Gregory2, Diego Gianolio2, William Brondyk1, Pradeep Dhal1,
Aharon Cohen1, Bing Wang1
1

Genzyme - A Sanofi company, 2Sanofi Oncology

Keywords:
ADC stability quantitation affinity cytotoxin
Novel aspects:
Novel method for quantitation of ADCs based on affinity capture of ADC, release of the ADC bound drug by digestion andquantitation of the released free drug by mass spectrometry.
Abstract:
Potency and specificity are desirable attributes of a successful drug. These qualities are implemented by antibody drug conjugate（ADC）constructs, where potent cytotoxic agents（drugs）are linked to antibodies with high specificity for the surface of targeted
cells. The stability of the ADC as an intact entity is therefore of particular importance.
ADCs are typically quantified by ELISA after development of antibodies against either the antibody or the attached drug. However,in neither case is it assured that the ADC measured by ELISA is intact.
We therefore developed a mass spectrometry based method for quantitation of ADCs based on affinity capture of the ADC,
followed by thorough washing to remove non-specifically bound material including free drug, and subsequent release of the ADC
bound drug by digestion. The resulting free drug was quantified by mass spectrometry.
This work flow differs from a recently developed workflow by Xu et al 2011 in several respects. The here presented affinity capture
does not require access to high purity antigen, and the quantitation method measures the drug released and extracted from the
ADC directly by mass spectrometry, as opposed to deglycosylation and reduction of the IgG with subsequent mass spectrometricdetermination of the drug antibody ratio（DAR）values of the light and heavy chains.
Data will be shown that demonstrate the linearity of the approach and application hereof for ADC stability studies.
It should be underlined that in addition to measuring the ADC bound drug directly, this approach will be of particular value forcomparative developments of novel ADCs, where different antibodies are linked to an array of drugs by different cleavable drug
linkers, as development of individual ELISA kits for each ADC would be time consuming and expensive. The here presented
workflow can seamlessly be altered for quantitation of any new cleavable linker based ADC.
Reference
Xu et al 2011, Anal. Biochem. 412 56-66

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 29: The Advances in Biological Mass Spectrometry in Drug Discovery andDevelopment: Current State of the Art and Challenges

PWe-135
11:10 － 12:20

Method for the direct injection and on-line sample clean-up of wholemethanolic extracts from dried blood spots

Reiko Iizuka1, Motoji Oshikata1, Paul Rainville2, Jennifer Simeone2, Thomas Wheat2, Robert Plumb2
1

Nihon Waters, Tokyo, Japan, 2Waters, Milford, MA

Keywords:
DBS, On-line sample clean-up, At-column dilution, 2 D LC/MS/MS, Bioanalysis
Novel aspects:
Ability to inject whole organic DBS methanolic extracts with on-line sample clean up in a single injection.
Abstract:
Introduction
The collection of blood onto Guthrie-type filter paper has recently been demonstrated and is referred to as dried blood spots（DBS）. This sample preparation method has been demonstrated to reduce both cost and animal usage. Most often the analyteof interest is extracted from the DBS with neat methanol and must be diluted with water prior to LC/MS/MS analysis. This dilution
step therefore influences assay sensitivity. In this work, we present the use of 2 D LC/MS/MS coupled with at-column dilution（ACD）for the analysis of rosuvastatin from DBS extracted with neat methanol. This approach enabled maximum sampling of thewhole extract onto the 2 D LC/MS/MS system and on-line sample clean up in a single injection.
Methods
An 80 µL aliquot of the methanolic DBS extract was injected onto the 2 D LC/MS/MS system. ACD was performed using a 50 µL
mixer prior to the trapping column. Following sample loading onto the trap column for 0.5 minutes, the trap column was washedwith 20 percent acetonitrile for 0.5 min. The valve was then switched and the sample forward flushed onto the analytical column.
Rosuvastatin was eluted from the analytical column at 500 µL/min under a linear gradient from 5 -55 percent acetonitrile in 5 min.
Mass spectrometry was performed on a Xevo TQS operating in positive elctrospray ionization mode and in MRM acquisition
mode.
Preliminary Data
An assay was developed by utilizing a heart-cut configuration and at-column dilution for the methanolic extract of rosuvastatin
DBS. The at-column dilution of the rosuvastatin DBS allowed for more sample to be loaded onto the system, producing greaterpeak height and improved peak shape, thus producing a low LLOQ for the assay. The assay proved linear for 2.5 orders ofmagnitude with all residual values less than 9 %, producing an assay within bioanalytical validation criteria as defined by the FDA.
The LLOQ of the assay was determined to be 0.1 ng/mL with a signal to noise value of greater than 5 ：1. Carryover was evaluatedby injection of a matrix blank following the ULOQ of 50 ng/mL. We observed no detected analyte in the matrix blank. Interferences
both from the DBS card as well as the matrix where reduced via the heart cutting method as well as contamination of the MS
source. Based on the results of this study, this method shows promise for other bioanalytical assays whereby sample（s）
consisting of a high organic component can be directly injected and on-line clean up performed in a single injection.

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 29: The Advances in Biological Mass Spectrometry in Drug Discovery andDevelopment: Current State of the Art and Challenges

PWe-136
13:30 － 14:40

Metabolite analysis using a novel relationship and database driven
software platform approach for screening and understanding metabolism

Etsuko Suzuki1, Motoji Oshikata1, Mark Wrona2, David Deans3, Jeff Goshawk3,
Debadeep Bhattacharya2, Joanne Mather2, Robert Plumb2
1

Nihon Waters, Tokyo, Japan, 2Waters Corporation, Milford, MA, 3Waters, Manchester, UK

Keywords:
Metabolite identification, Database, Network, UPLC, QTofNovel aspects:
A networked platform integrating advances in multi-parametric use of data to identify drug related material and disseminate to
global organisations
Abstract:
Introduction
Informatics is increasingly becoming the key component in driving knowledge generation from DMPK LC/MS studies. Current
software platforms are generally limited to single experiment analysis types and although these approaches continue to serve us
well, the majority of these platforms were designed for linear experimental designs. As analytical methods deployed in DMPK
have delivered improvements in data quality and throughput , researchers are focusing on ways to increase efficiency by deriving
greater value from their complex datasets. The approach defined is a software system that uses the latest computing hardware（64-bit, multi-core capabilities）, latest generation hardware（UPLC/MS Qtof platforms）and software（scientific library data
management）to accurately screen, confidently interrogate datasets and report the data in a highly customizable format.
Methods
Metabolite analysis for several compounds was performed using a prototype acquisition and processing software. Central to thisinformatics platform is a scientific library which stores information related to compounds of interest providing a starting point fordeveloping methods as well as capturing the results of the analysis. From here samples were automatically processed to provide
a comprehensive list of metabolites which can then be interrogated to suit both the analytical and reporting needs of the user.
We will discuss the use of data properties such as； mass accuracy, decimal mass analysis, isotopic pattern and fragment
correlation analysis to drive a filtering process that enables unbiased identification of drug related components and allows theuser to establish and map metabolite relationships.
Preliminary Data
Data set was processed and directly compared with the current generation of metabolite screening software. Data was processed
and analyzed using newly integrated Apex peak picking algorithms, taking advantage of the multicore processing capabilities ofmodern GPU based technology which has shown an improvement of > 3 fold in the time required for extraction and processing
raw data. Additionally, processing is centralized around the built in scientific library functionality which manages both compoundand metabolite identification specific criteria. This advanced decision making results in a transparent user experience which
automatically adapts to the unique chemical properties of a compound allowing users to generate metabolite relationships within
a single LCMS software package as opposed to current methodology relies heavily on linear single compound, single dataset,single answer approaches. With a scientific library underpinning, we are able to both build and retrieve metabolite relationshipinformation across datasets and samples that were historically difficult to manage as a single report. Stronger integration of insilico tools, metabolite prediction, isotopic pattern analysis and more flexible mass defect filtering have been used in order tomore confidently assign drug related materials. In this presentation we will demonstrate how drug metabolism departments will
have the ability to manage instrument and server assets remotely across the network, this network based infrastructure also
facilities scientifically sharing and disseminating data and knowledge across labs and across networks. DMPK datasets have beenprocessed for both generating qualitative structural and relationship pathways as well as for quantitative analysis and putting thedatasets into meaningful context within a single, integrated processing and reporting environment.

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 29: The Advances in Biological Mass Spectrometry in Drug Discovery andDevelopment: Current State of the Art and Challenges

PWe-137
11:10 － 12:20

HILIC-UHPLC-MS/MS determination of entecavir in rat plasma, urine andplasma ultrafiltrate using various sample preparation techniques

Lucie Novakova1, Hana Vlckova1, Tomas Gottvald1, Jaroslav Janak1, Frantisek Trejtnar2,
Jana Mandikova2, Petr Solich1

Charles University in Prague, Faculty of Pharmacy in Hradec Kralove, Department of analytical chemistry, 2Departmentof Pharmacology and Toxicology, Faculty of Pharmacy, Charles University in Prague, Heyrovskeho 1203, 500 05 Hradec
Kralove, Czech Republic

1

Keywords:
entecavir, UHPLC, HILIC, MS/MS, biological material
Novel aspects:
A new HILIC UHPLC-MS/MS method for the determination of entecavir in various biological material was developed and validated.
Several approaches were used to reduced matrix effects.
Abstract:
Entecavir is a novel deoxyguanosine nucleotide antiviral agent with activity against hepatitis B virus（HBV）. The agent possessesa polar structure, which might cause difficulties, when determined by conventional LC-MS methods. In this study RP-UHPLC method
using BEH C18 stationary phase with mostly aqueous binary mobile phase composed of（ 4 ：96）acetonitrile/0.01% formic acid and
HILIC-UHPLC method using BEH Amide stationary phase with binary mobile phase composed of acetonitrile/ 5 mM ammonium
acetate pH 4.0（75：25）at flow-rate 0.3 ml/min were compared in terms of sensitivity, linearity, repeatability and accuracy. The
effect of various mobile phase additives（formic acid, acetic acid, ammonium acetate and ammonium formate at both acidic andbasic pH）was carefully studied in both RP and HILIC method.
HILIC-UHPLC-MS/MS method demonstrated more favorable results and moreover, it provided more convenient conditions forstraightforward coupling with solid phase extraction-based（SPE）sample preparation techniques. Several biological materials（raturine, rat plasma and plasma ultrafiltrate of rats）were examined using various SPE-based extraction techniques（RP-SPE, ionexchange SPE and microextraction by packed sorbent（MEPS）approach）.
The sensitivity of developed UHPLC-MS/MS method was relatively higher compared to previously published works. Method
validation in terms of linearity, accuracy, precision, selectivity and matrix effects was performed for each of biological material. By
the combination of several approaches, that enable to reduce matrix effects（relatively selective SPE sample preparation step,
efficient UHPLC separation, HILIC mode enabling different selectivity and high sensitivity, quantitation using selective SRM modeand SIL-IS with C 13 N 15 labeling and finally sample dilution）, resulting method provided accurate results with negligible matrix
effects and still high sensitivity sufficient for the determination of entecavir in rat urine, plasma and plasma ultrafiltrate samples inspite of the dilution step during sample preparation.
The authors gratefully acknowledge the financial support of research projects of Charles University in Prague UNCE 17/2012 andPRVOUK.

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 29: The Advances in Biological Mass Spectrometry in Drug Discovery andDevelopment: Current State of the Art and Challenges

PWe-138
13:30 － 14:40

Determining The Pharmacokinetics And Metabolic Fate Of A Novel AntiCancer Compound Using LC/MS/MS With Comprehensive Data Collection

Shunya Sasaki1, Motoji Oshikata1, Joanne Mather2, Robert Plumb3, Ian Wilson4

1
Nihon Waters, Tokyo, Japan, 2Waters Corporation, Milford, MA, 3Imperial College, London, UK, 4AstraZeneca,
Macclesfield, UK

Keywords:
qualitative and quantitative analysis, Tandem quadrupole MS, Q-Tof, Bioanalsyis, Metabolite identification
Novel aspects:
Quantification over a wide dynamic range using a hybrid QToF instrument with simultaneous acquisition of qualitative data.
Abstract:
Introduction
The aim of preclinical drug metabolism is to determine the safety profile of a drug candidate. As part of the process it is necessary
to quantify the compound or active moiety and provide information on the metabolic fate of the molecule. In this paper we
present a comparison of the benefits and challenges for the simultaneous acquisition of qualitative and quantitative LC/MS/MS
data using either a QToF or tandem quadrupole instrumentation. The throughput, sensitivity and spectral quality were comparedusing plasma samples derived from oral administration of a novel oncology compound to the rat and dog. The samples weredeprotinated and analysed by accurate mass LC/MS and tandem quadrupole LC/MS/MS.

Methods
Plasma was collected following the oral administration of a novel oncology compound over four weeks and processed via protein
removal with a phospholipid preparation plate. The chromatography was performed on a 2.1 x 50 mm 1.7μm C18 column wasmaintained at 50 °C and eluted with a basic aqueous methanol 5 minutes gradient at 600μL/min. The column eluent wasmonitored by positive electrospray TOF MS, across mass range 50 1200 m/z and on a tandem quadrupole MS using MRM and data
dependent MS. The compound was quantified using a stable-label isotope and authentic standard. The metabolites weredetected and identified by either accurate mass and comparison of the fragment ion spectra or via data directed MS/MS on thetandem quadrupole.

Preliminary Data
The tandem quadrupole mass spectrometer was used to simultaneously acquire LC/MS data in two modes； i）MRM to acquire
quantitative data for the analytes and ii）data directd MS/MS to obtain qualitative data for metabolite detection. The QToF wasoperated in alternating low and elevated energy mode allowing the simultaneous collection of both precursor and product ion
data. The QTof was operated with a resolution of 20,000 FWHM and a data collection rate of 20 spectra-per-second. This resolution
allowed the use of a narrow mass window for data processing； in this case it was possible to employ a 0.5mDa window. The datacollected from both instruments showed the presence of four major metabolites； two hydroxylated metabolites, a demethylated
metabolite and a dealkylated metabolite. The Cmax for the metabolites occurred at 3 hours with all the metabolites being
cleared by 24hrs. The high degree of selectivity of the QTof produced excellent signal/noise resulting in an LOD of 50pg/mL linear
over 4 orders of magnitude. The metabolites were detected by the use of the accurate mass precursor and the structure
elucidated using the accurate mass fragment ions produced in the high collision energy data. The limit of detection for the tandem
quadrupole mass spectrometer in MRM mode was determined to be 10pg/mL. Whilst the triple quadrupole mass spectrometer
showed greater sensitivity for quantification, the full scan data used for metabolite detection and identification was approximately
50-fold less sensitive than the QToF. The specificity of the tandem quadrupole instrument for metabolite detection was increasedby using a data directed approach； here the MRM transition for a series of potential metabolites using knowledge of the parent
fragmentation pattern and potential biotransformations. When a potentially metabolite related peak was detected the instrument
automatically collected MS/MS data during the trailing edge of the chromatographic peak.

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 29: The Advances in Biological Mass Spectrometry in Drug Discovery andDevelopment: Current State of the Art and Challenges

PWe-139
11:10 － 12:20

Development of a Highly Sensitive Methodology for Quantitative
Determination of Fexofenadine in Microdose Studies by Multiple Injection
Method Using UHPLC-MS/MS

Yukari Tanaka1,2, Yutaka Yoshikawa2, Hiyoyuki Yasui2
1

SHIONOGI & CO., LTD., Osaka, Japan, 2Kyoto Pharmaceutical University, Kyoto, Japan

Keywords:
multiple injection method, ultra-performance LC-MS/MS, fexofenadine, microdose study, high sensitivity
Novel aspects:
This study suggests that HSMIM has an ultra high sensitivity and high-throughput for determining by LC-MS/MS using a simple
sample preparation in PK and microdose studies.
Abstract:
Introduction

Recently, the trend has been toward lower efficacy doses because of the synthesis of many compounds with strong activity,
quantitative methods utilizing LC-MS/MS with higher sensitivity are required. LC-MS/MS with high sensitivity and selectivity is themost informative method with respect to structural analysis of drug metabolites and determination of drug concentrations inbiological samples. Fexofenadine, a non-radiolabeled compound, is an antihistamine drug that was orally administrated at a doseof 100 µg/man/day in the first microdose clinical study in Japan in 2005； subsequently, the concentration of fexofenadine in humanplasma was measured by LC-MS/MS. The lower limit of quantification（LLOQ）was 10 pg/ml when the concentration of
fexofenadine in human plasma, prepared by solid-phase extraction, was determined using LC-MS/MS. Previous studies havefrequently used the protein precipitation method using an organic solvent for sample treatment and the LLOQs in the“ng/ml“
range to determine the concentration of fexofenadine in biological samples by LC-MS/MS. However, the drug concentration ofsamples in a microdose study cannot be measured at an LLOQ of only ng/ml because of lack of sensitivity. In case of employing
solid-phase extraction or liquid-liquid extraction for sample preparation, it is possible to set the LLOQ at pg/ml, but total sample
analysis is required, which demands increased time as the procedure of sample preparation is quite complex. In pharmacokinetic（PK）studies with many samples, solid-phase extraction method is disadvantageous, as it is more expensive than the protein
precipitation method. In this work, fexofenadine was selected as a model compound, and we developed and validated the novel
method, ultra high-sensitivity quantitative method of multiple injection method（HSMIM）, for both rat and human plasma samples
without an internal standard compound by using simple sample treatment procedures like the protein precipitation method.
Furthermore, we demonstrated the analytical time required by using the ultra-HPLC（UHPLC）technique.
Methods

The UHPLC system used was Nexera（Shimadzu Corporation, Kyoto, Japan）. The mass spectrometry-based detection was
performed on an API 5000 triple quadrupole mass spectrometer（AB SCIEX, Foster City, CA, USA）. The mass spectrometer was
operated in the multiple reaction monitoring（MRM）mode using positive-ion electrospray ionization. HSMIM consists of 2 steps.
Step 1 involves injection and condensation： the same compound is continuously injected into a column and trapped at thecolumn head under an isocratic condition of low organic solvent concentration. Step 2 involves elution： the compound trappedat the column head is eluted and introduced into the mass spectrometer.
Results

The calibration curves of fexofenadine prepared using rat plasma and determined by HSMIM were fairly linear over theconcentration range of 5 -5000 pg/mL. The intra-day precisions of fexofenadine were <8.7%, and the accuracies ranged from 95.2%
to 99.3%. The inter-day precisions were <8.4%, and the accuracies ranged from 98.6% to 102.6%, respectively. The concentrating
effect of fexofenadine on the column was investigated by HSMIM by using rat plasma samples. Consequently, we confirmed thatthe peak area of fexofenadine in HSMIM showed an approximately 5 -fold increase compared to the conventional method. In
addition, reduction of the analytical time and the sharper peak were achieved by applying the UHPLC technique to HSMIM. We
were able to set up a high-throughput analytical method in which the total analytical time was approximately only 2 min per
sample, and the retention time of fexofenadine was about 0.6 min. The concentration in rat plasma after oral administration of 100µg/kg fexofenadine（ 1 /100th of the efficacy dose in rat）was determined by this validated method. As a result, the plasma
concentrations of all samples could be adequately measured using this method.

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 29: The Advances in Biological Mass Spectrometry in Drug Discovery andDevelopment: Current State of the Art and Challenges

PWe-140
13:30 － 14:40

Mass spectrometric analysis of ginsenosides in Panax ginseng root andcallus using pseudo two-dimensional liquid chromatogram

Yumiko Ueno1, Issey Osaka2, Hirotaka Hisatomi1, Shu Taira3, Hideya Kawasaki1, Ryuichi Arakawa1

1
Kansai University,Suita,Japan, 2Yokohama City University, Yokohama, Japan, 3Japan Advanced Institute of Science andTechnology, Nomi, Japan

Keywords:
pseudo 2 D chromatogram, LC-MS, Orbitrap, ginsenosides, Panax ginseng
Novel aspects:
A rapid and simple method for creating a pseudo 2 D chromatogram using conventional LC-MS was developed and applied toanalysis of ginsenosides in the calli and root of Panax ginseng.
Abstract:
Introduction
Panax ginseng C.A. Meyer is one of the most famous herbal products available and the root of Panax ginseng is used in sometraditional Chinese medicine treatments. The root contains many bioactive compounds of which its major class is glycosides,
which includes ginsenosides. It is important to understand what effective combinations and appropriate amounts of glycosides
should be used in drug discovery and pharmacology. Recently, on-line two-dimensional（ 2 D）liquid chromatography（LCxLC）
methods that use two different columns with different chromatographic behaviors have been developed. But it takes longer times
to determine the LC conditions and create the LCxLC chromatograms. Therefore we developed a rapid and simple method for
creating a pseudo 2 D chromatogram using conventional LC-MS for analysis of ginsenosides.
Methods
The samples were extracts of the calli and the root of Panax ginseng（Zen-Noh, Japan）. Dried 500 mg samples were prepared bysonication in 70 % aqueous methanol. The sample solution was centrifuged and the obtained solution was measured by LC-MS（Exactive, Thermofisher Scientific）. LC-MS chromatography data of retention time（RT）, m/z, and ion intensities were digitalizedusing the software program SIEVE（ver.1.2, Thermofisher Scientific）. The noise level of the ion intensity was set to an appropriate
value to remove insignificant peaks. The LC-MS chromatography data were extracted with each use of the reverse phase and
HILIC columns. A scatter diagram of the retention times for the pseudo 2 D chromatogram was created on the basis of the m/z
values.
Results
The standard solution of seven ginsenosides was analyzed using LC-MS with the reverse phase or HILIC column. We found thatginsenosides Rg 1 and Re were only separated by the HILIC column. In contrast, the Rg 1 and Rf isomers were only separated by
the reverse phase column. A pseudo 2 D chromatogram was created from the two chromatograms to solve the problem of
separation. The RT values for the HILIC and reverse phase chromatograms were plotted along the abscissa and the ordinate of thepseudo 2 D chromatogram, respectively. The pseudo 2 D chromatogram showed that the plots for Rg 1 and Rf, which were eluted
together by HILIC LC-MS, and the plots for Rg 1 and Re, which were eluted together by reverse phase LC-MS, could be separated
and visualized. The pharmacological properties of callus A and the root of Panax ginseng are different, possibly because of thedifferent amounts and types of ginsenosides that they possess. Therefore, it is significant when analyzing their pharmacologicalproperties to determine the components that are present in their extracts rapidly. Extracts derived from the calli and roots ofPanax ginseng were analyzed by the pseudo 2 D chromatogram. The differences in the components found when comparing thecallus and root, or callus and another callus that were cultured under different culture conditions, were distinguished bycrosschecking their pseudo 2 D chromatograms against one another. We found that the components contained in the calli andtheir quantities were altered by the culture conditions in which the calli were grown. The pseudo 2 D chromatogram is expected
to be a useful method for visualizing complex component patterns found in glycosides and unknown compounds in foods.

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 29: The Advances in Biological Mass Spectrometry in Drug Discovery andDevelopment: Current State of the Art and Challenges

PWe-141
11:10 － 12:20

Metabolic site elucidation of glucuronide metabolites using ion mobility
and molecular modeling

Motoji Oshikata1, Masayo Yabu1, Yutaka Takahashi2,3, Mitsuo Takayama3

1
Nihon Waters, Tokyo, Japan, 2MS Solution, Tokyo, Japan, 3Graduate School of Nanobioscience, Yokohama City
University, Yokohama, Japan

Keywords:
Ion mobility spectrometry, Metabolite identification, glucuronide metabolites, Positional isomers
Novel aspects:
Ion mobility technology delivers the new approach for structure elucidation of glucuronide metabolites based on comparison ofcollision cross-section of metabolites.
Abstract:[Introduction]
The identification of drug metabolites is a critical component in drug discovery and development process. It supports the selectionof species for the toxicology study, and provides information on the potential to form pharmacologically active metabolites or
metabolites with toxicological consequence in human. Tandem mass spectrometry coupled with liquid chromatography（LC/MS/MS）has commonly been used as the first step of metabolite structure elucidation. However, collision-induced dissociation（CID）
often fails to identify the site of biotransformation. Especially product ions of glucuronide metabolites are lacking in information
about biotransformation site, because glucuronic acid moiety as a neutral fragment is easily loosed from precursor ion. In suchcase, metabolite isolation is needed and NMR is typically employed to obtain structure information, and this approach can be
including time consuming process. Ion mobility spectrometry（IMS）has the ability to separate isomeric species such as
glucuronide metabolites, rapidly（msec）based on difference in collision cross-section（W）in the gas-phase, thus providing
specific information on ionic configuration, i.e., the position of the glucuronidation.
[Method]
Glucuronide metabolite isomers of raloxifene and ezetimibe were used as model compounds. Experiments were performed usinga hybrid quadrupole/ion mobility/orthogonal acceleration time-of-flight Synapt G 2 HDMS instrument coupled with ultra
performance liquid chromatography（UPLC-IMS-MS, Waters Corp., Milford, MA）. Ion mobility separation was performed at apressure of 3.2 mbar nitrogen with a wave velocity of 650 m/sec and travelling wave amplitude of 40 V was used. Theoretical Ω
values were calculated using DriftScorp（Waters Corp., Milford, MA）, and compared to the travelling wave derived Ω values. Ion
mobility calibration was undertaken using eight singly charged polyalanine ions.
[Results and discussion]
Raloxifene- 4 ’-gluclonide（Rxf- 4 -G）and raloxifene- 6 -glucuronide（Rxf- 6 -G）showed different UPLC chromatographic elution time.
However, the MS spectra and breakdown curve of product ions showed all the identical patterns, therefore the identification of
metabolic site was difficult for these metabolites. The W values of Rxf- 4 -G and Rxf- 6 -G were measured by UPLC-IMS-MS.
Theoretically calculated W value for Rxf- 4 -G and Rxf- 6 -G were 176.6 Å2 and 180.7 Å2, respectively. Collision cross-section values
derived from travelling wave ion mobility were in very close to the theoretically derived values, thus allowing accurate
identification of the glucuronide metabolites, even those with a collision cross-section difference of less than 1 Å2.
Further, phenolic glucuronide of ezetimibe（Eze-P-G）and benzylic glucuronide of ezetimibe（Eze-B-G）were also separated with
UPLC. MS/MS analysis of these ezetimibe glucuronides gave characteristic structure information for each compound. Product ion
spectrum of Eze-P-G included a peak corresponding to the loss of water molecule from benzyl hydroxyl group moiety. On theother hand, the product ions from Eze-B-G did not show the peak of water loss because glucuronic acid is adducted to the benzylhydroxyl group. In such case, MS/MS analysis provides information for elucidating the structure of isomeric metabolites. The major
product ion from both glucuronides of ezetimibe is m/z 392 corresponding to neutral loss of both water and glucuronic acid fromprecursor ion. The IMS driftgram of m/z 392 suggested the presence of geometric（cis/trans）isomers. The intensity ratio of cisand trans-isomers were different between Eze-P-G and Eze-B-G. This may be originating from the difference of conformation foreach glucuronide.
In conclusion, IMS technology was a powerful tool for structure identification of isomeric species, such as glucuronide metabolite,
without additional analytical techniques and chemical synthesis. Additionally, IMS technology suggested a possibility for providing
information of relationship between conformation of gas-phase molecule and fragmentation mechanisms.

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 29: The Advances in Biological Mass Spectrometry in Drug Discovery andDevelopment: Current State of the Art and Challenges

PWe-142
13:30 － 14:40

Mimicking of the phase I metabolism of drugs by photocatalytic reactions

Miina Ruokolainen, Tiina Sikanen, Petri Kylli, Risto Kostiainen, Tapio Kotiaho
University of Helsinki, Helsinki, Finland

Keywords:
Titanium dioxide, Photocatalysis, Oxidation, Phase I metabolism
Novel aspects:
Titanium dioxide photocatalytic reactions for mimicking phase I metabolism of drugs.
Abstract:
The metabolism of a drug candidate has to be investigated early in the preclinical phase of drug development in order to predictthe potential risks. Drug metabolism is studied in vitro using hepatocytes, microsomes or recombinant enzymes and theexperiments are extensive, laborious and expensive. Due to these drawbacks, methods such as electrochemistry（EC）, Fenton
reaction, EC-Fenton reaction and metalloporphyrin systems have been studied as alternatives to in vitro metabolism studies.1-3
The reaction products of these oxidation methods have been shown to resemble the products of oxidative drug metabolism
reactions, which are the most important phase I metabolism pathways. Also another oxidative system, namely titanium dioxide（TiO2）photocatalysis, has been recently shown to produce reaction products which correlate with metabolites produced in vivo
and with human liver microsomes in vitro.4 TiO2 is able to catalyze both reductive and oxidative reactions when exposed to UV
light having high enough energy to excite electrons from the valence band to the conduction band of TiO 2. The aim of our study is
to further investigate the feasibility of a TiO2 photocatalytic system for imitation of phase I oxidative metabolism, compare it toelectrochemical oxidation and develop a simple generic photocatalytic method using TiO2 particles.
Photocatalytic reactions were carried out in liquid phase using titanium dioxide Degussa P25 particles. The reaction mixture wasmagnetically stirred during exposure to UV light. Titanium dioxide particles were removed from the reaction mixture bycentrifugation and the supernatant was collected for mass spectrometric analysis. The reaction products were analyzed using
appropriate ionization technique, electrospray ionization or atmospheric pressure photoionization, according to the properties of
the analyte. TiO 2 photocatalytic reaction products of the selected drug molecules were compared with in vitro and in vivo
metabolites and with electrochemical oxidation products found in literature.
The photocatalytic method is simple and relatively rapid. The preliminary results suggest that TiO2 photocatalytic reactions havepotential for imitation of phase I metabolism. Photocatalytic reactions could be particularly useful for production of metabolites inlarger scale than is possible using in vitro methods.
References：
1. Jurva U et al. Rapid Commun. Mass Spectrom. 2003, 17： 800-810
2. Johansson T et al. Rapid Commun. Mass Spectrom. 2007, 21： 2323-2331
3. Lohmann W and Karst U, Anal. Bioanal. Chem. 2008, 391： 79-96
4. Nissila T et al. Lab Chip 2011, 11： 1470-1476

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 29: The Advances in Biological Mass Spectrometry in Drug Discovery andDevelopment: Current State of the Art and Challenges

PWe-143
11:10 － 12:20

Streamlining the Metabolite Identification Workflow Using High Resolution
QTOF Data and Mass-MetaSite.

Lester C Taylor1, Ismael Zamora2, Gabriele Cruciani2
1

Agilent Technologies, 2Molecular Discovery Ltd

Keywords:
Predictive metabolite identification.
Novel aspects:
Metabolite identification based on matching MS/MS data and analysis of the potential interactions of drug compounds in thecytochrome P450 cavity and their chemical reactivity towards oxidation
Abstract:
Introduction：
The availability of high resolution accurate mass QTOF data has revolutionized the metabolite identification field. Nowadays, anexpert can generate an unprecedented amount of accurate data for each chromatographic peak in order to assign the chemical
structure of metabolites. Therefore, sophisticated data processing software is needed to help transform this huge volume of data
into information that can be shared in an efficient manner.
This presentation demonstrates the use of Mass-MetaSite integrated with MassHunter file format, to identify drug related material
peaks in the chromatogram, assign chemical structures for each of the peaks found.
Results：
The Mass-MetaSite process consists of two steps. The first step identifies Drug Related Material using the chromatogram obtained
from the QTOF MS and MSMS methodology in order to find chromatographic peaks that are related to the parent compound. It
reports 2 types of peak： a）peaks that correspond to a mass shift from the parent corresponding to one or more metabolic
reactions（phase I and II）that are set prior the calculation within the software and b）unknown metabolites, these are observed
peaks but do not correspond to any of the list of known metabolic transformations.
In the second step the chemical structures giving rise to the peaks are assigned. In order to perform this operation, the software
produces a set of theoretical fragments（structure and mass）for the parent and the metabolites and the masses for that fragment
list compared with the actual fragment ions found in the MS and MSMS spectra. From this comparison the software is able tolocalize the region in the molecule where the metabolic reaction may take place. In the case that the fragment analysis cannot
specify a single atom in the parent that produce the metabolic reaction, a markush representation of the metabolites compatible
with the mass spectra data is shown to the user and in order to prioritize among the different potential structural solutions the
MetaSite Site of Metabolism（SoM）algorithm 1,2 is applied. The SoM prediction is used to predict the regioselectivity of themetabolic reaction only for phase I metabolism and it is based on an analysis of the potential interactions of the compounds inthe cytochrome P450 cavity and the chemical reactivity towards oxidation.
Discussion：
We present an integration of QTOF high resolution accurate mass measurements with software that processes the data in orderto suggest peaks and structural assignment of each peak to the metabolite identification expert via the automatic Mass-MetaSite
batch process and the reporting system based on the WebMetabase application. The computational tool performs a systematic
analysis of the fragmentation by breaking bonds to elucidate the structure of the metabolite and report to the expert thesuggested structural fragments. In addition, when the mass spectral data is not enough to localize a definitive structure of themetabolite, the MetaSite SoM prediction algorithm is applied to suggest a potential metabolite that is in agreement with thespectral data.
The WebMetabase tool collects the data from the automatic procedure in Mass-MetaSite, and presents the data to the expert for
approval, keeping all the fragments in a database. WebMetabase also allows the analysis of experiments with multiple samples,for example performing a kinetic analysis enabling the possibility to keep both qualitative and quantitative data in a single system.
References:
1. Cruciani G. et al.（2005）MetaSite： Understanding Metabolism in Human Cytochromes from the
Perspective of the Chemist. J. Med. Chem., 48, 6970-6979.
2. Enhanced metabolite identification with MS（E）and a semi-automated software for structural elucidation. Bonn B, Leandersson
C, Fontaine F, Zamora I. Rapid Commun Mass Spectrom. 2010 Nov 15；24（21）：3127-38.

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 29: The Advances in Biological Mass Spectrometry in Drug Discovery andDevelopment: Current State of the Art and Challenges

PWe-144
13:30 － 14:40

The development of multistatin method for determination of statins, their
metabolites and interconversion products in human serum

Hana Vlckova, Karolina Skraskova, Pavel Svoboda, Helena Tomsikova, Petr Solich, Lucie Novakova
Charles University in Prague, Faculty of Pharmacy, Hradec Kralove, Czech Republic

Keywords:
statins, UHPLC-ESI-MS/MS, sample pretreatment, MEPS, SPE
Novel aspects:
A novel method for determination of 17 analytes（statins, their metabolites and interconversion products）was developed andvalidated. Two sample pretreatment techniques（MEPS, SPE）were compared.
Abstract:
Statins decrease the levels of total cholesterol, low-density lipoprotein cholesterol and plasma triglycerides due to inhibition of
microsomal 3 -hydroxy- 3 methylglutaryl-coenzym A （HMG-CoA） reductase. They are used for the treatment ofhypercholesterolemia in clinical practice. Recently anticancer effect was discovered for selected statins.
The aim of this project was to develop and validate UHPLC-MS/MS method and convenient sample preparation procedure forsimultaneous determination of statins（atorvastatin, rosuvastatin, simvastatin, lovastatin, pravastatin, fluvastatin and pitavastatin）,
their metabolites and interconversion products in human serum. As the sample preparation technique solid phase extraction andmicroextraction by packed sorbent were used and compared.
Suitable conditions for UHPLC/MS/MS determination of 17 analytes were found. Analytical column Acquity UPLC BEH C18（1.7 µm,
2.1 x 50 mm）and gradient elution were used. Optimized composition of mobile phase was acetonitrile：0.5 mM AmAc pH 4.0 atflow-rate 0.3 ml/min. Electrospray ionization（ESI）in polarity switching mode was employed as an ionization technique.
Quantification of all analytes was performed using SRM（selected reaction monitoring）. Collision energy and cone voltage were
optimized individually for each analyte.
Novel analytical method enabled simultaneous determination of 17 analytes within 10.5 minutes. The validation data indicatedgood linearity（r > 0.999 for allanalytes）, sensitivity（LOQ 5 ng/ml for most analytes）, repeatability of retention time（< 1.0 %RSD for all analytes）and repeatability of peak area（< 7.0 % for all analytes）.
The universal UHPLC-MS/MS method for simultaneous determination of statins, their metabolites and interconversion products
was developed and validated. Suitable sample preparation technique was chosen, developed and validated. This method wasapplied to the real sample of human serum and used for the monitoring of drug levels in human serum of patients treated bystatins（especially atorvastatin and rosuvastatin）.
The authors gratefully acknowledge financial support of the MSMT of Czech republic - FRVSNo. 894/2012.

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 29: The Advances in Biological Mass Spectrometry in Drug Discovery andDevelopment: Current State of the Art and Challenges

PWe-145
11:10 － 12:20

Electrochemical generation of oxidative and reductive metabolites of
selected drugs and their UHPLC/MS/MS characterization

Michal Holcapek1, Robert Jirasko1, Veronika Jedlickova1, Tomas Mikysek1, Milan Nobilis2,
Lenka Skalova2
1

University of Pardubice, Czech Repubic, 2Faculty of Pharmacy, Charles University, Hradec Kralove, Czech Republic

Keywords:
Drug metabolism； Phase I and II metabolites； UHPLC/MS； Electrochemistr； Anthelmintics.
Novel aspects:
Electrochemical generation of oxidative and reductive metabolites for anthelmintics and other selected drugs, their UHPLC/MS/MS
characterization and comparison with in vitro and in vivo experiments.
Abstract:
The study of drug metabolism is a crucial task in the pharmaceutical research. The detection and structure elucidation of Phase I
and II metabolites of new drugs is an important step in the drug research and development. In vitro and in vivo experiments are
performed for the simulation of oxidative or reductive metabolism using cell cultures and laboratory animals, but the on-line or offline electrochemical generation of metabolites can be also useful for this purpose. Glassy carbon or precious metal（Pt, Au, Ag）
electrodes are used as working electrodes in the electrochemical generation of oxidative products, while the mercury electrode ispreferred for the generation of reductive products. The novel laboratory-made design of high-capacity electrochemical cell will be
described.
In our experiment, the combination of UHPLC separation with subsequent high mass accuracy measurements in full scan andtandem mass spectra modes using QqTOF analyzer is used for the identification and structural characterization of reductive
metabolites of anthelmintic drug flubendazole. The carbonyl reduction leads to the formation of a chiral center, which requires thechiral separation is necessary for the determination of both enantiomers. On the other hand, individual glucuronides of thesephase I reductive metabolites are diastereizomers and can be separated using reverse-phase UHPLC.
Concerning the data interpretation, the elemental composition of present metabolites is determined based on the combinationof high mass accuracy, specific mass defect, characteristic neutral loss scans, the comparison of fragmentation with the parent
drug or relative retention shifts. UHPLC/MS/MS detection of chromophore-containing metabolites in the complex mixture is
improved by the parallel UV detection. Advanced software tools are applied for the metabolite identification using the comparisonof the blank chromatogram with the electrochemically treated samples. Moreover, the multistage mass spectra measurement
using an ion trap based analyzer with the direct infusion introduction of selected metabolites after their isolation provides useful
information about their fragmentation patterns. The similar approach is used for the characterization of oxidative products ofwidespread hypolipidemic drug atorvastatin. The addition of glucuronic or sulphuric acid together with a suitable catalyzer for thegeneration of corresponding conjugates of phase I metabolites is further studied. Electrochemically generated products arecompared with in vivo and in vitro experiments. Finally, the possibility to semi-preparative isolation of drug metabolite standards
and their usage for the chromatographic quantitation or their NMR characterization are discussed.
Financial support of this project was provided by the Czech Science Foundation（No. P205/10/0217 and No. P206/12/P065）.

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 29: The Advances in Biological Mass Spectrometry in Drug Discovery andDevelopment: Current State of the Art and Challenges

PWe-146
13:30 － 14:40

In-depth pharmaco-phosphoproteomics using meter-long monolithic
columns to evaluate molecular-targeting drugs

Masaki Wakabayashi1, Ayaka Sato1, Mio Iwasaki1, Naoyuki Sugiyama2, Yasushi Ishihama1
1

Kyoto University, Kyoto, Japan, 2Keio University, Tsuruoka, Japan

Keywords:
Pharmaco-phosphoproteomics, dimethyl labeling, monolithic columns, drug discovery
Novel aspects:
Pharmaco-phosphoproteomic approach with the combination of meter-long monolithic columns and stable isotope dimethyl
labeling enabled to discover and validate novel kinase-targeted drugs in a high-throughput manner.
Abstract:
In current cancer treatment, many“molecular-targeted drugs“ are clinically utilized and have become major choices ofchemotherapy due to the effectiveness instead of traditional anti-cancer agents, and about half of molecular-targeted drugs were
designed to target oncogenic kinases which cause aberrant cellular signaling. Protein phosphorylation is well known to regulate
diverse cell functions, and therefore, the abnormal profiles are closely related to the onset and development of various diseases
including cancers. Thus, the agents which suppress specific protein phosphorylation are thought to be potential candidates foranti-tumor drugs. In order to evaluate the drug candidates efficiently, we should have a grasp of the whole network consisting ofkinase-regulated phosphorylation and the alterations in pathological states. In addition, high-throughput method is required forscreening candidates. From the above-mentioned points of view, pharmaco-phosphoproteomic approach is currently the mostsuitable for evaluating kinase-targeted anti-cancer drugs.
Recent advances in phosphopeptide enrichment methods, isotope-labeling strategies for quantitation, MS technologies such as
sensitivity and scan speed, and computational algorithms for determination of peptide sequence and phosphorylation sites, aremaking revolutionary changes in phosphoproteome analysis. Although it is not so difficult to identify/quantify tens of thousands ofphosphopeptides by the current approaches, the long analysis time as well as huge amount of samples are inevitably required. In
this study, we introduced a stable-isotope dimethyl labeling not only for quantitation but also for enhancing the identification
efficiency. In addition, we employed a meter-scale monolithic silica capillary column to provide extremely high identification rate
against time.
With dimethyl labeling, 1824 phosphorylated peptides were identified from 600 µg of proteins, whereas 1654 phosphopeptides
were identified without dimethyl labeling. In dimethylated samples, we observed more multiply phosphorylated peptides andmore phosphopeptides with C-terminal lysine residues, compared with non-labeled samples. These results would be caused bythat dimethyl labeling possibly stabilizes positive charges of N-terminal and C-terminal lysyl amino groups and enhances theionization efficiency, especially for multiply phosphorylated peptides which is difficult to detect in positive ion mode. Furthermore,
phosphopeptide enrichment efficiency with titania-based hydroxy acid-modified metal oxide chromatography（Ti-HAMMOC）was
improved from 95 to 98%. This small change was quite important because a very small amount of non-phosphorylated peptides
dramatically affects the detection limit of phosphopeptides. These results clearly demonstrated that dimethyl labeling is effective
not only for quantitation but also for identification efficiency.
In order to perform extremely sensitive and timesaving analysis, we employed a one-dimensional LC-MS system with meter-scalemonolithic silica capillary columns and combined two database search algorithms： MascotTM and ProteinPilotTM. As a result, using
125 µg of cell lysates, we have successfully identified about 10,000 phosphopeptides which would be the highest coverage in asingle shotgun analysis and, compared to a conventional particle-packed column system, 3 -fold increase in MS sensitivity wasobserved due to the reduced co-elution of digested peptides.
Finally, we combined the above-mentioned techniques to realize high-throughput, comprehensive, and precisely quantitative
phosphoproteome analysis, and applied to quantitate the phosphorylation profiles between the cells in basal and drug-stimulated
states. A wide range of small molecules, mainly anti-cancer drugs, were administrated to various cancer cell lines, and thephosphorylation profiles were comprehensively quantified and integrated. From these results, we performed cluster analysis to
extract the drug-type or cell-type specific alteration, and additionally estimated the unknown signaling networks related to antitumor effect. The integrated database is very useful for hunting and validating novel molecular-targeted drugs.

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 29: The Advances in Biological Mass Spectrometry in Drug Discovery andDevelopment: Current State of the Art and Challenges

PWe-147
11:10 － 12:20

A Semi-Automated Method for Sequencing Oligonucleotides using ISD
and Pseudo-MS3 on a MALDI-Ion Trap-TOF Mass Spectrometer

Matthew E Openshaw1, Omar Belgacem1, Marco Smith2
1

Shimadzu, Manchester, UK, 2GlaxoSmithKline, Stevenage, UK

Keywords:
MALDI, in-source decay（ISD）, oligonucleotides, ion trap-TOF
Novel aspects:
In-source decay（ISD）and pseudo-MS 3 sequencing of modified oligonucleotides on a MALDI-ion-trap-TOF mass spectrometer
Abstract:
The advantages of MALDI-TOF for the analysis of oligonucleotides include the high sample throughput and the ability to confirm
the molecular weight of the intact sample. As a result, MALDI-TOF has been widely accepted for this application. Anotheradvantage of performing oligonucleotide analysis on a MALDI-TOF instrument is the ability to generate sequence information
using in-source decay（ISD）, an experiment which can be performed even on the simplest MALDI instrument configuration i.e.
linear mode. However, analysis in linear mode, particularly ISD analysis which involves the use of elevated laser powers, typicallylimits resolution and mass accuracy.
In the case of unmodified RNA oligonucleotides, this may not be a significant problem as the smallest mass difference between
unmodified residues is several mass units. However, for oligonucleotides being developed for use as therapeutics, the RNA
nucleosides may be modified to increase stability through resistance to degradation. In the case of 2 ’-O-methyl
phosphorothioate-modified RNA oligonucleotides, the mass difference between residues can be as small as 1 Da（modified C =336 Da, modified U = 335 Da）. In such cases, the lower mass accuracy of linear mode ISD analysis may not be sufficient tounambiguously determine the sequence.
To overcome this, we have performed ISD analysis of the modified oligonucleotides in reflectron mode on a MALDI-ion trap-TOF
mass spectrometer. An advantage of the ion trap-TOF instrument is that high resolution and mass accuracy can be achieved
across all modes of MS, even when using higher laser power. As a result, monoisotopic resolution was observed for the ISD
fragments. One limitation of this approach on this instrument is that fragment ion sensitivity decreases with increasing mass and it
was not possible to sequence the entire oligonucleotide in one direction. However, in 2 of the 3 samples studied, sequencing
from both ends of the oligonucleotide allowed the complete sequence to be determined.
Sequencing was performed in a semi-automated manner using software originally developed for copolymer analysis. By specifying
the formula of the different ’components’ of the oligonucleotide（e.g. 4 modified nucleosides, modified phosphate group, end
groups, adduct）, the software is used to calculate the masses of various computations within user-defined limits. The high massaccuracy of the MALDI-ion trap-TOF and the observed isotopic pattern can be used by the user to eliminate incorrectcompositions. In this proof-of-principle work, sequencing is performed in a sequential manner, increasing the sequence one
residue at a time. In future, this process could be fully automated. Using this approach, the composition of the 3 ’- and 5 ’-ends
could be proposed based on the first fragments detected. However, the sequence order of this 3 - 4 residue tag could not be
determined from the ISD spectrum due to the complexity of the spectrum below 1000 m/z. To overcome this, we performed
pseudo-MS 3 i.e. MS/MS of an ISD fragment, in an attempt to determine the sequence of the terminal residues.

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 29: The Advances in Biological Mass Spectrometry in Drug Discovery andDevelopment: Current State of the Art and Challenges

PWe-148
13:30 － 14:40

Caco-2 PERMEABILITY STUDIES AND DETERMINATION BY LC/MSMS OF
MEMANTINE

Banu Erdem1, Seval Korkmaz2, Murat Onul3, Engin Bayram3

Marmara University Faculty of Engineering Department of Bioengineering, 2Abdi Ibrahim Pharmaceuticals Inc., Istanbul,
Turkey, 3Ant Teknik Cih. Ltd. Sti.,Istanbul, Turkey

1

Keywords:
memantine, permeability,caco- 2 cell line, LCMSMS
Novel aspects:
BCS Classification of Memantine API’s used for development of new generic formulations was identified and Caco- 2 studies ofMemantine was used first time to prepare of its biowaiver file.
Abstract:
Memantine is the first in a novel class of Alzheimer’s disease medications acting on the glutamatergic system by blocking NMDA
glutamate receptors. Despite years of research, there is little evidence of effect in mild to moderate Alzheimer’s disease.
We aimed the study to show BCS Class of Memantine for its biowaiver file.
Caco- 2 Permeability Studies were performed in the Cell Culture and In Vitro Screening Laboratory of Abdi Ibrahim
Pharmaceuticals R&D Center.
Caco- 2 cells（ECACC）were seeded at a density of 8.000 cells/well on tissue culture-treated polycarbonate filters（area 0,143
cm 2 ）in Corning 96 well Transwell plates（Corning Costar）. The culture medium（EMEM）, pH 7.4 containing 10 % fetal bovine
serum and 1 % non-essential amino acids（Sigma）, added with penicillin- streptomycin solution（Sigma）was changed every day
and cell cultures were incubated at a temperature of 37ºC in an atmosphere of 95% relative humidity, 10% CO2. Filters were used
for transport studies 21 days after seeding.
Viability and integrity tests were performed by Lucifer yellow test and RTCA（Real Time Cell Analyzer）.
Samples were added into wells at 100 uM concentrations in HBSS（Hank’s Balance Salt Solution）containing HEPES buffer at Fassif
Mod condition. While incubating cells at 37 ºC, aliquots were taken from both chambers at the end of 30- 60- 90 and 120 minutes.
Samples diluated in double distillated water and concentrations of compound in the samples were measured by LC-MSMS
instrument（Shimadzu LCMS-8030）.
Two different API were used from two different companies and permeability constant of API A was found（Paap 1,283x10-5 cm/s）
and B was found（Paap 6.63x10-6 cm/s）and it is BCS classification was shown as Class I. The difference between two permeability
constants of Memantine API’s could be related to its particule size and polymorphs.

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 29: The Advances in Biological Mass Spectrometry in Drug Discovery andDevelopment: Current State of the Art and Challenges

PWe-149
11:10 － 12:20

Development of mass sepctrometric method for pharmacokinetic study ofHerceptin

Jin Nyoung Choi1, Jeong Won Kang1, Do Young Choi1, Gyu-Tae Park2, Soyong Jang2, Kwang Pyo Kim1
1

Konkuk University, Seoul, Korea, 2CKD Reasearch Institute, Yongin, Korea

Keywords:
Herceptin,HPLC-MS/MS,pharmacokinetics
Novel aspects:
We developed a senstive and quantitative mass spectrometric method for Herceptin without using Ab for the potential applicationof pharmacokinetic studies for protein drugs.
Abstract:
The therapeutic monoclonal antibody drug Trastuzumab（INN； trade name Herceptin）is widely used for treating metastatic
breast cancer patients with overexpression of HER 2 on the tumor. In this study, a sensitive and specific high performance liquid
chromatography-tandem mass spectrometry（HPLC-MS/MS）method was developed and validated for the pharmacokinetic assay
system with an enzyme-linked immunosorbent assay（ELISA）system.
Blood sampling was repeated 3 times each for 15 min, 30 min, 1 hr, 2 hr, 4 hr, 8 hr, 1 day, 2 day, 4 day, 6 day, 9 day, 12 day,15 day and 30 day after dose. To precisely and quantitatively measure the absolute levels of Herceptin in serum, the proteolytic
peptides with Herceptin-specific amino acid sequences were synthesized with incorporated stable isotopes, as ideal internal
standards to mimic native peptides formed by proteolysis. The tryptic peptides of serum with standard peptides were subject toanalysis with linear trap quadrupole mass spectrometer equipped with a nano-electrospray ion source and quantified by selectedreaction monitoring（SRM）mode. The method was sensitive with the lowest limit of quantification（LLOQ）at 0.5ng/ml serum. The
dynamic range of the analysis was from 250 fmol/ul to 1 pmol/ul（r2=0.99）.

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 29: The Advances in Biological Mass Spectrometry in Drug Discovery andDevelopment: Current State of the Art and Challenges

PWe-150
13:30 － 14:40

Caco-2 PERMEABILITY STUDIES AND DETERMINATION BY LC/MSMS OF
THIOCTIC ACID

Murat Onul1, Seval Korkmaz2, Banu Erdem3

1
Ant Teknik, Istanbul, Turkey, 2Abdi Ibrahim Pharmaceuticals Inc., Istanbul, Turkey, 3Marmara University Faculty ofEngineering, Departmant of Bioengineering, Istanbul, Turkey

Keywords:
thioctic acid, lipoic acid, Caco- 2 cell line, permeability, LCMSMS
Novel aspects:
Usage of LC/MSMS techniques to achieve formulation studies of thioctic acid by improving its permeability results obtained from
Caco- 2 studies.
Abstract:
Lipoic acid (LA) also known as Thioctic acid and α-lipoic acid is an organosulfur compound derived from octanoic acid. LA
contains two vicinal sulfur atoms（at C 6 and C 8 ）attached by a disulfide bond and is thus considered to be oxidized（althougheither sulfur atom can exist in higher oxidation states）. The carbon atom at C 6 is chiral and the molecule exists as twoenantiomers R-（+）-lipoic acid（RLA）and S-（-）-lipoic acid（SLA）and as a racemic mixture R/S-lipoic acid（R/S-LA）. Only the R-（+）-enantiomer exists in nature and is an essential cofactor of four mitochondrial enzyme complexes. Endogenously synthesized RLA
is essential for life and aerobic metabolism. Both RLA and R/S-LA are available as over-the-counter nutritional supplements andhave been used nutritionally and clinically since the 1950s for various diseases and conditions. LA appears physically as a yellow
solid and structurally contains a terminal carboxylic acid and a terminal dithiolane ring.
The aim of current study was to show efficacy of Caco- 2 permeability studies for testing of permeability characteristic ofimmediate release solid oral dosage formulations of formulations of thioctic acid by comparing with permeability characteristic of
its API. Permeability Studies were performed in the Cell Culture and In Vitro Screening Laboratory of Abdi Ibrahim Pharmaceuticals
R&D Center.
Caco- 2 cells（ECACC）were seeded at a density of 8.000 cells/well on tissue culture-treated polycarbonate filters（area 0,143
cm 2 ）in Corning 96 well Transwell plates（Corning Costar）. The culture medium（EMEM）, pH 7.4 containing 10 % fetal bovine
serum and 1 % non-essential amino acids（Sigma）, added with penicillin- streptomycin solution（Sigma）was changed every day
and cell cultures were incubated at a temperature of 37ºC in an atmosphere of 95% relative humidity, 10% CO2. Filters were used
for transport studies 21 days after seeding.
Viability and integrity tests were performed by Lucifer yellow test and RTCA（Real Time Cell Analyzer）.
Samples were added into wells at 100 uM concentrations in HBSS（Hank’s Balance Salt Solution）containing HEPES buffer at Fassif
Mod condition. While incubating cells at 37 ºC, aliquots were taken from both chambers at the end of 30- 60- 90 and 120 minutes.
Samples diluated in double distillated water and concentrations of compound in the samples were measured by LC-MSMS
instrument（Shimadzu LCMS-8030）.
Caco- 2 permeability characteristic of thioctic acid and its solid oral dosage form were determined by LC/MSMS analysis
techniques.

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 29: The Advances in Biological Mass Spectrometry in Drug Discovery andDevelopment: Current State of the Art and Challenges

PWe-151
11:10 － 12:20

Chemical modificomics: Alternative analytical platform for efficientbiomarker discovery

Tomoyuki Oe

Tohoku University, Sendai, Japan

Keywords:
proteomics, chemical modifications, omics
Novel aspects:
Significance of searching the chemical modifications on a specific target protein has been demonstrated as a possible novel omics
strategy.
Abstract:
Recent strategies in biomarker discovery have relied on mass spectrometry-based global proteomics. However, biomarker
discovery from the entire proteome is analogous to finding a needle in a haystack. Accordingly, we have attempted to establish a
novel omics strategy to facilitate biomarker discovery. The strategy focuses on only an individual peptide or protein, that is closely
related to the targeted disease. Chemical modifications of the selected peptide or protein are exhaustively screened. We call thisapproach chemical modificomics. Proteins and peptides are exposed to higher chemical stress during certain physiological events,
i.e. increased oxidative stress in degenerative aging disease and higher glucose stress in diabetes mellitus. Therefore, the resulting
chemical modifications can provide meaningful information about biological events. In other words, the modifications serve as
dosimeters for stress exposure and as diagnostic and therapeutic markers. However, bioactive peptides or proteins associatedwith specific diseases have been quantified almost exclusively by immunoassay-based procedures, with no concern for possible
minute chemical modifications. Hence, critical information could be overlooked or considerable misunderstandings could occur, if
the unidentified modifications alter protein functions. Here, I introduce a novel strategy based on a combination of immunoaffinity
clean-up and mass spectrometric identification to screen chemical modifications of a specific target peptide or protein.
Angiotensins: Alternative approach for cardiovascular diseases
Angiotensin II（Ang II）, the major bioactive peptide in the renin-angiotensin system, is involved in the regulation of cardiovascularhomeostasis and the development of various cardiovascular diseases including hypertension, atherosclerosis, and heart failure.
Although the contribution of Ang II in each disease remains unclear, oxidative stress is considered a central mechanism, owing to
the involvement of reactive oxygen species in numerous signaling pathways of Ang II. However, there has been little attention
given to the potential oxidative modifications of Ang II. We found a novel chemical modification of Ang II generated underoxidative conditions. The structure was unique and similar to Ang A, which exhibits a strong vasoconstrictive effect and elevatedconcentrations in end-stage renal failure. The structural similarity warrants further investigation of its biological activity.
Keratins: Non-invasive assessment of skin aging
Keratins, the main constituents of human skin, have recently been recognized as major target proteins of chemical modifications,since skin is a main barrier of the body against environmental stresses such as UV and chemical agents. However, because of the
difficulties associated with keratins’ insolubility and handling, few studies have investigated chemical modification of keratins. We
have developed a robust method to screen keratin modifications as a combination of non-invasive sampling（tape stripping）,
simple clean-up, and tryptic digestion（filter-aided sample preparation）, and high-throughput MALDI-TOF/MS analysis. Using themethod, we have identified sites modified by oxidation with H2O2, and found that Met259, 262, 296, and 469 in K 1 were good candidate
skin aging markers.  
Serum albumin： The most promising global stress marker
Human serum albumin（HSA）, the most abundant blood plasma protein, has several important functions including maintaining
osmotic pressure and serving as a carrier for hydrophobic compounds. Since HSA is circulated through the entire body, thisprotein is likely subjected to many chemical stresses. We have optimized a robust method to obtain 100% sequence coverage,
thereby enabling exhaustive screening of all the chemical modifications of HSA. Through screening of drug-HSA and chemical-HSA
adducts, the strategy is expected to contribute to toxicity and risk assessments involving early-stage drug candidates andenvironmental chemicals, respectively.
References［ 1 ］Goto, Kojima, Shitamichi, Lee, Oe： Anal. Methods, accepted, DOI： C 2 AY05841C.
［ 2 ］Lee, Goto, Oe： Chem. Res. Toxicol., 21, 2237（2008）.
［ 3 ］Lee, Takahashi, Goto, Oe： Chem. Res. Toxicol., 23, 1771（2010）.
［ 4 ］Lee, Miyamoto, Goto, Oe： J. Proteomics, 75, 435（2011）.

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 29: The Advances in Biological Mass Spectrometry in Drug Discovery andDevelopment: Current State of the Art and Challenges

PWe-152
13:30 － 14:40

Metal Modified Nanoparticle as a Probe for Quantitative Analysis of
Clodronate Drug by MALDI-MS in Human Plasma

Wei Hsu1, Yi-Chi Ho1, Mei-Chun Tseng1, An-Kai Su1, Ping-Yu Lin1, Huan-Ting Wu2, Chun-Cheng Lin2,
Ming-Ren Fuh3, Yu-Ju Chen1

1
Institute of Chemistry, Academia Sinica, Taipei, Taiwan, 2Department of Chemistry, Tsing Hua University, Hsinchu,
Taiwan, 3Department of Chemistry, Soochow University, Taipei, Taiwan

Keywords:
clodronate, magnetic nanoparticle, quantification, drug development, sample preparation
Novel aspects:
The metal-chelated nanoparticle assay provided effective and rapid extraction and detection of clodronate from human plasma by
MALDI-TOF MS.
Abstract:
Clodronate is a bisphosphonates medicine, which is chosen to alleviate bone resorption and tumor bone diseases. In
pharmacokinetic studies, many researchers have been reported for metabolism of clodronate in human biofluid. According to thereports, the pharmacokinetic studies of the clodoenate metabolism pointed that the metabolic concentration was ranged from0.05 to 3 ug/mL in 48-hour duration after taking the medication. Therefore, it is important to develop an effective and rapid assay
to evaluate the pharmacokinetic studies of the clodronate in the bio-fluid. However, due to the matrix complexity in biologicalmatrix, these clinical specimens, such as blood serum, plasma, urine, saliva and etc, generally require a sequence of sample
treatment steps prior to analysis. In addition, the bisphosphonates is strong ionic compound leading to hydrophilic characteristicthat causes the difficulty of extracting it from bio-fluids.
Thus, several methods for extraction of clodronate have been reported. However, these conventional sample pretreatment
methods such as calcium precipitation and solid /liquid phase extraction usually required derivation method that is tedious andtime-consuming. With the charge affinity between the bisphophonate group and transition metal ion, we reported a magnetic
metal-chelating ligand by covalent conjugation of nitrilotriactic acid（NTA）on the magnetic nanoparticle（MNP）for simultaneousenrichment and detection of clodronate. The several different metalions were studied, and the Zn +2 -NTA showed the best
efficiency. Without the need of sample derivation and elution, the magnetic NTA nanoparticle demonstrated an efficient metalchelated probe compatible to MALDI MS analysis. Attributing to the high surface-to-volume ratio of Zn +2 -NTA@MNP and theadjusting PH value of plasma for modification the charge state of clodronate, the extraction of clodronate can be finished rapidly
about 10 min with an excellent efficiency. We also use seed-layer preparation method and spiked internal standard to reducesignal fluctuation in MALDI-TOF analysis for quantitation of clodornate in human plasma. Comparing with the drug metabolism
and pharmacokinetic（DMPK）study, the LOQ of nanoprobe-based affinity mass spectrometry（NBAMS）assay is 0.04 ug/mL which
is better than it of the traditional GC/MS analysis. Good linear range（R2=0.9992）was also obtained from 0.04~ 4 ug/mL in plasma.
In addition, the intra-day and the inter-day precisions showed coefficients of variance（CV）from a range of 4.1 to 9.5% and 3.5 to
10.5%, respectively. The accuracy of intra-day and inter-day were found to be from a range of 2.5% to 12.2% and 2.8% to 17.5%,
respectively. In conclusion, we demonstrated that a metal modified nanoparticle combined with MALDI-TOF MS is a simple, rapid,
reproducible and accurate platform for simultaneous enrichment, detection and quantification of small molecular drugs inpharmaceutical metabolism study.

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 29: The Advances in Biological Mass Spectrometry in Drug Discovery andDevelopment: Current State of the Art and Challenges

PWe-153
11:10 － 12:20

Caco-2 PERMEABILITY STUDIES AND DETERMINATION BY LC/MSMS OF
TRIMEBUTINE

Murat Yayla1, Seval Korkmaz2, Banu Erdem3, Murat ONUL4

1
Ant Teknik, Ankara, Turkey, 2Abdi Ibrahim Pharmaceutical Inc.,Turkey, 3Marmara University Faculty of Engineering,
Department of Bioengineering, Istanbul, Turkey, 4Ant Teknik, Istanbul, Turkey

Keywords:
trimebutine, Caco- 2 cell line, permeability, LCMSMS
Novel aspects:
Usage of LC/MSMS techniques to improve formulation studies of trimebutine by improving its permeability results obtained from
Caco- 2 studies.
Abstract:
Trimebutine is a drug with antimuscarinic and weak mu opioid agonist effects. The maleic acid salt of trimebutine is marketed
under the trademark of Debridat, Recutin, Polybutin, or Modulon for treatment of irritable bowel syndrome and othergastrointestinal disorders. The major product from drug metabolism of trimebutine in human beings is nor-trimebutine, whichcomes from removal of one of the methyl groups attached to nitrogen. Both Trimebutine and its metabolite are commerciallyavailable.
The aim of current study to identify Caco- 2 permeability characteristics of trimebutine immediate release solid oral dosage formand suspension form by comparing with permeability chaecteristics of its API to obtain better generic formulation.
Caco- 2 Permeability Studies were performed in the Cell Culture and In Vitro Screening Laboratory of Abdi Ibrahim
Pharmaceuticals R&D Center.
Caco- 2 cells（ECACC）were seeded at a density of 8.000 cells/well on tissue culture-treated polycarbonate filters（area 0,143
cm 2 ）in Corning 96 well Transwell plates（Corning Costar）. The culture medium（EMEM）, pH 7.4 containing 10 % fetal bovine
serum and 1 % non-essential amino acids（Sigma）, added with penicillin- streptomycin solution（Sigma）was changed every day
and cell cultures were incubated at a temperature of 37ºC in an atmosphere of 95% relative humidity, 10% CO2. Filters were used
for transport studies 21 days after seeding.
Viability and integrity tests were performed by Lucifer yellow test and RTCA（Real Time Cell Analyzer）.
Samples were added into wells at 100 uM concentrations in HBSS（Hank’s Balance Salt Solution）containing HEPES buffer at Fassif
Mod condition. While incubating cells at 37 ºC, aliquots were taken from both chambers at the end of 30- 60- 90 and 120 minutes.
Samples diluated in double distillated water and concentrations of compound in the samples were measured by LC-MSMS
instrument（Shimadzu LCMS-8030）.
Caco- 2 permeability characteristic of trimebutine and its oral dosage forms were determined by LC/MSMS analysis techniques.

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 29: The Advances in Biological Mass Spectrometry in Drug Discovery andDevelopment: Current State of the Art and Challenges

PWe-154
13:30 － 14:40

Caco-2 PERMEABILITY STUDIES AS A NEW ALTERNATIVE MODEL TO
BIOEQUIVALENCE AND BIOWAIVER TESTS

Seval Korkmaz

Abdi Ibrahim Pharmaceuticals, Istanbul, TURKEY

Keywords:
Caco- 2 cell line, permeability, bioequivalence, biowaiver
Novel aspects:
A novel developed in vitro Caco- 2 permeability studies will be presented as the alternative method to bioequvalence andbiowaiver studies..
Abstract:
Frequently, bioequivalence studies of novel developed immediate release oral generic drug formulations can be failed in spite of
their successful in vitro results. That dramatic results are related inconcideration of its permeability characteristics according to
BCS（Biopharmaceutical Classification System）. The scientific concept of the BCS was incorporated in the regulation of the USA（Guidance for Industry. Waiver of In Vivo Bioavailability and Bioequivalence Studies for Immediate-Release Solid Oral Dosage
Forms Based on a Biopharmaceutics Classification System in the Europena Union regulation（EMEA Committee for medical
products for human use CHMP BCS-based biowaiver）and also in the World Health Organization（WHO）. The BCS-based
biowaiver approach is meant to reduce in vivo bioequivalence studies, i.e., it may represent a surrogate for in vivo bioequivalence.
Caco- 2 permeability study is already been used to test permeability characteristics of API（Active Pharmaceutical Ingradient）andits solid oral dosage formulations. A new method was developed to show in vitro permeability characteristics of generic solid oral
drug formulations by comparing with original/ reference drug under the same condition（FaSSIF）with in vivo bioequivalence tests.
Because the results of current developed method were shown similarity with clinical bioequivalence test, it can be proposed as
an alternative method for biowaiver files and in vivo bioequivalnece tests（Its patent application has been done）. It has beenexerted on usage of Caco- 2 study results to determine of IVIVC（in vitro in vivo correlation）.

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 29: The Advances in Biological Mass Spectrometry in Drug Discovery andDevelopment: Current State of the Art and Challenges

PWe-156
13:30 － 14:40

Selectivity test: Potential Interfering substances evaluation during LDTDMS/MS quantification

Alex Birsan, Serge Auger, Annick Dion-Fortier, Pierre Picard
Phytronix Technologies, Quebec, Canada

Keywords:
Small Molecules, Quantitative Analysis, LDTD-MS/MS, Selectivity test
Novel aspects:
Evaluation of potentially matrix interferences substances on drug’s quantification using a none chromatographic approach： LDTDMS/MS.
Abstract:
Introduction
The Laser Diode Thermal Desorption is a rapid analysis approach in which samples are thermally desorbed by a laser diode.
Molecules are channeled, using a carrier gas, to a corona discharge region for ionization prior detection via a mass spectrometer.
Drugs of interest as well as other potential interfering substances are desorbed at the same time during analysis. To verify thepotentially interference effect, the selectivity test is perform using the following molecule（Caffeine, Ibuprofen, Nicotine, Warfarinand Acetaminophen）. They were spiked at the maximum expected concentration in QC sample of a drug of interest（Dextrorphan）. All QC are compared to the reference to evaluate the potential effect.
MethodThe following potentially interfering compounds were spiked, at 1 and 10 times the Cmax value reported in literature, in a plasma
QC sample containing Dextrorphan：Caffeine at 6 mcg/ml, Ibuprofen at 35 mcg/ml, Nicotine at 30 ng/ml, Warfarin at 340 ng/ml
and Acetaminophen at 150 mcg/ml. Those compounds were spiked individually in plasma QC. These potential drug interferences
are evaluated on active compound extracts using three different extraction techniques. Protein precipitation： 25 uL of plasma, 50
uL of IS in Acetonitrile and 25 uL of water saturated in NaCl. Liquid-Liquid extraction： 25 uL of plasma, 25 uL NaOH 0.1 N, 10 uL of IS
in ACN and 100 uL of EtAc. Solid phase extraction is performed on Oasis HLB（ 1 cc）cartridge.
Results
Linearity of Dextrorphan expressed by r2=0.9986 cover a range between 2.5 to 500 ng/ml for the protein precipitation approach. QC
samples spiked at 100 ng/ml are compared with potentially interfering substances QC in a protein precipitation extraction.
Variations are within 96.5 to 110.4 % range. Liquid-Liquid extraction linearity shows r 2 =0.9967 over the same dynamic range.
Interfering QC variations are within 94.6 to 108.6%. Solid phase extraction linearity shows r2=0.9976 over the same dynamic range.
Interfering QC variations are within 85.6 to 106.8 % . No analytical difference is observed between the low and high level ofinterfering substances. Qualification of the method in term of linearity, reproducibility and accuracy meet validation requirements.
All measurements of QC fall within the +/- 15% of the expected value which demonstrate the method selectivity on this potential
interfering substances test.

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 29: The Advances in Biological Mass Spectrometry in Drug Discovery andDevelopment: Current State of the Art and Challenges

PWe-157
11:10 － 12:20

Intact Monoclonal Antibody Characterization Using A Bench-Top Orbitrap
Mass Spectrometer

Zhiqi Hao1, Yi Zhang1, Dave Horn1, Shiaw-Lin Wu2, Patrick K Bennett1, Andreas F Huhmer1
1

Thermo Fisher Scientific, 2Barnett Institute, Northeastern University, Boston, MA

Keywords:
Orbitrap, Q Exactive, monoclonal antibody, top-down
Novel aspects:
Intact monoclonal antibody characterization using novel scan functions on a bench-top Orbitrap mass spectrometer.
Abstract:
Introduction
Monoclonal antibodies（mAbs）are increasingly developed and utilized for the detection and treatment of diseases including
cancer. Due to the heterogeneity of mAb products, thorough characterization is necessary for their reproducible as well as safe
production. Among the analytical tools used for the analysis of therapeutic mAb, mass spectrometry（MS）, especially highresolution and accurate mass MS, has become more and more important in providing accurate information on various protein
properties, such as intact molecular mass, glycosylation form distribution, amino acid sequence, post-translational modifications,minor impurities due to sample processing and high order structure, etc. In this study, an orbitrap based LC-MS workflow solution
was developed for robust, accurate and comprehensive mAb characterization at intact protein level.
Methods
Two different mAbs were analyzed. Intact or reduced mAb samples were desalted, and the light and heavy chains separatedusing a ProSwift reverse-phase monolithic column. Proteins eluted from the column were analyzed using a bench-top Q Exactive
quadrupole Orbitrap or a prototype second generation Exactive Orbitrap mass spectrometer. Top-down MSMS was performed
using high energy collision dissociation with a unique spectrum multiplexing feature（msx HCD）in the Q Exctive instrument. Full
MS spectra of intact or reduced mAb were analyzed using Protein Deconvolution 1.0 that utilizes the ReSpect algorithm formolecular mass determination. The masses were compared to the expected masses with the various combinations of commonly
found glycoforms. The top-down msx HCD spectra were analyzed using ProSight PC 2.0.
Results
The full MS spectra of the mAbs show a complete charge envelope distribution of the mAb, with each charge state revealing five
major glycosylation forms that are baseline separated. The measured ppm error on average molecular mass for data generated in
30 experiments using two different instruments is 6.9 ppm with a standard deviation of 6.4 ppm. Using the 140 K resolution setting,the isotopic peaks of the light chain was baseline resolved over the 1 min elution time, which resulted in monoisotopic molecularmass determination with an error of less than 5 ppm. For the top 5 glycoforms, the relative abundance reproducibility was withina few percent. These results present the mass accuracy and reproducibility for mAb intact mass measurement using the benchtop Orbitrap MS instruments in conjunction with the deconvolution software. To obtain amino acid sequence, top-down MS/MS
was applied to the reduced mAb samples using the msx HCD approach. In this data acquisition mode, fragment ions produced
from several individual HCD events, each on a precursor of a different charge state of the reduced mAb, were detected together
in the Orbitrap mass analyzer. Besides the improved throughput from spectrum multiplexing, the advanced signal processing
provides improved resolution and higher Orbitrap scan speeds, which is critical for on-line protein top-down sequencing. High
resolution, information rich spectra were generated on the one minute LC elution time for reduced mAb samples. For the lightchain, over 40% sequence coverage was achieved, including the N-terminal variable region, with a mass error of less than 5 ppm
for fragment ions. Results from this study indicate that both precise mass measurement and extensive, high confidence sequence
information can be obtained for intact mAb using this workflow solution that combines high resolution MS, fast chromatography,
high throughput msx HCD and accurate data analysis.

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 29: The Advances in Biological Mass Spectrometry in Drug Discovery andDevelopment: Current State of the Art and Challenges

PWe-158
13:30 － 14:40

Novel approaches in the imitation of oxidative drug metabolism byelectrochemistry-mass spectrometry

Hjalmar P Permentier1, Eslam Nouri-Nigjeh1,2, Andries P Bruins1, Rainer Bischoff1

1
University of Groningen, Groningen, The Netherlands, 2New York State Center of Excellence in Bioinformatics and Life
Sciences, Buffalo, USA

Keywords:
electrochemistry, drug metabolism, cytochrome P450, stable isotope labeling
Novel aspects:
Electrochemical synthesis of specific drug metabolites with（on-line）mass spectrometric detection and characterization allows
imitation and insight into Cytochrome P450 oxidation reactions
Abstract:
The combination of electrochemistry（EC）with mass spectrometry（MS）forms a powerful analytical tool which finds applicationsin the modification and characterization drug compounds as well as proteins.
Instrumental methods for the in vitro mimicry of oxidative drug metabolism by Cytochrome P450 enzymes in the body are of greatinterest for drug development and testing. EC-MS as an analytical technique has unique applications in the field of drug
metabolism and may help speed up drug development, first by allowing rapid assessment and prediction of potential metabolites
formed in the body, and second by providing a system for the synthesis of sufficient material for subsequent toxicity tests.
In our approach, electrochemical methods are employed to induce either direct oxidation or the formation of intermediate
reactive（oxygen）species. The model compound in this study is lidocaine, a local anesthetic, which is metabolized in the liver by
CYP450 isoenzymes to its N-oxide and to several hydroxylated and N-dealkylated forms. In an electrochemical cell, direct oxidation
at glassy carbon electrodes is very efficient and selective for the N-dealkylation reaction, but is not capable of the other importantoxidation reactions such as aromatic hydroxylation. Alternatively, reactive oxygen species can be electrochemically produced in acontrolled way by oxygen reduction or by the electrochemically induced Fenton reaction. Strict control of instrumental
parameters, such as potential, square-wave pulsing with variable cycle times, electrode material and solvent conditions, are used
to tune the type and distribution of oxidation products, and we have developed a set of conditions which will allow reliable andcontrolled oxidative modification of model drug compounds, based on the available functional groups and desired oxidation
products.
The oxidation products of drug compounds are often isobaric, due to introduction of an oxygen atom in different places in themolecule. The use of stable isotope-labeled reagents（ 18 O-labeled water, or hydrogen peroxide）and specific fragmentation
methods（thermal decomposition of N-oxides during APCI）enabled the correct assignment of isobaric oxidation products. In
addition, the stable isotope method allowed the identification of the source of the oxygen atom in each product（molecularoxygen, water or hydrogen peroxide）, which provides valuable insight in the electrochemical and chemical reaction mechanisms.
The mechanisms proposed for the EC-induced modifications are compared with the known CYP450 catalyzed reactions.

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 29: The Advances in Biological Mass Spectrometry in Drug Discovery andDevelopment: Current State of the Art and Challenges

PWe-159
11:10 － 12:20

Analysis of lipid composition in stimulated human cancer cells by LC-MS/
MS following treatment with protolichesterinic acid isolated from Cetraria
islandica

Finnur F Eiriksson1,2, Baldur B Sigurdsson2, Sesselja S Omarsdottir1, Helga M Ogmundsdottir1,
Margret Thorsteinsdottir1,2
1

University of Iceland, Reykjavik, Iceland, 2ArcticMass, Reykjavik, Iceland

Keywords:
LC-MS/MS, Eicosanoids, Lipoxygenase, Lichen, Chemometry.
Novel aspects:
Analysis of the effects of protolichesterinic acid isolated from Cetraria islandica by targeted lipidomics in stimulated human cancer
cells.
Abstract:
Cancer cells show differences from healthy normal cells in their metabolism, which contribute to their survival and growth. Lipids
have numerous functions in biological processes, structural as well as regulatory. Fatty acid synthase（FAS）is an essential enzyme
for cancer cells and a high level of FAS is a characteristic of many human malignancies. Hydroxyeicosatetraenoic acids（HETEs）
the products of lipoxygenase（LOX）pathways have been implicated in cancer development. Protolichesterinic acid（PA）, a
secondary metabolite of lichens, is a potent inhibitor of 5 - and 12-LOX and may also have FAS inhibitory activity. The aim of thiswork was to develop a liquid chromatography tandem mass spectrometry（LC-MS/MS）method for evaluation of lipid compositionin stimulated human cancer cell lines, before and after treatment with PA. PA was purified from a petroleum ether extract of
Cetraria islandica with preparative high performance liquid chromatography（HPLC）. A LC-MS/MS method was developed andoptimized for quantification of palmitic acid, HETEs and LTB4, utilizing a chemometric approach. Seven variables； type of column,
flow rate, initial amount of organic solvent, type of organic solvent, gradient slope, collision energy and capillary voltage were
optimized by D-optimal design and related to sensitivity, retention time and resolution utilizing PLS-regression. Different sample
preparation methods； solid phase extraction, protein precipitation and liquid-liquid extraction were tested and parametersoptimized. The amount of HETEs and the derivative, LTB4 in both cultured cancer cells and cultured medium were analyzed withthe optimized LC-MS/MS method after stimulation with calcium ionophore（A23187）and treatment with PA. Preparative HPLC was
successfully used for crude-purification to obtain one of the major secondary metabolites in the lichen Cetraria islandica（L.）Ach.,
PA and its tautomer lichesterinic acid. NMR and analytical HPLC were used to verify the purity and identification of PA. The
identification was confirmed and the purity was evaluated as 99.60%. Palmitic acid could not be successfully analyzed because ofcontamination problems and extraordinary precautions need be taken to achieve accurate analysis of palmitic acid and to avoid
fatty acid contamination. Identification and optimization of significant experimental factors influencing the response for the LC-MS/
MS method were done by design of experiments（DoE）. The sensitivity was significantly influenced by flow rate, collision energy,
gradient slope, organic phase and type of column. Interaction between gradient and organic phase was significant which indicatedthat these variables could not be independently controlled to obtain optimum conditions. A LC-MS/MS method was developed
and validated for quantification of the LOX pathway products； LTB4, 5 - and 12-HETE. An intraday validation assessment showedthat the quantitative determination was linear for LTB4, 5 - and 12-HETE in the range tested（0.120-30 ng/mL）, and accuracy andprecision met the acceptance criteria with a coefficient of variations lower than 15%. Degree of interference was assessed and theconclusion was that no significant interfering peaks were detected. Stability of the analytes was established for 12 hours andstorage at 4 °C was found to be appropriate. Quantification of LTB4 and HETEs in the pancreatic cancer cell line Capan-2, before
stimulation with A23187 showed a weak signal（approximately 10-300 pg）for the analytes, suggesting the presence of theseanalytes in the cell samples. To confirm the presence of these lipids an optimization of the extraction method for lipids from thecells is needed and is in progress. This will include development of methods, ranging from the initial step of the cell membranerupture to the final sample preparation method prior to LC-MS/MS quantification of lipids in cultured tumor cells. The tumor cells
lines have been stimulated with A23187 and the amount of LTB4, 5 - and 12-HETE will be analyzed with the optimized LC-MS/MS.

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 29: The Advances in Biological Mass Spectrometry in Drug Discovery andDevelopment: Current State of the Art and Challenges

PWe-160
13:30 － 14:40

UHPLC-MS-MS with Precursor Ion Scanning, Neutral Loss Scanning andPolarity Switching for the Detection of Glutathione Conjugates of Reactive
Metabolites

Richard B van Breemen, Ke Huang

University of Illinois College of Pharmacy, Chicago, IL USA

Keywords:
drug metabolism, UHPLC-MS-MS, glutathione, fast triple quadrupole, stable isotope labeling
Novel aspects:
Unique combination of polarity switching, UHPLC, isotope labeling, precursor ion scanning, and constant neutral loss scanningwith an ultrafast triple quadrupole for the screening of complex mixtures for glutathione conjugates.
Abstract:
Drugs, compounds undergoing drug development, and natural products contained in botanical dietary supplements are
sometimes metabolized to electrophilic metabolites. These reactive metabolites can produce cytoxic or even genotoxic effects
such as oxidative stress, covalent modification of proteins and DNA. In vivo, reactive metabolites can be deactivated throughreaction with the endogenous nucleophile gluathione, and glutathione conjugates can be detected and characterized using
positive ion tandem mass spectrometry or negative ion tandem mass spectrometry. Drawing on this information, our laboratory（van Breemen et al., Comb Chem High Throughput Screen, 1999）reported a positive ion electrospray mass spectrometricscreening method for the rapid detection of reactive metabolites formed during in vitro incubations of drugs with human drug
metabolizing enzymes, enzymatic cofactors, and glutathione. Subsequently, Dieckhaus et al.（Chem Res Toxicol, 2005）reported theuse of negative ion electrospray tandem mass spectrometry for the detection of glutathione conjugates that did not form
abundant positive ions, and Yan and Caldwell（Anal Chem 2004）reported the use of dual intrinsic labeling of glutathione toeliminate false positive results during tandem mass spectrometric screening of complex matrixes for glutathione conjugates. Usinga new generation fast triple quadrupole（Shimadzu LCMS-8040）, we have succeeded in combining our original positive ion
screening approach with the negative ion method and the stable isotope labeling method into a fast and efficient UHPLC-MS-MS
assay. During a single UHPLC separation, positive ion electrospray constant neutral loss scanning for the elimination of acharacteristic gamma glutamyl group from an unlabeled region of glutathione is measured alternately with negative ion
electrospray precursor scanning for the characteristic glutathione anions of m/z 272 and its isotope labeled form of m/z 275. This
new assay detected more glutathione conjugates than was possible using either the positive ion method or the negative ion
method alone while eliminating false positive results. Implementation of UHPLC facilitated faster separation and detection of
glutathione conjugates than previous methods. The use of an ultrafast scanning triple quadrupole capable of rapid polarity
switching was essential not only because of the multiple types of MS/MS scans but also because of the UHPLC time scale which
was 10-fold faster than previous HPLC studies. This new glutathione conjugate detection method was validated using drugs suchas acetominophen that are known to form electrophilic quinone imine metabolites. Then, the method was used to screen popularbotanical dietary supplements that are used by menopausal women as alternatives to hormone replacement therapy. For
example, following incubation of a botanical extract prepared from Glycyrrhiza glabra（licorice）with human liver microsomes,
NADPH and glutathione, the natural product glabridin was identified as a constituent of licorice that can be metabolized toreactive intermediates that form glutathione conjugates. This new UHPLC-MS-MS approach to screen complex mixtures of drug
metabolites and botanical extracts for the potential to form electrophilic metabolites may be used to complement safety studies
of dietary supplements and drugs under development.

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 30: Data Processing and Informatics for SIMS

PWe-161
11:10 － 12:20

Comparison of titanate nanosheets by G-SIMS and g-ogram

Ichiro Mihara1, Satoka Aoyagi2, Keizo Nakagawa3
1

Kuraray CO., LTD, Tokyo, Japan, 2Shimane University, Shimane, Japan, 3The University of Tokushima, Tokushima, Japan

Keywords:
G-SIMS, g-ogram, TOF-SIMS, Titanate nanosheet
Novel aspects:
Fragment ions from organic-inorganic complex samples were characterized by G-SIMS and g-ogram to evaluate their chemical
structures in nanolayers.
Abstract:
Nanostructure evaluation is important for developing highly controlled nanomaterials. Layerd titanate nanosheets having a lamellar
mesostructure were analyzed using time-of-flight secondary ion mass spectroscopy（ToF-SIMS）in order to evaluate detailed
chemical structures. Although ToF-SIMS is a useful method for evaluating complex organic and inorganic materials, it is often
difficult to interpret ToF-SIMS spectra of complex samples due to intricate fragment ions mainly from organic materials. Recently
Gentle-SIMS（G-SIMS）and g-ogram have been developed in order to interpret complicated secondary ions in static-SIMS spectra.
Secondary ions are classified based on ratio of static-SIMS spectra obtained with two primary ion conditions causing different
degrees of fragmentation. The g-ogram is a derivative technique from G-SIMS and provides useful information on origins ofsecondary ions.
In this study, G-SIMS and g-ogram were applied to organic-inorganic complex to characterize secondary ions related to organic
materials and to distinct molecular ions and more intact fragment ions from fragment ions of larger molecules. Several layered
titanate nanosheets samples produced were prepared using different surfactants, dodecanediamine（DDA）, laurylamine（LA）and
hexylamine（HA）, as templates to produce lamellar structures. TIPT + DDA + TEOA, TIPT + LA and TIPT + HA samples wereprepared using tetraisopropyl orthotitanate（TIPT）, triethanolamine（TEOA）and DDA, and were prepared using TIPT andsurfactants, laurylamine（LA）and hexylamine（HA）, respectively. As a result, secondary ions related to titanate compounds weredetected in the titanate nanosheet samples having lamellar mesostructures though they were not detected in TIPT samples
without surfactants. Especially in TIPT + DDA + TEOA sample the surfactant molecular ion was also observed, and therefore it isindicated that robust lamellar mesostructure of titanate compounds and the surfactant is produced by reactions related to TIPT,
DDA and TEOA. It is also suggested that organic-inorganic materials which have large hydrocarbon groups can be analyzed using
G-SIMS.

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 30: Data Processing and Informatics for SIMS

PWe-162
13:30 － 14:40

Adaptive Thresholding for Three-Dimensional Wavelet Denoising of TOFSIMS Images : Toward Digital Staining of Pathological Specimens

Koichi Tanji, Manabu Komatsu, Hiroyuki Hashimoto
Canon Inc. Tokyo, Japan

Keywords:
three-dimensional wavelet analysis； adaptive thresholding
Novel aspects:
We propose an adaptive three-dimensional wavelet algorithm for rapidly denoising data, and it determines the wavelet thresholdbased on an optimum thresholding by gradually changing a temporarily set threshold.
Abstract:
This report presents a novel noise reduction algorithm for spatially distributed mass spectrometric data. In recent years, mass
spectrometry imaging has received considerable attention, owing to its capability to monitor many molecules at the same time.
Mass spectral peaks corresponding to particular molecules can be used as biomarkers in disease diagnosis. We have recently
focused on time-of-flight secondary ion mass spectrometry（TOF-SIMS）, which enables two-dimensional mass spectrometry
imaging of components with sub-micrometer resolution. In mass spectrometry, noise reduction is crucial for identifying weak
peaks. Up to now, various methods for denoising mass spectra have been proposed. Because of the high spatial resolution of
TOF-SIMS, the two-dimensional distribution of components, which is reconstructed by using specific m/z peaks, can be treated ascontinuous. Therefore, TOF-SIMS data have, in essence, three-dimensions. A difficulty in TOF-SIMS, however, is that extremely
large amounts of data are generated, especially in measurements on pathological tissue samples. Therefore, a fast algorithm isnecessary for the practical application of noise reduction to TOF-SIMS data.
We have recently reported an adaptive three-dimensional wavelet algorithm for rapidly denoising data acquired by TOF-SIMS as
follows. In the computation, the TOF-SIMS data are stored in three-dimensional space, where the xy-plane corresponds to themeasured area and the z-axis corresponds to the m/z mass spectrum. Reconstructed images for specific m/z peaks along thez-axis indicate the spatial distributions of components such as protein and lipid. In this algorithm, two different basis functions areapplied to the xy-plane and the z-axis, and three-dimensional wavelet shrinkage and reconstruction is performed. One basis
function is suitable for continuous data such as the distribution of components, and the other is suitable for discrete data such asa mass spectra. This method has resulted in marked noise reduction of TOF-SIMS data, while it still had arbitrariness indetermining the threshold of wavelet analysis.
In this study, we propose an adaptive thresholding algorithm for wavelet denoising. In addition to the different basis functions, we
determine the wavelet threshold based on an optimum thresholding by gradually changing a temporarily set threshold to evaluate
the effect of the threshold on the noise reduction. To evaluate the effect on the noise reduction, the amount of change in signal
before and after the noise reduction is estimated from the amount of change in the standard deviation of the signal. The results
show that the noise is reduced without loss of the original signals and that the signal-to-noise ratio is substantially improved by
the noise reduction algorithm.

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 43: Novel Proteomics Methodologies

PWe-163
11:10 － 12:20

Elucidating the aberrant PK Profiles of Monoclonal Antibody byProteomics Approaches

Wai Siang Law1, A P Warren1, P Lloyd1, J Sims1, J Balthasar2, Carsten Krantz1

1
Novartis Pharma AG, Basel, Switzerland, 2Department of Pharmaceutical Sciences School of Pharmacy andPharmaceutical Sciences University at Buffalo, The State University of New York, Buffalo, New York 14260

Keywords:
Monoclonal antibody, pharmacokinetic, tissue analysis,
Novel aspects:
Immunocapture technique based on anti-human antibody to pull down both the drug and target to perform a multiplxed PK/PD
analysis.
Abstract:
Monoclonal antibodies（mAbs）have emerged as one of the most important drug classes in the biopharmaceutical industry. An
understanding of the factors which can affect the pharmacokinetic（PK）behavior, particularly the elimination mechanisms ispivotal during their pre-clinical and clinical evaluation. Drug ABC exhibits a rapid clearance in Cynomolgus monkey. Deposition ofthis mAb in lymph node and spleen is observed by recent in-house immunohistochemistry study. However, there is little
information available on the extent of ABC accumulation in tissue, the stoichiometry of target and drug ABC deposition and theimpact of such accumulation on its PK profile. The purpose of this project is to establish liquid chromatography coupled withmass spectrometry（LC-MS）and the-state-of-art proteomics approaches to quantify drug ABC in serum and tissue samples. In
order to increase further the selectivity and sensitivity of LC-MS/MS, an immunocapture technique based on anti-human antibody
that allows efficient extraction of human mAb from Cynomolgus monkey serum and tissue is developed. Combined with LC-MS/
MS, a multiplexed PK/PD assay within a single analysis is generated

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 43: Novel Proteomics Methodologies

PWe-165
11:10 － 12:20

Mass spectrometry-based non-invasive proteomic analysis of human skinkeratins for discovery of biomarkers of oxidative skin damage

Seon Hwa Lee, Kohei Miyamoto, Takaaki Goto, Tomoyuki Oe
Tohoku University, Sendai, Japan

Keywords:
keratins, oxidative skin damage
Novel aspects:
We have developed the first MS-based approach to detecting oxidative modifications in keratins obtained directly from humanskin and found the potential biomarkers of oxidative skin damage.
Abstract:
Human skin is one of the major barriers of our body against environmental stimuli such as UV and industrial chemicals. Therefore,
as the main constituent of the skin, keratins have recently been recognized as the major oxidative target proteins. However,
because of the difficulties associated with their insolubility and handling, there have been lack of studies to identify the sites ofoxidation in keratins. Here, we introduce a combination of non-invasive sampling, simple clean-up, efficient digestion method, andmass spectrometric（MS）analysis to screen keratin modifications. Human skin proteins were obtained from the upper arm withadhesive tapes. After the tape stripping, one-fourth of the tape was placed into a tube containing sodium dodecyl sulfate（SDS）
buffer and butylated hydroxytoluene（BHT）. Proteins on the gluey surface were then solubilized by scraping using a grinding
plastic pestle. After the centrifugation, the supernatant was used for the following proteomic analysis. SDS, DTT and IAA were
removed at every step by centrifugation with a filter unit（>30 K）. The resulted protein mixture was digested by trypsin on thefilter unit and the tryptic peptides as filtrate were then analyzed by MALDI-TOF/MS, LC-ESI/MS, and MS/MS. From the human skinproteins obtained by tape stripping, keratines have been identified as major proteins（82 out of 101 peptide peaks）. Especially,
K 1 and K10 have been found as the predominant keratins with sequence coverages of approximately 45%. Most of the keratin
methionine sites detected in our experiment were found to be partially oxidized in intact human skin samples. Among them,specific sites, such as Met259, Met262, Met296, and Met469 located in the α-helical rod domain of K1, were shown to be the mostsusceptible to oxidation with hydrogen peroxide. Thus, their relative oxidation levels were increased in a dose- or time-dependantmanner upon treatment with hydrogen peroxide in vitro or in vivo, respectively. Therefore, we believe that this approach would
provide significant information of the oxidative modifications on keratins that could be useful as biomarkers of the skin damage.
Supported by KOSÉ Cosmetology Grant from The Cosmetology Research Foundation, Japan（to T.O.）.
Reference： Lee SH, Miyamoto K, Goto T, Oe T： J. Proteomics, 75, 435-49（2011）.

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 43: Novel Proteomics Methodologies

PWe-166
13:30 － 14:40

RELATIVE QUANTITATION OF TMT-LABELED PROTEOMES FOCUS ON
QUANTITATIVE PRECISION AND ACCURACY

Rosa I Viner1, Michaela Scigelova2, Martin Zeller2, Madalina Oppermann2, Thomas Moehring2,
Vlad Zabrouskov1
1

Thermo Fisher Scientific, San Jose, USA, 2Thermo Fisher Scientific, Bremen, Germany

Keywords:
MS3, TMT Quantification, Orbitrap Elite
Novel aspects:
Improvements in TMT based quantification using MS 3 HCD and Orbitrap Elite
Abstract:
Introduction: Isobaric tagging methods involving ’differential’ isotope labeling using chemical tags are universally applicableapproaches measuring relative amounts of proteins across two or more different samples. The main focus of the presented work
was on assessing the quantitative precision and accuracy for a TMT sixplex-labeled complex proteome sample using latest ion
trap-Orbitrap hybrid instrumentation.
Methods: Six aliquots of a digest of 9 proteins labeled each with one of the TMT sixplex reagents and mixed to obtain the final
ratio 10 ： 1 ： 10 ： 2 ： 10 ： 1.5. Such sample was added to the background of a TMT sixplex-labeled E. coli lysate digest. Peptides
were analyzed using nano-LC coupled to the Orbitrap Elite. A data dependent method（Top10 method）, as well as an MS 3 based method where MS 3 multipole collision cell fragmentation was triggered on a strong fragment ion from a particular m/z
range of an ion trap fragmentation spectrum（MS 3 method）were evaluated. Quantitative precision was expressed as protein
ratio variability. Quantitative accuracy was presented as a deviation of a measured
ratio value from the expected value.
Results: The percentage of peptides whose fragmentation spectra contained all six reporter ions（quantifiable peptides）exceeded
90% for any sample load tested. The variability was lower than 10% for approximately 90% of the quantified proteins at a 500 ng
sample load, and for about 70 % at a 20 ng sample load. The Orbitrap detection at resolution settings 15,000 corresponds toeffective resolution >27,000 for m/z 126-130.Consequently, a very tight mass tolerance（+/- 10 ppm）could be used effectively
filtering most reporter ion interferences. For higher ratios（theoretical ratio 10： 1 ）the median peptide ratio increased from 5.64
（Top10 method）to 7.97（MS 3 method）.
Conclusion: An improved mass spectrometer design resulted in a high percentage of quantifiable peptides. The quantitative
precision is signal intensity-dependent. Very high resolution effectively eliminated isobaric interferences in the reporter ion region.
The impact of peptide precursor co-isolation issue on quantitative accuracy was significantly reduced with MS 3 method.

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 43: Novel Proteomics Methodologies

PWe-167
11:10 － 12:20

Mobility Assisted Data Independent Label-Free LC-MS Analysis of
Citrobacter rodentium Infected Mouse Colon

Maki Terasaki1, Chris Hughes2, Johannes P Vissers2, James Collins3, Gad Frankel3
1

Nihon Waters K. K., Tokyo, Japan, 2Waters, Manchester , UK, 3Imperial College, London, UK

Keywords:
Label-Free, Quantitation, Ion-Mobility, Data-Independent Analysis
Novel aspects:
Comparative analysis and classification of Citrobacter rodentium infected mice by Mobility Assisted Data Independent Label-Free
LC-MS
Abstract:
Introduction
Citrobacter rodentium is a murine specific pathogen used to study human infection with enteropathogenic andenterohaemhorragic E. coli. C. rodentium colonises the gut via formation of attaching and effacing lesions, causing transmissiblecolonic hyperplasia and a self-limiting disease in C57Bl/ 6 mice. In this presentation, we describe the use of quantitative
proteomics experiments to determine changes to the murine colonic proteome at two key time points of infection, day 8（bacterial colonisation peak）and day 14（colonic hyperplasia peak）. We used liquid chromatography coupled with ion mobility
and mass spectrometry to determine relative abundance changes to the colonic proteome. Subsequent experiments focused on
the effects of bacterial mutants in key virulence determinants on protein expression in the mouse colon.
Methods
Six week old mice were infected with C. rodentium or administered PBS as a control. At day 8, or day 14 post infection, mice（ 4infected, 4 control）were euthanized, 6 cm of colon removed and pooled（ 2 colons per sample）for protein extraction andtrypsin digestion. Peptides were reversed phase separated, using a nanoscale LC system, configured with a trap column and a 75
µm analytical column. A 90 min, 300 nL/min gradient was performed with each sample analysed in triplicate. Data were acquired in
HDMSE mode where raising the collision energy following ion mobility separation produced fragments that exhibit the same drift
time as their precursor. Precursors and fragments were correlated and searched using post processing software.
Abstract
Initial experiments were conducted to determine the optimum extraction procedure of proteins from mouse colon. Proteins wereextracted either using 5 % Rapigest, Molloy-like lysis buffer or Molloy-like lysis buffer in the absence of CHAPS. Samples weredigested with trypsin and it was determined, from the number of proteins identified, approximately 850 from 1 μg of starting
material, that the extraction method based on Molloy-like lysis buffer was the most optimal. For the comparative study, eightmouse colon tryptic digest samples（four infected, four controls）were each analysed in triplicate and random order, with theamount of material loaded for these experiments reduced to around 100 ng. The resulting data were searched with the UniProt
mouse database using a dedicated data independent search algorithm and a total of 2852 proteins comprising at least 1
proteotypic peptide were identified from the entire sample set, with approximately 1000 proteins from 10,000 non-redundant
peptides being identified in each injection and over 1250 proteins in two out of the three injections for each sample. Injections of
50 ng E. Coli tryptic digest were made at various points to ensure that performance of the LCMS system was maintained
throughout the entire experiment. Reproducibility was demonstrated with over 800 proteins consistently identified in each of the
QC injections. Further interrogation of the dataset allowed for the calculation of the relative in-sample abundance for eachidentified protein. Thereafter, unsupervised hierarchical clustering of the relative abundance calculated values demonstrated aclear classification of C. rodentium infected and uninfected mice and afforded identification of both host and pathogenic infection
related proteins.

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 43: Novel Proteomics Methodologies

PWe-168
13:30 － 14:40

A method for isolation of endogenous disulfide-containing peptides froma biological sample.

Caroline D Pereira1, Naoto Minamino2, Toshifumi Takao1
1Institute for Protein Research, Osaka University, Osaka, Japan, 2National Cardiovascular Center, Osaka, Japan

Keywords:
Disulfide bond, Pituitary, 18O-labeling
Novel aspects:
A method for isolation of endogenous disulfide-containing peptides
Abstract:
A disulfide bond is formed between two cysteine residues and deeply involved in protein folding and expression of biologicalactivity. In human and E. coli sequence databases, at least 80% of proteins and peptides contain multiple cysteine residues. The
fact that many of bio-active peptides have disulfide bonds prompted us to develop a method for selectively isolating these
peptides from a complex mixture, which may lead to discovery of a novel bio-active peptide in a biological sample.
Pituitary produces many biologically active compounds such as various peptide hormones, which are produced by several
enzymatic cleavages from large precursor proteins, despite that the modes of processing have not been fully understood.
In order to isolate only disulfide-containing peptides, a method using a hetero-bifunctional cross-linker（SPDP： N-succinimidyl 3 （ 2 -pyridyldithio）propionate）linked to aminopropyl resin has been developed. The conditions and efficiency of the isolation
were examined using synthetic peptides and a tryptic digest of bovine serum albumin, a 69-kDa protein containing 17 disulfide
bonds. The protein was reduced under denaturating conditions, cleaved with trypsin, and directly applied to the above resin. The
eluate from the resin with a reducing agent was found to predominantly contain peptides with Cys residue（s）, while peptides
without Cys were observed in the flow-through.
Peptides in a biological sample are always susceptible to degradation during isolation, which mainly owes to the action of various
enzymes present in the sample. We here successfully applied the 18 O-labeling method which allows for discrimination ofendogenous peptides from the degradation products that are produced during isolation and purification, i.e. the decomposed
peptides are distinctively labeled with 18O as the result of hydrolysis in 18O water. Pituitary tissue from Rattus norvegicus was put
into 18O water and boiled for 10 min to deactivate enzymes present in the sample. Peptides were extracted with 0.25% aqueous
acetic acid. The preliminary analysis of a crude extract with MALDI-MS/MS gave non-labeled and 18 O-labeled peptide signals,
which could be differentiated by the shift in mass of 2 or 4 Da.
A combination of the above two methods should be useful for discovering endogenous disulfide-containing peptides in pituitary
tissue, which might be candidates of bioactive peptides with unique structures formed by disulfide bond（s）.

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 43: Novel Proteomics Methodologies

PWe-169
11:10 － 12:20

A Targeted Quantitative Phosphoproteomic Approach using iTRAQLabelled Synthetic Peptides as Internal Standards

Andre C Mueller, Leonhard Heinz, Kumaran Kandasamy, Manuela Bruckner, Christoph L Baumann,
Giulio Superti-Furga, Keiryn L Bennett
CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences/Vienna/Austria

Keywords:
Quantitative phosphoproteomics, iTRAQ, targeted analysis, synthetic phosphopeptide library
Novel aspects:
The use of iTRAQ-labelled synthetic phosphopeptides in a pseudo-selected reaction monitoring ’targeted approach’ as a robust
validation method to study signalling networks.
Abstract:
Mass spectrometry（MS）is widely-used to study posttranslational modifications of proteins. Phosphorylation of serine, threonine
and tyrosine residues are amongst the most-studied and best-understood of all protein modifications. Continuous improvements
in phosphopeptide enrichment strategies and advances in sensitivity, accuracy and speed of MS instrumentation allow theidentification of thousands of phosphorylation sites in a high-throughput manner. Phosphoproteomic approaches are used to
study phosphorylation events within cellular signalling pathways in the context of innate or adaptive immunity, cancerous cells or
tissues. To infer a biological significance for a given scenario, comparative quantitative proteomic approaches are necessary toenable the detection of regulated sites and determine the fold-change in phosphorylation. The use of the iTRAQ 4 -plex
technology allows the multiplexing of 4 samples in a single mass spectrometric analysis. Thus, the comparison of the relative
abundance of a peptide/protein or modified peptide/protein across states is facilitated.
A global phosphoproteomic analysis was conducted to determine the regulatory effects of a newly-identified negative regulator（referred to as ’LiMIT’）of pro-inflammatory toll-like receptor（TLR）signalling in murine macrophages. Stable RAW264.7 cell lines
with lentivirally-transduced shRNAs either targeting ’LiMIT’ expression or non-targeting negative control RAW264.7 cell lines
expressing endogenous levels of ’LiMIT’ were used for two independent experiments. Lipopolysaccharide（LPS）activated the TLR/
MAPK pathway, and a relative comparison of the unstimulated cells with 10 and 20 minute post LPS-stimulation was achieved with
the iTRAQ 4 -plex labelling strategy. The fourth iTRAQ channel was used for a reciprocal cross comparison of non-stimulated LiMIT
knockdown versus LiMIT wild-type and vice versa. One hundred micrograms of total protein per iTRAQ channel was prepared,
and the tryptic peptides labelled with the 4 -plex reagent. A modified IMAC strategy based on Ficarro et al.1 was utilised to enrich
iTRAQ-labelled phosphopeptides. The resultant samples were fractionated with reversed-phase chromatography at pH 10. Ten
and fifty fractions were collected for the phosphoproteome and the phospho-depleted proteome, respectively. Samples wereanalysed in duplicate on an LTQ Orbitrap Velos mass spectrometer using a hybrid multi-stage activation-collision-induced
dissociation/higher-energy collision-induced dissociation（MSA-CID/HCD）method for phosphopeptide analysis. The phosphodepleted proteome was analysed by HCD alone.
The data was searched against the murine SwissProt database with MASCOT and evaluated with Proteome Discoverer. Over 3,400
annotated phosphopeptides were grouped into more than 1,900 protein groups. Manual validation is still ongoing； however,preliminary results have identified at least 91 proteins showing the effect of LiMIT knockdown on the regulation ofphosphorylation； with 26 proteins belonging to the MAPK/TLR network, e.g., ERK or Raf1.
The reproducibility of MS identification and iTRAQ quantitation can vary strongly in data-dependent acquisition mode andtherefore a pseudo-selected reaction monitoring（SRM）’targeted approach’ has the potential to provide improved peptide
identification and strengthen statistics. From a selected list of differentially-regulated tryptic phosphopeptides, synthetic versions
will be produced. These phosphopeptides will be used as universal internal standards for inter-experimental comparison andconcurrent peptide validation. The envisaged workflow will consist of：（i）purification of crude synthetic phosphopeptides；（ii）
validation of the peptides by MS； and（iii）refinement of the MS method for optimisation of peptide fragment ion sequences andconcomitant iTRAQ quantitation（dynamic range, ion statistics）. Following proof-of-concept, the ’phosphoproteomic analysis will
be repeated with an extended library of iTRAQ-labelled, standard peptides multiplexed with the three cellular states（LPS
stimulation at t = 0, 10 and 20 minutes）for the LiMIT knockdown and the control experiment. The ultimate objective of thetargeted approach is to implement a generic, technical platform that enables a fast, robust and normalised quantitative proteomic
analysis across different experiments.
1

Ficarro, S. B., et al.（2009）Anal. Chem., 81, 4566-4575

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 43: Novel Proteomics Methodologies

PWe-170
13:30 － 14:40

The study of aging processes using in vitro glycation experiments applied
to low density lipoproteins using MALDI Mass Spectrometry

Omar Belgacem1, Matthew Openshaw1, Grazyna Sobald2, Katharina Pock3, Gerald Stuebiger4

Shimadzu, Manchester, UK, 2Dept. of Nuclear Medicine, Medical University Vienna, Austria, 3Octapharma
Pharmazeutika Produktionsges.m.b.H, Vienna, Austria, 4Center of Biomolecular Medicine and Pharmacology, Medical
University Vienna, Austria
1

Keywords:
MALDI, Glycation, Aging, Lipoproteins, Bioinformatic
Novel aspects:
The detection of glycation sites for Low Density Liporproteins that are located near binding receptors. The detection of unusual
amino acid modifications that could be an indication of aging.
Abstract:
Introduction
The aging and oxidative processes are widely known to induce all sorts of health problems that are now causing a huge stress on
the healthcare system of any modern country. The glycation process, also known as Maillard adductions, is involved in not only
human disease and pathology but also in food processing and pharmaceutical formulations. We have developed over the years a
methodology to specifically detect glycation sites on various standard proteins in order to be able to look at real samples such as
human serum albumin and Low density lipoproteins（LDL）in vivo. We have also tried to link our findings with LDL lipids profiles.
Methods
Standard proteins and peptides（BSA, Substance P）were used for the method development and the generation of MSMS
signatures at the modified peptide level. Glycated（g）, glycoxidated（gox）lipoproteins samples were generated in vitro using avery soft glycation process mimicking physiological conditions. The lipoproteins were obtained by ultracentrifugation of normal
human plasma. Proteins were then digested with trypsin. Peptides were separated by HPLC and sequence mapping was
performed by MALDI-MS/MS. Mass spectra were recorded using MALDI-QIT-TOF-MS/MS platforms in order to generate the MSMS
signatures of the modified peptides. MALDI-TOF-RTOF was used for the LC-MALDI mapping. Data were processed using asoftware equipped with a database of user-defined post-translational modifications（PTM-Finder TM ）. The Phospholipids（PLs）
were isolated by directly extracting the lipids from the LDL particles using a solvent extraction method. PLs profiles were thendirectly analyzed by MALDI-MS.
Results
We have designed a method allowing us to specifically detect glycated peptides out of a very complex mixture. This was done by
first generating MSMS signature specific to peptides that are glycated with glucose, lactose and maltose. These signatures, which
consisted in characteristic neutral losses（-162, -120 and-36 Da）, were independent of the peptide chain but directly linked to thesugar moiety attached to the amino acid backbone. We incorporated these losses into a bioinformatic platform in order tospecifically detect glycated peptides. This was done by using Mascot searches in combination with an MSMS screening module（PTM Finder software）. The g- and gox-LDL samples were digested with trypsin and separated through a micro-column in order
to avoid any ion suppression effect. Following digestion and LC- separation, it was interesting to note that out of 357 lysine
residues； only two of them were glycated. We were able to cover 33 % of the whole amino acid sequence which account for
approximately 120 potential lysine glycation sites. The 2 detected glycations sites are located near binding receptors which may
explain some biological dysfunction in diabetic patients. Another interesting aspect is the large number of peptides（MSMS
spectra）that did not match any known amino acid sequence of the Apo-B-100. We suspect that these peptides were modified but
not glycated and therefore could not be detected using standard bioinformatics approach（Mascot, PTM search etc）. These
peptides are now in the heart of a new investigation where the aim is to detect peptides that reacted with highly reactive lipids
species within the LDL particles during the oxidation/glycation process. Ultimately the quantifications of such peptides could be astrong indicators of the general health of the patient.

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 43: Novel Proteomics Methodologies

PWe-171
11:10 － 12:20

Phosphoprotein analysis using a combination of an online monolithic
trypsin-immobilized enzyme reactor and TiO2 enrichment column withtandem mass spectrometry

Kun Cho1, Hyo Jin Hwang2, Jisun Yoo1, Jin Young Kim1, Young Hwan Kim1, Han Bin Oh2
1

Korea Basic Science Institute, Ochang, South Korea, 2Department of Chemistry, Sogang University, Seoul, South Korea

Keywords:
IMER, TiO2, Phosphoprotein, Tandem mass spectrometry
Novel aspects:
We demonstrated the online based combined applications for analysis of phosphoproteins.
Abstract:
Post-translational modifications（PTMs）of proteins entail a change of proteins in chemical compositions by proteolytic cleavage
or by addition of functional groups at certain amino acid residues. Many researchers have identified the involvement of thephosphorylation PTMs in many biological pathways or processes. However, the analysis of phosphorylated PTMs is still aformidable task in the biological and technical aspects due to several reasons such as structural homogeneities and its low
relative abundances. To overcome the drawbacks, we demonstrated the online based combined applications for analysis ofphosphoproteins. The system is composed of three of main part, such as online phosphoprotein digestion using trypsin
immobilized enzyme reactor（IMER）, online phosphoprotein enrichment using TiO2 column, and dual tandem mass spectrometry
with collisionally activated dissociation（CAD）and electron transfer dissociation（ETD）. Phosphoproteins were generated fromprotein standard mix including a-casein, b-casein, ovalbumin, BSA, and Ribonuclease B. Our online based system demonstrated
that is the powerful tool for analysis of phosphoproteins, which are easy to use and higher recovery of phosphoprotein.

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 43: Novel Proteomics Methodologies

PWe-172
13:30 － 14:40

Label-Free Phosphopeptide Quantitation and Occupancy Determinationfrom a Single Protein Using LC-MALDI

Oliver Drews, Anja Resemann, Waltraud Evers, Eckhard Belau, Asperger Arndt, Detlev Suckau
Bruker Daltonik GmbH, Bremen, Germany

Keywords:
Occupancy of phosphorylation site, label free quantitation
Novel aspects:
A new approach to quantitatively assign phosphorylation sites was developed combining a dephosphorylation strategy with labelfree LC-MS.
Abstract:
Introduction:
Posttranslational modifications in general and protein phosphorylation in particular have gained much attention in proteomics inrecent years. Most work focuses on establishing catalogues of phosphopeptides in a proteome of interest and determining
whether a certain peptide is phosphorylated or not, but this research stops short of checking for the occupancy of individual
residues while verifying multiple phosphorylation sites. It is understood , the degree of phosphorylation at various sites in a single
protein can strongly effect its function.
Here, we describe a new approach for the quantitative assessment of phosphorylation occupancy at various previously known
target sites within a single isolated protein resembling the situation after immunoprecipitation of a protein in question.
Methods:
The protein KAPCA_HUMAN, containing 10 known phosphorylation sites, was used to quantitatively determine the occupancy ofall sites. After EDTA-removal, the sample was trypsinized and split into two aliquots. One aliquot was enzymatically dephosphorylated in contrast to the other aliquot, so that a native（N）and a dephosphorylated（D）peptide aliquot were obtained.
Aliquots were analyzed in replicates using nanoflow LC-MALDI-TOF/TOF for peptide identification and the label-free quantificationof the relative abundance of the free, unphosphorylated peptides. The intensity of the D-peptides was assumed to be 100 % free
peptide. By subtracting the lower intensity of the N- peptides（ 1 -x）, the occupancy x［%］could be determined.
Results:
KAPCA_HUMAN（cAMP-dependent protein kinase catalytic subunit alpha）contains 10 phosphorylation sites of which 8 arepartially and 2 are stoichiometrically phosphorylated. The protein was obtained in highly purified form from FW Herberg, Kassel.
For the determination of the occupancy, 3 replicate LC runs of 1 pmol of each the native（N）and the dephosphorylated（D）
tryptic digest were performed. MALDI-MS/MS and Mascot searching provided peptide sequence verification. The native sample
yielded pairs of phosphorylated and non-phosphorylated peptides with the same sequence. Unique to this method, only the nonphosphorylated peptides were used for the occupancy determination and the use of LC equipment was not required.
The occupancy of the phosphorylated residues was calculated by（ 1 - N/（N+D））*100［ %］, where N and D represent theabundance of the native and the dephosphorylated peptide, respectively. As expected, the peptides containing the
stoichiometrically phosphorylated residues were detected only as phosphopeptides in the N aliquot confirming 100%
phosphorylation. Three other sites were non-stoichiometrically phosphorylated at Ser10（93 %）, Ser139（37 %）, and Ser259（23 %）.
For 5 residues, phosphorylation was not detectable, implicating that the degree of phosphorylation at these 5 sites was lessthan 2% . The abundance of these non-phosphorylated peptides in the D and N aliquots were equally abundant within thequantification error tolerance. The coefficients of variation（CVs）of the label-free dephosphorylation-LC-MALDI method are withina range of 10 to 30 % in agreement with established and cost intensive isotope labeled reference methods（WD Lehmann）.
An interesting side aspect of this work was finding that trypsin cleavage was impaired near phosphorylation sites, changing thedigestion characteristics depending on the modification state. This can effect any peptide based quantitative proteomics strategy
and thus discourages quantitative high-throughput shotgun phosphoproteomics work.

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 43: Novel Proteomics Methodologies

PWe-173
11:10 － 12:20

Ion-Trap Quantitative Proteomics of Glycoproteome from Pancreatic
Cancer Patients based on the Label-Free and iTRAQ Approaches

Anna Drabik1, Piotr Suder1, Marek Sierzega2, Radoslaw Pach2, Jan Kulig2, Anna Bodzon-Kulakowska1,
Jerzy Silberring1,3
1
3

AGH University of Science and Technology, Krakow, Poland, 2Jagiellonian University Medical College, Krakow, Poland,
Polish Academy of Sciences, Gliwice, Poland

Keywords:
ion trap, glycoproteome, label-free, iTRAQ, pancreatic cancer
Novel aspects:
The obtained results bring insight into quantitative proteomics using ion-trap MS. Described data show differences in theglycoproteomes between pancreatic cancer patients after gemcitabine treatment, indicating responders vs. non-responders.
Abstract:
In order to manage the requirements of current biological and medical studies, designed to understand molecular and cellularmechanisms, the“omics“ techniques have evolved into sophisticated and advanced tools. Whereas most of the initial proteomic
studies were focused on proteins identification, modern mass spectrometry（MS）technology provides useful platforms toinvestigate quantitative changes in protein components. Quantitative proteomics can be successfully used in characterizing
alterations in protein abundances as a consequence of pathophysiology or progressing treatment of a disease. This is based on
an assumption that such differences represent differential protein expression originated from particular conditions of the biologicalsystem.
Proteins gained from different samples should be digested by proteolytic enzyme, and separately labeled at their amino groups
with one of the Isobaric Tags for Relative and Absolute Quantification（iTRAQ）. The resulting labeled peptides are then combined
and fractionated by multidimensional chromatography, and quantitatively analyzed by MS. The intensities of these fragments areused for quantification of individual peptides representing various proteins. Originally, iTRAQ was devoted for measurements by
the MALDI TOF/TOF method, followed by other linear quadrupole-based analyzers. Our studies show how to overcome themajor obstacle of this method, and enable the use of an ion-trap instrument to minimize the cut-off at the low m/z range.
Another presented approach is based on the label-free technology, which allows for quantitation of peptides using spectral
characteristics, such as peak area or frequency of peptide fragment peak during nanoLC-MS/MS analysis, named spectra counting.
Peptide peak intensity or spectral counting is measured for individual nanoLC-MS/MS runs, and changes in protein abundances
are calculated via direct comparison between subsequent analyses. Spectral counting facilitates faster scan rates, higher intensity,and rapid MS to MS/MS switches, while peak integration benefits from constant retention time gained from the nanoLC systemand high accuracy mass analyzers. The data processing pipelines typically include data normalization, retention time integration,
peak detection, identification, matching, quantification, and statistical analysis. The main obstacle of this approach is the necessity
to gain sufficient reproducibility of the retention times for corresponding peaks.
Presented studies based on quantitative analysis of peptides obtained from standard protein tryptic digest were analyzed with the
Ion-Trap Amazon ETD（Bruker Daltonics, Germany）mass spectrometer combined with Proxeon nanoLC system. Nano-LC
chromatography used for the experiments consisted of two orthogonal separation techniques, Hydrophilic Interaction Liquid
Chromatography（HILIC）and Reversed Phase（RP）chromatography. Such an approach provided an evidence, that an ion-trap
instrument is able to perform both iTRAQ and Label-Free based quantitative analysis. Based on this knowledge, we analyzed sera
obtained from pancreatic cancer patients after gemcitabine treatment. Quantitative analysis of glycoproteins fraction after Lectin
Affinity Chromatography（LAC）was carried out following HILIC and RP nanoLC separations. The obtained peptides were analyzed
using MS/MS.
The results suggest, that individual peptides in complex biological systems might be quantified subsequently to sufficient sample
separation. However, special concern should be devoted to the selection of the appropriate quantitation approach according to
the needs of sample quality and experimental design.
This work was supported by the grant from The Polish Ministry of Science and Higher Education N N403 086039.

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 43: Novel Proteomics Methodologies

PWe-174
13:30 － 14:40

Screening of Oxidative Stress induced LDL-modifications using (LC-)
MALDI-MS(/MS) based Lipoproteomics

Gerald Stübiger1, Ulrike Resch1, Michael Wuczkowski2, Helmut Sinzinger1, Omar Belgacem3
1

Medical University Vienna, Vienna, Austria, 2Technoclone GmbH, Vienna, Austria, 3Shimadzu, Manchester, UK

Keywords:
Atherosclerosis, Oxidative stress, Lipoprotein modifications, Clinical diagnostics, MALDI-QIT-TOF-MSMS
Novel aspects:
A novel software assisted LC-MALDI-MS/MS based platform with potential for clinical monitoring of lipoprotein modifications is
introdced.
Abstract:
Introduction
Oxidative modification of low-density lipoprotein（LDL）induced by reactive oxygen species and/or elevated glucose levels is
considered a major risk factor of cardiovascular disease（CVD）and atherosclerosis. Our aim is to identify the modifications ofoxidized LDL（OxLDL）directly at the constituent phospholipid（PL）and amino acid（AA）sequence levels（e.g. of the ApoB-100
protein）. For this purpose we introduce a lipoproteomics approach using（LC-）MALDI-MS（/MS）combined with an intelligent
software based screening tool.
Methods
Lipoproteins were isolated from normal human plasma by preparative ultracentrifugation and modified by diverse reactive oxygen
species and lipid peroxidation products（e.g. MDA, HNE）in the presence or absence of sugars（e.g. glucose）. PLs were extracted
and directly analyzed by MALDI-MS. The apolipoproteins were digested with endoproteinases（e.g. trypsin）, peptides wereseparated by HPLC and sequence mapping was performed based on MALDI-MS/MS. Mass spectra were recorded using MALDITOF-RTOF- and MALDI-QIT-TOF-MS（/MS）platforms, respectively. Data were processed and searched for specific MS/MS
signatures based on a parameterizable database and software module（PTM-FinderTM, Shimadzu）.
Preliminary Data
LDL oxidation increased MDA levels（> 30 nmol/mg protein）and signal intensity of LPCs（e.g. LPC16： 0 ）but decreased signal
intensity of sn- 2 PUFA containing PCs（e.g. PAPC, PLPC）indicating for lipid peroxidation and increased activity of lipolytic enzymes（e.g. Lp-PLA 2 ）. Moreover, large MS/MS dataset（more than 400 MS/MS spectra per LDL type）were monitored to identify user
defined modifications at the AA level. Based on this approach in glycated LDL（gLDL）two distinct glycation sites located within
the LDL-R binding domain of ApoB-100 were found. Furthermore, additional sets of MS/MS spectra from other OxLDL samples（e.g.
Cu-LDL, HOCl-LDL）were screened for characteristic protein carbonylation products（e.g. Michael adducts）.

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 43: Novel Proteomics Methodologies

PWe-176
13:30 － 14:40

In-depth human proteome analysis by one-shot LC-MS/MS with meterscale monolithic silica capillary columns

Mio Iwasaki1, Masaki Wakabayashi1, Naoyuki Sugiyama2, Nobuo Tanaka3, Yasushi Ishihama1
1

Kyoto University, 2Keio University, Yamagata, Japan, 3GL Sciences, Tokyo, Japan

Keywords:
Human proteome / LC-MS/MS / HeLa cell / monolithic silica column
Novel aspects:
This ’one-shot’ proteomics using one-dimensional separation by 600 cm monolithic silica column showed a possibility to unveil
entire human proteome with small sample amount.
Abstract:<Introduction>
Shotgun proteomics based on capillary liquid chromatography coupled with tandem mass spectrometry（LC-MS/MS）has beenplaying important roles for analyzing protein samples. A rapid progress has been made in mass spectrometric technologies suchas high sensitivity and high scan speed, resulting in improvement of the identification efficiency of proteome. However, it is still
difficult to analyze the entire proteome of mammalian cells.
In shotgun proteomics, protein digests from cells have a variety of peptides with extremely high complexity and the wide
concentration range. That is why various kinds of multidimensional separations have been employed for reducing sample
complexities and improving identification efficiency of proteome. However, this approach has demerits of increased sample
amount and long analysis time.
An alternative approach to improve the identification efficiency in shotgun proteome analysis is to increase the separation
efficiency in one-dimensional reversed phase liquid chromatography. Monolithic silica columns offer high-efficiency separation withlong column formats because of their high permeability［ref. 1 ］. We have recently been developing one-dimensional LC-MS/MS
systems with meter-scale monolithic silica columns for E. coli and human proteome analysis.
With triplicate LC-MS/MS analyses（41 hours gradient）using 350 cm long monolithic silica columns, we identified 2,602 proteins for
the E. coli cells, in which the equivalent number of genes was detected by DNA microarray analysis［ref. 2 ］. For humanproteome analysis, 5,970 proteins were identified for the HeLa cells by quadruplicate LC-MS/MS analyses（ 8 hours gradient）using
400 cm long monolithic silica column, which is the best result yet reported without the use of exhaustive pre-fractionation［ref. 3 ］.
Compared to a conventional particle-packed column system, 5 -fold improvement in MS responses was observed by the reduced
co-elution of peptides using improved separation efficiency of monolithic silica column system for the sample of both species.
However, we did not observe dynamic range extension for the identified human peptides as our E. coli proteome analyses,
suggesting that current analytical technologies are still not adequate for highly complex human peptide mixtures. Our objective isto improve one-dimensional separation efficiency to achieve the identification of entire human proteome with small sample
amount. In this study, we used HeLa protein digest and examined one-dimensional LC separation with a 600 cm long monolithic
silica column.
<Results and discussion>
Using the 600 cm long monolithic silica column, we evaluated the effect of the gradient time for the analysis of HeLa cell lysates.
As expected, the number of identified proteins was remarkably increased as the gradient time became longer. Consequently,
36,701 non-redundant peptides from 7,112 proteins were identified by the single LC-MS/MS analysis of 15 μg sample with 60 hour
gradient time. In total, 59,182 non-redundant peptides from 9,500 proteins were identified by the combined LC-MS/MS analyses
using 60 μg injected sample and the total analysis time of less than 7 days. Because this system does not need any additional
experimental fractionations prior to LC-MS/MS, this ’one-shot’ proteomics approach allows simplifying the workflow of shotgun
proteomics and minimizing the sample amount as well as the total analysis time even when the shallow gradient was applied.
References［ 1 ］Miyamoto, K. et al., Anal Chem, 80（22）, 2008.
［ 2 ］Iwasaki, M. et al., Anal Chem, 82（ 7 ）, 2010.
［ 3 ］Iwasaki, M. et al., J Chromatogr A, 1228, 2012.

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 43: Novel Proteomics Methodologies

PWe-177
11:10 － 12:20

Automated Quality Control of Biopharmaceutical N-/C-Terminal
Sequences by Routine Top-Down Mass Spectrometry

Rainer Paape2, Anja Resemann2, Lars Vorwerg2, Jens Hoehndorf2, Detlev Suckau2, Hank Wang1
1

Bruker Daltonics Inc., Beijing, China, 2Bruker Daltonik GmbH, Bremen, Germany

Keywords:
Automated sequence validation in recombinant protein QC, top-down analysis, in-source-decay using MALDI-TOF/TOF, N- andC-terminal sequencing, TDS-MALDI
Novel aspects:
Automated sequence validation of MALDI-TDSdata for routinely assigning the status of N- and C-termini of biopharmaceuticalproteins.
Abstract:
Introduction:
During production of recombinant proteins, e.g., for pharmaceutical application, fast and reliable QC methods are needed. Mass
spectrometric analysis of tryptic protein digests , also known as“bottom-up“ analysis, is typically used for that purpose providing
for high sequence coverage of the target protein. However, terminal sequences are not safely covered by this approach. Topdown sequencing（TDS）analysis such as In-Source-Decay（ISD）using MALDI-TOF/TOF instrumentation produces N- andC-terminal fragments covering up to 80 amino acids from either end. Sample preparation, spectrum acquisition and interpretationof the TDS-MALDI data only take minutes to assign N- and C-termini on a routine base.
Methods:
The proteins bovine RNAse B, chicken lysozyme, bovine carbonic anhydrase, BSA and the 116 kDa -galactosidase were analysed
by MALDI-TDS following standard procedures. A software method was developed for fast and simple validation of the terminal
sequences of isolated proteins with MALDI-Top-Down-Sequencing （MALDI-TDS） using ISD. ISD spectra were matched
automatically with the theoretical TDS-spectra calculated from suggested sequence candidates. The quality of the match wasscored. The acquired ISD spectrum, the best matching protein sequence, and the detailed N- and C-terminal sequence assignment
were assembled as a PDF report for further manual analysis or documentation.
Results:
Protein ISD-spectra were automatically matched against a set of expected protein sequences that also included possible terminalor side chain modifications. The software calculated a, c, y and z+ 2 ions for each protein sequence and matched the theoreticalfragments against the spectrum. A scoring algorithm identified the best match. The results were assembled in an in-depth PDF
document that displayed the annotated ISD spectrum together with the protein sequence for the best matching protein in asequence coverage map. For all other protein sequence candidates the information about a confirmed N- or C-terminus is listed
as well. The reports are collected in a common report repository and include assignment of modifications in the range covered by
the termini analysis（e. g. residues 1 -80 from both sides）.
Using a batch process it was possible to match a series of spectra against a series of protein sequences.
The automatic interpretation of TDS spectra was achieved. Terminal truncations, modifications such as acetylation or
pyroglutamylation were detected in various investigated proteins as well as terminal sequence extensions. MALDI-TDS permitted
assigning the status of protein termini even in a fully automated fashion.

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 43: Novel Proteomics Methodologies

PWe-178
13:30 － 14:40

Evaluation of targeted proteomic approaches on a Q-TOF system

Carsten Funke, Stepahnie Kasper, Wolfgang Jabs, Carsten Baessmann
Bruker Daltonik GmbH

Keywords:
Targeted proteomics, high mass accuracy, QTOF, quantitiation
Novel aspects:
The targeted quantification efficiency of a Q-TOF system is determined using proteomic standards covering a broad concentration
range.
Abstract:
Mass spectrometry-based quantitative proteomics is a popular tool for biomarker discovery. Advances in the speed of separationof proteomics samples and the quality of mass spectrometric detection also enable the subsequent validation of discovered
protein biomarkers in complex biological matrices using targeted approaches. These biomarker validation approaches heavily rely
on methods exactly targeting and measuring the quantity of specific peptides and proteins in complex proteomic samples.
Typically, SRM and MRM（single- and multi-reaction monitoring）approaches are applied for validation tasks. Nevertheless, thosemethods require detailed target knowledge and do not allow re-examining of acquired data.
We describe here targeted proteomics approaches using the high resolution and accuracy achievable with an Ultrahigh Resolution（UHR）Q-TOF system. Different data acquisition methods are used for targeted quantitative proteomics, namely MS-based
acquisition, broad band collision induced dissociation（CID）, and middle band CID.
Target protein quantification is done in a complex E.coli background（500ng）spiked with different amounts of a proteomic
standard, which spans a concentration range of 6 decades in total（UPS-2, Sigma）. Protein identification information, including
m/z values and retention times, used as targets for quantification is obtained out of the equimolar proteomic standard（UPS-1,
Sigma）containing the same set of proteins as the dynamic range mixture. Tryptic peptides are separated on a nanoUHPLC system（U3000 RSLCnano, Dionex）and data acquisition is done using an Ultrahigh Resolution（UHR）-TOF（maXis impact, Bruker
Daltonics）equipped with a Captive Spray ionization MS source.
Peptide targets for quantitative proteomics are obtained out of an equimolar proteomic standard by a data-dependent MS/MS
approach. Results of different identification runs are combined and reveal successful identification of all 48 proteins. Creation ofhigh resolution extracted ion chromatograms used for quantification is done based on the information of identified peptides withregard to m/z values and retention. Quantification of the target peptides spiked into E.coli background based on pure MS dataacquisition reveals reliable results of targeted proteomics. In total 28 proteins were significantly regulated（p<0.05）demonstratinga high quantification efficiency from 500 fmol down to the amolar range. Results of targeted proteomics approaches using broadband and middle band CID will not only provide quantitative but also qualitative data in single runs. Quantification efficiency withregard to the coverage of a high dynamic range of these methods will be compared to the pure MS-based acquisition.

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 43: Novel Proteomics Methodologies

PWe-179
11:10 － 12:20

Electron Transfer Dissociation differentiates the isobaric deamidation
products Aspartate and IsoAspartate.

Andrea Kiehne1, Ralf Hartmer1, Laxmi Adhikary2, Anand Khedkar2, Harish Iyer2, Raju Mukherjee2
1

Bruker Daltonik GmbH, 2Biocon

Keywords:
ETD, ion trap, isoaspartate
Novel aspects:
ESI-Trap ETD fragmentation provides a simple method of distinguishing asparagine deamidation and aspartate isomerization inbiopharmaceutical antibodies
Abstract:
Biopharmaceuticals, in particular monoclonal antibodies, are set to revolutionize healthcare of the future. It is expected that by2014 five of the top ten best selling pharmaceuticals will be derived from proteins or peptides. Degradation of these protein drugs
results in reduced potency and increased immunogenicity. Deamidation of asparagine is a major cause of protein degradation andto protein folding disorders such as Alzheimer’s disease. Aspartic acid formed by nonenzymatic deamidation of asparagines often
isomerizes to isoaspartic acid. Accumulation of isoaspartic acid initiates protein aggregation and protein degradation. With CID
fragmentation it is impossible to discriminate aspartate from isoaspartate residues. However, ETD fragmentation produces asignature backbone cleavage which can be used to differentiate Asp and IsoAsp containing peptides.
Monoclonal antibodies were incubated in 4 M guanidium chloride, with 50 mM Tris-HCl and 0.5M EDTA, ph 8, and reduced with 10
mM dithothreitol for 15 min, followed by carbamidomethylation with 20 mM iodoacetamide for 30 min. The resultant heavy andlight chains were separated and purified via size exclusion chromatography（TSK-GEL G400SW column, Tosoh Bioscience）inpreparation for protease digestion. The tryptic peptides were separated using a C18（4.6 mm_250 mm, Grace-Vydac）reversephased column and injected online to an ESI ion trap mass spectrometer（HCTultra ETD, Bruker Daltonics）. For collision-induced
dissociation（CID）the trap was operated in autoMS/MS（UltraScan）mode. For ETD low-energy electrons were generated byelectron impact of methane and transferred to fluoranthene to generate negatively charged fluoranthene ions.
Tryptic peptides were separated on a reversed-phase C18 column and detected online through ESI-MS/MS. The tryptic peptides
were identified from the MS profile. The peptides containing asparagine were specifically investigated for a change in mass andretention time. An increase in mass of 1 Da was detected in two of the asparagine-containing peptides from the heavy chain
constant region. The reversed-phase HPLC method was able to separate three related species from both Asn containing peptides,
these were the original Asn containing peptides and the suspected Asp and IsoAsp containing peptides. Examination of the b andy type ions from the CID MSMS spectra of the suspected Asp and Iso Asp peptides conclusively confirmed the inclusion of anincreased mass of 1 Da which enabled identification of the site of deamidation. However, due to the isobaric nature of thepeptides, CID fragmentation products from the Asp and IsoAsp containing peptides were found to be identical and therefore CID
was unable to differentiate between the aspartate isomers.
Formation of isoaspartate results in an extra methylene unit in the peptide backbone. Under ETD fragmentation, identical c and z
type ions were found in the spectra of Asp and IsoAsp peptides. However, the isoaspartyl peptides also displayed a uniquefragmentation signature（Cn + 58 Da and zl-1 - 57 Da）which was not observed in the Asp peptides. These results demonstrate that
ETD fragmentation is a convenient method for monitoring and differentiating site-specific asparagine deamidation and aspartate
isomerization in a single MSMS experiment performed during routine peptide mapping of biopharmaceutical antibodies.

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 43: Novel Proteomics Methodologies

PWe-180
13:30 － 14:40

Identification and quantification of concentration-dependent biomarkers
in MCF-7/BOS cells exposed to 17beta-estradiol by 2-D DIGE and labelfree proteomics

Mike Collodoro, Pascale Lemaire, Gauthier Eppe, Virginie Bertrand, Rowan Dobson,
Gabriel Mazzucchelli, Joelle Widart, Edwin De Pauw, Marie-Claire A Gillet
Liege University, Liege, Belgium

Keywords:
Biomarkers, estrogens, food-chain security, proteomics, MCF- 7 /BOS
Novel aspects:
2 D-DIGE and label free proteomics coupled with PCA analysis allowed the discovery of estrogen activity biomarkers that can beused for rapid screening of estrogenic activity of xenobiotics.
Abstract:
The rapid screening of xenoestrogens prior to heavy analytical techniques is an important step in environmental pollution
monitoring and food chain safety.
We report the identification of biomarkers resulting from the exposure of MCF- 7 /BOS cells to 17beta-estradiol（E 2 ）. The
biomarkers were identified from the cytosolic fractions of cells treated for 24 h with mitogenic concentrations of 1, 30 and 500 pMof 17beta-estradiol.The biomarkers were identified using 2 independent and complementary techniques, 2 -D DIGE / MALDI-TOF
peptide mass fingerprint, and 2 -D UPLC-ESI MS/MS. To enable the further addition of new chemicals in the study, the 2 D-DIGE
experiment were performed using, instead of the classical internal standard, a reference gel. A robust PCA analysis allowed tofilter the biological variability and recover a dose dependence variation. Five biomarkers were up-regulated proteins, HSP 74, EF2,
FKBP4, EF 1 and GDIB and one was a down-regulated protein, K 2 C8. Three of these proteins, EF2, FKBP 4 and K 2 C 8 are
implicated in a network centred on the estrogen receptors ESR 1 and ESR 2 as well as on AKT1. After the discovery phase, threebiomarkers were selected as a signature for the response to the presence of estrogens. They were monitored using SRM after
incubation of MCF- 7 /BOS in the presence of E 2 for confirmation or selected xenoestrogens. Daidzein, coumestrol andenterolactone induced an up-regulation of EF 2 and FKPB 4 proteins, while tamoxifen and resveratrol induced a down-regulation.
The exposure of all phytoestrogens induced the down-regulation of K 2 C8. These markers form a preliminary molecular signaturethat can be used when testing the estrogenic activity of new chemicals, xenobiotics, either pure or in mixtures extracted fromfood chain or environmental samples.

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 43: Novel Proteomics Methodologies

PWe-181
11:10 － 12:20

Quantitative phosphoproteome analysis of apoptotic human cell culturesusing SILAC combined with complementary ESI-MS techniques

Yumiko Matsuyama1, Benjamin Mueller2, Wolfgang Jabs3, Carsten Baesmann3, Stephanie Kaspar3,
Karsten Niehaus4, Thomas Noll2, Raimund Hoffrogge2

1
Bruker Daltonics K.K., Yokohama, Japan, 2Institute of Cell Culture Technology, Faculty of Technology, Bielefeld
University, Germany, 3Bruker Daltonik GmbH, Bremen, Germany, 4Proteome and Metabolome Research, Faculty ofBiology, Bielefeld University, Germany

Keywords:
Quantification of phosphoproteome, apoptosis, SILAC, combined approach using of ETC on ion trap and ESI-UHR-TOF
Novel aspects:
First quantitative MS-analysis of apoptosis-related phosphoproteome and first application of complementary ESI-MS techniques forthe quantitative analysis of phosphopeptides.
Abstract:
Introduction
Apoptosis, the main form of programmed cell death, plays an important role in multicellular organisms as it is involved in modelingof tissue structures, elimination of mutated and damaged cells and homeostasis. Hence, a dysfunction can lead to severe diseases,
such as cancer, autoimmune diseases or neurodegenerative disorders. Furthermore, in bioreactor cultivations of animal cells
apoptosis negatively affects the production of biopharmaceuticals.
As protein phosphorylations are of great importance for apoptosis regulation and induction, the elucidation of changes in thephosphoproteome would lead to a better understanding of apoptosis-related diseases and new solutions for handling animal cells
in bioreactor cultivations. Here, we present a comprehensive approach combining SILAC and complementary ESI-MS techniques
for identification and quantification of apoptosis-involved protein phosphorylations.
Methods
Cultivation of Jurkat ACC 282 cells was carried out in shaking flasks in chemically-defined, serum-free medium. Three of six
replicates were grown in heavy medium, containing stable isotope labeled lysine and arginine, and induced for apoptosis with 100
uM etoposide while the other three replicates grown in light medium were left untreated. 4 h after apoptosis induction cells fromtreated and untreated cultures were harvested and mixed followed by protein extraction. 3 mg of total protein extract wereseparated by SDS-PAGE. The gel was split into 10 horizontal strips. Each strip was cut out, in-gel digested with trypsin andenriched for phosphopeptides via IMAC. The samples were analyzed by nanoLC coupled to an ESI（CID/ETD）-ion trap and an
ESI-UHR-TOF, respectively.
Results
Previous experiments of apoptosis induction in Jurkat cells showed that 100 uM etoposide led to activation of the apoptotic
execution phase after 5 hours. In order to see changes of the phosphoproteome during the induction phase of apoptosis asample point of 4 hours after etoposide treatment was chosen. At this time point, comparing treated and untreated cells no
difference in viability and viable cell density was observed. XX h after induction clear signs of apoptosis were observed. While theuntreated cells continued to growth to a viable cell density of XXXXX the treated cultures dropped to a VCD of XXX and a viabilityof XXX（ 9 X % in untreated cells）.
Our method gives access to more than 2000 quantifiable phosphopeptides, including numerous apoptosis-related proteins. The
use of complementary MS-techniques combines the advantage of ETD and CID fragmentation supplied by the utilized ESI-ion trapwith the strength in quantification offered by an ESI-ultra high resolution-TOF. In test runs analyzing complex samples we could
show an increase of about 50% in phosphopeptide identification by the additional use of ETD-fragmentation.
The elucidation of changes in the phosphoproteome of apoptotic human cell cultures will lead to new strategies for the
suppression of apoptosis in production processes of biopharmaceuticals. Since phosphorylations are involved in early events ofapoptosis induction, manipulations focusing on apoptosis-related phosphorylations would lead to a more effective inhibition of
apoptosis.

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 43: Novel Proteomics Methodologies

PWe-182
13:30 － 14:40

Validating a novel Parkinson’s Disease SRM-based assay using automatedsample preparation

Christine A Jelinek1, Liana Rosenthal1, Yan Jia1, Kevin W Meyer2, Ted Dawson1, Robert J Cotter1

1
Johns Hopkins School of Medicine, Baltimore, United States of America, 2Perfinity Biosciences, West Lafayette, UnitedStates of America

Keywords:
post-translational modifications, parkinson’s disease, selected reaction monitoring, mass spectrometric assay, biomarker
Novel aspects:
automating front-end protoemic sample preparation for an SRM-based novel Parkinson’s Disease diagnostic assay increases
throughput but decreases assay analysis time and experimental variability
Abstract:
Parkinson’s Disease is the second most common neurodegerative disorder world-wide. Given that early intervention is associatedwith improved quality of life and lifespan for afflicted patients, it is especially unfornate that currently there is no biomarker ordiagnostic test available for Parkinson’s Disease（PD）. To address the need for a PD diagnostic, we have developed a selectedreaction monitoring（SRM）assay to monitor alpha-synuclein post-trannslational modificaitons associated with PD pathophysiology.
Using Selected Reaction Monitoring-mass spectrometry, six phosphorylated, two nitrated alpha-synuclein peptides, and theircorresponding tryptic peptides will be measured in cerebrospinal fluid of PD patients with and without cognitive impairment andhealthy controls. Our screen will help determine if modified alpha-synuclein contributes to the pathophysiology of PD and/or if theselected alpha-synuclein peptides could serve as clinical diagnostics. Unlike assays that require multiple manual sample
preparation steps, the sample preparation for our developed assay occurs with the Perfinity Workstation. Replacing manual
preparative steps with column-mediated digestion, desalting, and Reverse Phase-HPLC separation has greatly improved the
reproducibility and throughput of our SRM-assay--critically important features for a clinical assay.
The SRM platform used during assay development and validation consists of a Perfinity Workstation（Perfinity Biosciences,
Indiana）placed on-line to a triple quadrapole mass spectrometer. The SRM-software Skyline directed iterative assay development.
To facilitate quantitation of the sixteen alpha-synuclein peptides, three signature apomyoglobin peptide standards have been
added to samples as external standards. The sixteen alpha-synuclein peptides have been defined by a minimum of threetransitionions. Selected transitions were chosen both for uniqueness and relative intensity. The assay protocol is as follows： using
the Perfinity workstation, 200 microliters of individual CSF obtained during lumbar puncture was diluted 1 ： 5 in .1% Rapidgest, 50
microliters of which was then injected, digested on-column in six minutes, desalted on-column, and then subjected to 30 minute
LC-SRM analysis.
Using SRM-MS placed in-line with a Perfinity Workstation to construct a PD-diagnostic assay, we have successfully screened 10control subjects using our workflow. As anticipated, SRM-MS displays the sensitivity and specificity needed to detect low
abundant candidate biomarkers in complexbiological fluid like CSF. For each of the twenty CSF samples we have screened using
the developed assay（ten patient samples acquired in duplicate）, all nineteen monitored peptides were detected in sufficientabundance so as to facilitate downstream quantitation. Furthermore, replacing traditional bench-top sample preparation with the
Perfinity Workstation has ensured that the technique is both high through-put and highly reproducible. Using the Perfinity
Workstation for our sample preparation ensures analysis is completed in under 45 minutes for each patient sample. Analysis time
now includes all sample preparation required for SRM-based mass spectrometry（even tryptic digestion）. Additionally, for our ten
patient control subject cohort, our assay generated %cvs for all peptides of under10%. Analysis of the six minute tryptic digestion
using the uv-vis detector of the Perfinity HPLC suggests that tryptic digestion efficiency for CSF is approximately 90%. For the work
here, our developed SRM-MS assay will be used to screen a small clinically relevant PD-patient cohort. The intended cohort willconsist of five Parkinson’s patients without dementia, five Parkinson’s patients with dementia, and five healthy control subjects
allrandomly selected for screening in triplicate. Area under the curve measurements of the most intense transition/peptide will
facilitate the quantitative approach taken.

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 43: Novel Proteomics Methodologies

PWe-183
11:10 － 12:20

In vitro phosphorylation to manipulate the abundance profiles inproteomic samples

Shunsuke Takagi1, Haruna Imamura1, Masaki Wakabayashi1, Naoyuki Sugiyama2, Yasushi Ishihama1
1

Kyoto University, Kyoto, Japan, 2Keio University, Yamagata, Japan

Keywords:
LC-MS/MS, phosphoproteomics, in vitro phosphorylation, Low abundant protein, in-depth proteome analysis
Novel aspects:
Manipulate the proteome abundance in the sample by using in vitro kinase reaction followed by phosphoproteomics, in order toachieve the in-depth proteome analysis.
Abstract:
Shotgun proteomics based on LC-MS/MS has been widely used for analyzing proteome profiles in cells of interest. Currently
thousands of proteins can be easily identified from single LC-MS/MS run, even when highly complex samples such as cell lysates
are analyzed. However, it is still problematic to identify very low abundant proteins in the presence of high abundant proteins. In
order to solve this problem, pre-fractionation approaches prior to LC-MS/MS have been employed. However, we cannot avoid thetrade-off between the higher coverage by multidimensional separation and the longer analysis time by the increased number offractions.
Here we introduced a novel approach to manipulate the proteome abundance in the sample by using in vitro kinase reaction
followed by phosphoproteomics.
HeLa cells were dissolved in 12 mM sodium deoxycholate and 12 mM sodium lauroylsarcosinate, followed by trypsin digestion［ 1 ］. The digested peptides were treated with phosphatase to remove the endogenous phosphorylations. The dephosphorylated
peptides were phosphorylated by spiked kinases in vitro. Finally, the phosphorylated peptides were enriched by HAMMOC（hydroxy acid-modified metal oxide chromatography）with titania［ 2 ］and analyzed by nanoLC-MS/MS using ABSciex TripleTOF
5600. The MS was equipped with a Dionex UltiMate 3000 RSLCnano pump and an HTC-PAL autosampler. LC column was packed inhouse with ReproSil-Pur C18-AQ（ 3 μm particle size, 120 A pore size, 100 μm i.d., 15 cm long）. The injection volume was 5 μL,
and the flow rate was 500 nL/min. The mobile phases consisted of（A）0.5 % acetic acid and（B）0.5 % acetic acid and 80 %acetonitrile. A three-step linear gradient of 5 -10% B in 5 min, 10-40% B in 60 min, 40-100% B in 5 min, and 100% B for 10 min was
employed for the peptide elution.
Since each kinase has phosphorylation motifs to the substrate sequence, it phosphorylates proteins or digested peptides
selectively, and we can extract the substrate proteins/peptides by using the phosphoryl group as the target of chemoaffinity
purification based on phosphoryl group-metal oxide interaction. Consequently, peptides with the motif sequence are enriched andthe rest of peptides are discarded. Thus, the complexity in samples is drastically reduced, enabling to identify low abundant
proteins more efficiently.
In this study, 4 Ser/Thr kinases（Ck 1 d, Ck 2 a2, Erk1, Erk 2 ）and 2 Tyr kinases（EPHB1, JAK 2 ）were employed. Each kinase wasreacted with 100 μg of HeLa cell digests and triplicate analyses were done. As a result, we identified 2542 non-redundant proteins.
Among them, 310 proteins were not identified by the conventional approach［ref 3 ］（4634 proteins were identified）. Because Tyr
kinases are generally less selective, more proteins were identified（754 and 2287 proteins from Ser/Thr kinases and Tyr kinases,
respectively）. On the other hand, the percentage of Ser/Thr kinase-derived proteins identified exclusively by this approach was
higher than that of Tyr kinase-derived proteins. Although further optimization is required to increase the substrate proteins, thisapproach would be useful to manipulate the proteome profile of the samples to achieve the in-depth proteome analysis.

References
1 ）Masuda, T. Tomita, M. & Ishihama, Y. Phase transfer surfactant-aided trypsin digestion for membrane proteome analysis. J
Proteome Res, 2008 Feb； 7 （ 2 ）：731-40.
2 ）Sugiyama, N. Masuda, T. Shinoda, K. Nakamura, A. Tomita, M. & Ishihama, Y. Phosphopeptide enrichment by aliphatic hydroxyacid-modified metal oxide chromatography for nano-LC-MS/MS in proteomics applications. Mol Cell Proteomics, 2007 Jun；6 （ 6 ）：1103- 93 ）Iwasaki, M. Sugiyama, N. Tanaka, N. & Ishihama, Y. Human proteome analysis by using reversed phase monolithic silica
capillary columns with enhanced sensitivity. J Chromatogr A. 2012 Mar 9 ；1228：292-7.

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 43: Novel Proteomics Methodologies

PWe-184
13:30 － 14:40

Identification of kinase substrates by siRNA-induced kinase knock-down
coupled with quantitative phosphoproteomics

Ayaka Sato1, Sayaka Iwano1, Wei-Chi Ku1, Masaki Wakabayashi1, Naoyuki Sugiyama2,
Fumiko Toyoshima1, Yasushi Ishihama1
1

Kyoto University, Kyoto, Japan, 2Keio University, Yamagata, Japan

Keywords:
phosphoproteomics / LC-MS/MS / siRNA / kinase knockdown
Novel aspects:
Kinase substrates are identified with siRNA and quantitative phosphoproteomics.
Abstract:
Protein phosphorylation, one of the most ubiquitous post-translational modifications, is a key module in protein kinase-mediatedcellular signal transduction, regulating a wide range of cell functions. It has been extensively identified by shotgun
phosphoproteomics with highly selective phosphopeptide enrichment. However, for most of the phosphorylation sites, we do notknow which kinase is responsible in vivo. Therefore, we developed a novel approach to identify kinase substrates by knocking
down the kinase followed by phosphoproteomic analysis to quantify the phosphorylation level of substrate candidates. As amodel case, we employed serine threonine protein kinase PCTAIRE- 1 （PCTK 1 ）to be knocked down by siRNA, and quantitative
phosphoproteome analysis was carried out.
HeLa cells with or without PCTK 1 siRNA were prepared. After lysing the cells, proteins were extracted and digested by trypsin
after reductive alkylation. The resultant sample was desalted using reversed-phase StageTips with C18 disk membranes.
Phosphopeptide enrichment was carried out by hydroxy acid-modified metal oxide chromatography（HAMMOC）with titania. An
AB SCIEX TripleTOF 5600 system（Foster City, CA, USA）equipped with a Dionex UltiMate 3000 RSLCnano pump and an HTC-PAL
autosampler（CTC Analytics, Zwingen, Switzerland）was employed for phosphopeptide analyses. ReproSil-Pur 120 C18-AQ
materials（ 3 mm, Dr. Maisch, Ammerbuch, Germany）were packed into a self-pulled needle（150 mm length_100 mm i.d., 6 mm
opening）to prepare an analytical column needle with ’stone-arch’ frit. The injection volume was 5 mL and the flow rate was 500
nL/min. Mobile phases were（A）0.5% acetic acid in water and（B）a mixture of acetic acid（0.5%）, water（19.5%）and ACN（80%）.
The gradient condition was employed： 5 % to 10% B in 5 min, 10% to 40% B in 60 min, 40% to 100% B in 5 min and 100% B for 10
min. A spray voltage of 2400V was applied. The MS scan range was m/z 350-1400. The top ten precursor ions were selected for
subsequent MS/ MS scans.
At first, in order to investigate the influence of PCTK 1 on the signal transduction pathways, we prepared HeLa cells treated withsiRNA of PCTK1, confirming clear phenotypes. Then cells with overexpressed PCTK 1 as well as their control cells were additionallyprepared. These four samples were applied to quantify phosphoproteomics at label-free quantitation mode. As a result, 19
phosphopeptides were selected out as the downstream molecules regulated by PCTK 1 according to the quantitation criteria in
which the phosphorylation level was decreased in the siRNA samples, whereas increased in PCTK 1 -overexpressed samples. To
specify the direct substrate molecules of PCTK1, we performed in vitro kinase assay using recombinant PCTK 1 and HeLa cell
extracts. We successfully identified 81 phosphopeptides from 63 proteins. Motif extraction was carried out by Motif-X algorithm,
providing［.....SPP...］. By matching this motif with the 19 phosphopeptides, two candidates, cAMP-dependent protein kinase type1 -alpha regulatory subunit （KAP 0 ） and heat shock factor protein 1 （HSF 1 ）, were selected. Especially, the former
phosphopeptide was also identified as the in vitro substrate. It was found that the kinase knock-down together with in vitro kinase
assay and quantitative phosphoproteomics was a powerful tool to determine the in vivo kinase substrates. Since this approach
can be extended to larger-scale analysis, it will help to give more information about cellular phosphorylation network.

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 43: Novel Proteomics Methodologies

PWe-185
11:10 － 12:20

Comprehensive Targeted Quantitative Proteomics - Assessing
Quantitation Quality of Highly Multiplexed Assays

Christie L Hunter, Sean L Seymour

AB SCIEX, Foster City, CA, United States

Keywords:
SWATH acquisition, quantitative proteomics, data independent acquisition
Novel aspects:
Comprehensive quantitative proteomics analysis via data independent SWATH acquisition
Abstract:
The goal of quantitative proteomics is to both identify and quantify a broad range of proteins and peptides. The extreme
complexity and dynamic range of proteins in typical proteomic samples challenges traditional data dependent workflows byrequiring very high speed MS/MS acquisition to reproducibly and deeply interrogate the sample. The application of data
independent acquisition strategies to increase the reproducibility and comprehensiveness of data collection has been limited by
the speed of current mass spectrometers. Recent QqTOF innovations providing high speed acquisition of high resolution MS/MS
spectra have enabled a new data independent acquisition strategy. In this workflow（called MS/MSALL with SWATH™ Acquisition）,
the Q 1 quadrupole is stepped at 25 amu increments across the mass range of interest, passing a 25 amu window through into thecollision cell. The transmitted ions are fragmented and the resulting fragments are analyzed in the TOF MS Analyzer at highresolution. This is done in a looped fashion with an LC compatible cycle time, such that MS/MS spectra has been acquired on
every peptide in the sample. Because the fragment ions are collected at high resolution, high quality XICs can be generated postacquisition to produce the MRM-like data.
The utility of this workflow for highly multiplexed targeted quantification in complex proteome samples will be explored. Working
from peptide MS/MS spectral libraries, XICs for target proteins and peptides can be generated from this comprehensive dataset.
Initially, XICs to large #s of peptides and proteins were extracted and analyzed for quantitative reproducibility. Impact of various
acquisition conditions on quantitative specificity was explored and optimized. Finally, this technique will be compared to morecommon quantitative techniques such as MRM quantification.

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 43: Novel Proteomics Methodologies

PWe-186
13:30 － 14:40

Global profiling and quantification of histones, histone PTM’s and histonemodifying enzymes in mesenchymal stem cells using a multiplexed SRM
assay

Mei Ying Han1, Bryan Krastins2, Alejandra Garces2, Victoria V Lunyak3, Benjamin Blackwell3,
David Sarracino2, Amol Prakash2, Shadab Ahmad2, Maryann S Vogelsang2, Mary F Lopez2

Thermo Fisher Scientific, Yokohama, Japan, 2BRIMS, Thermo Fisher Scientific, Cambridge, MA, 3Buck Institute for
Research on Aging, Novato, CA

1

Keywords:
SRM assay, PTM, Profiling, quantification
Novel aspects:
Development of a multiplexed SRM assay combining histones, specific histone PTM’s and histone modification enzymes
Abstract:
Introduction
Histone post translational modifications（PTM’s）are a central theme in the regulation of gene expression. A rapidly growing list of
modifications confirms that they play fundamental roles in chromatin modeling processes. These processes are also thought toplay a role in stem cell development and senescence. To date, most studies in this area have been carried out by genomic
analysis, immunostaining, or top-down LC-MS/MS analysis, and as such are not fully quantitative. We have developed a workflow
for quantitative global profiling and targeted analysis of histones, histone PTM’s and histone modification enzymes using a
multiplexed SRM assay.
Methods
Samples
Mesenchymal stem cells were derived from human adipose tissue. Sample lysates were prepared from cell cultures at differentages（Self Renewing（SR）and Senescing（S））.
SRM assay development and analysis
SRM assays were developed on a TSQ Vantage triple quadrupole mass spectrometer（Thermo Fisher Scientific）, as previously
described. There were three technical replicates per sample, ie each sample was digested once and analyzed three times in the
SRM assay. Data were analyzed with Pinpoint™ and Ingenuity pathways analysis（IPA）.
Abstract
A multiplexed SRM assay for ten histone modification proteins and histone H 3 and gamma H 2 Ax sequences including PTM’s
identified in the literature was constructed. The assay was used to interrogate the S and SR mesenchymal stem cell extracts. Thisapproach was extremely fruitful and allowed us to confirm and quantify previously identified PTM’s occurring on histone H 3 andgamma H 2 Ax as well as differential expression of histone-modifying proteins that have not been previously reported inconjunction with either somatic or stem cell aging（replicative or genotoxic stress-induced senescence）.

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 43: Novel Proteomics Methodologies

PWe-187
11:10 － 12:20

Polyhedral approach to comprehensive quantitative analysis formembrane proteomics

Kazuaki Takafuji, Seiji Takashima, Akira Kikuchi
Osaka University, Osaka, Japan

Keywords:
Membrane proteomics Quantitative analysis SRM/MRM SIM
Novel aspects:
Polyhedral approach to comprehensive quantitative analysis
Abstract:
Proteomics analysis is one of the best methods available to study proteins comprehensively.
Recent progress allows to identify thousands of proteins all at once and even to quantitate the amounts of them. However,polytopic membrane proteins that possess several membrane spanning domains, such as transporters, have been omitted and/or
ignored by most of comprehensive proteomics analysis, because these proteins are very difficult to handle due to theirhydrophobic properties. Only few researches have focused on quantitation of transporters. Therefore, we have developed thecomprehensive quantitative proteomics approach for transporters in mammalian epithelial cells. Brush boarder membranevesicles（BBMVs）were isolated from mouse kidney cortex. Membrane proteins were extracted from BBMVs and digested. The digested
peptides were fractionated by chromatography and analyzed by Q-TOF mass spectrometers. Over one hundred transporters wereidentified and able to be quantitated. Thus, this procedure was performed on BBMVs and total membranes prepared from wildtype and diabetes model mice to compare expression levels of transporters. Alteration of the transporter-proteome wereobserved in the diabetes model mice, include sugar or drug transporters. This result indicates that the comprehensive quantitative
proteomics analysis became a useful tool to study polytopic membrane proteins.

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 43: Novel Proteomics Methodologies

PWe-188
13:30 － 14:40

Separation of polypeptides by isoelectric point focusing in electrosprayfriendly solution using multiple-junction capillary fractionator

Konstantin Chingin1, Juan Astorga-Wells1,2, Mohammad Pirmoradian Najafabadi1,2, Thorleif Lavold2,
Roman A Zubarev1,3

1
Karolinska Institutet, Stockholm, Sweden, 2Biomotif AB, Stockholm, Sweden, 3Science for Life Laboratory, Stockholm,
SwedenKeywords:
capillary isoelectric focusing, polypeptides, online proteomics, mass spectrometry, multidimensional analyses
Novel aspects:
OMJ-CIEF allows solution-phase separation by pI of both proteins and peptides, easy automated online sampling to MS, LC, CE
and other capillary-based analyses, and selective mobilization of focused fractions.
Abstract:
The growing popularity of recently introduced IPG-IEF fractionators（e.g., OFFGEL™ fractionator from Agilent）reflects the steadydemand for carrying separation of biological samples in solution phase. Direct analyte recovery from solution precludes the pitfalls
associated with gel methods, such as tedious and error-prone protocols for sample extraction and purification.
We present an online multiple-junction capillary isoelectric focusing fractionator（OMJ-CIEF）for the separation of biologicalmolecules in solution by pI. Owing to the capillary format of operation, OMJ-CIEF combines the merits of solution-phaseseparation with the possibility of automated online sampling to MS, LC, CE and other capillary-based analyses.
In OMJ-CIEF, the separation capillary is divided into seven equal sections joined with each other via tubular Nafion membrane
insertions. Each junction is communicated with its own external electrolytic buffer which is used both to supply electrical contact
and for solvent exchange. The performance of the fractionator was explored using protein and peptide samples covering broad pI
range（proteins： β-lactoglobulin A（pI 5.3）, hemoglobin A（pI 6.8）, myoglobin（pI 6.9-7.3）, α-chymotrypsinogen A（pI 9 ）,ribonuclease A（pI 9.5）, cytochrome C（pI 10-10.5）and lysozyme（pI 11.3）； peptides： tryptic digest of bovine serum albumin）.
Separation was carried in ionic and ampholytic buffers, including ammonium formate, ammonium hydroxide, histidine and arginine.
By maintaining electric potential across upstream segments of the capillary after the focusing stage, selective release ofdownstream analyte fractions could be achieved. The selective release mode circumvents the problem of peak broadening during
mobilization and enables convenient comprehensive sampling for orthogonal separation methods. Using single-component
ampholyte buffers with well-defined pI cut-off values, controlled separation of protein mixture into basic and acidic fractions wasdemonstrated.
The device is cheap and easy to fabricate in-house, simple in operation and straightforward in interfacing to hyphened analyticalplatforms. OMJ-CIEF has a potential of becoming a practical add-on unit in a wide range of bioanalytical set-ups, in particular as afirst-dimension separation in mass spectrometry based proteomics or as a preparative tool for analyte purification, fractionationand pre-concentration.

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 43: Novel Proteomics Methodologies

PWe-189
11:10 － 12:20

Comprehensive variability analysis of nutrients absorption mechanism insmall intestine by quantitative proteomics

Takashi Nishiyama, Shushi Nagamori, kazuaki Takafuji, Yoshikatsu Kanai
Osaka University Graduate School of Medicine, Suita, Japan

Keywords:
proteomics, membrane protein, transporter, nutrient absorption, sucrose
Novel aspects:
Overall alterations of membrane protein expression by nutrients were revealed by proteomics method.
Abstract:
Introduction
Small intestine is an important organ for absorption of nutrients； carbohydrate, amino acids, fat, minerals etc. In guts, nutrientsare digested by metabolic enzymes and absorbed by transporters. For example, sucrose is hydrolyzed to glucose and fructose by
sucrase and absorbed by glucose or fructose transporters. Proteins are digested into peptides and amino acids by peptidases andabsorbed by peptide or amino acid transporters. These proteins are express at the epithelial cells, especially brush border
membranes of small intestine. There is much evidence that the transporters are adaptively regulated by dietary levels of nutrients.
It is important to elucidate the regulation of nutrient absorption for understanding of physiological role or pathology variation ofsmall intestine.
To study the overview of mechanism for nutrient absorption, comprehensive expression analysis of membrane proteins is critical.
However, it is difficult to obtain comprehensive data of the expressions by conventional analytical methods, such as western blot
or microarray. Microarray is able to evaluate change of mRNA level comprehensively, yet correlation between expression levels ofmRNA and protein are not confirmed in many cases. Western blot is one of the most powerful tools to investigate alterations of aprotein expression level unless no antibody exists though it is practically impossible to prepare antibodies covers most of proteins.
Proteomics analysis is the best methods available to study proteins comprehensively. Recent progress allows to identify many
proteins and even to quantitate the amounts of them. However, polytopic membrane proteins that possess several membrane
spanning domains, such as transporters, have been omitted from most of proteomics analysis because of difficulties for handlingdue to their hydrophobic properties and relatively low level of the expression.
Therefore, we applied a quantitative proteomics approach developed specially for membrane proteins to evaluate thecomprehensive variability of proteins in brush boarder membranes of small intestine. Here, we analyzed membrane proteins ofintestines from the 3 groups of mice, sugar-drinking, artificial sweetener-drinking and water-drinking（control）groups for a model
study.
Method and Results
Mice were separated into 3 groups（sucrose group, sweetener group and control group）, and bred for 2 weeks under freetaking of low-carbohydrate diet and each test solutions. There were no significant differences of energy intake and body weight
among 3 groups. After 2 weeks, epithelial cells of small intestine（duodenum and jejunum）were isolated by scraping. Brushborder membrane vesicles（BBMV）were prepared by divalent cation method for the epithelial cells. Soluble proteins were
washed out from BBMV, and then membrane proteins were extracted from phospholipid bilayer membrane by using phasetransfer surfactant（PTS）method developed by Masuda et al specifically for extraction of membrane proteins. After the PTS
treatment, peptides were fractionated by two-dimensional reverse-phase
chromatography and desalted. The samples were analyzed by MSE method with Q-ToF type LC-MS（Waters, SYNAPT G 2HDMS）.
As the result, a total of 200 proteins were identified and 150 proteins were successful to quantitative analysis among 3 groups. In
the sucrose groups, there were marked alterations of expression levels of sugar transporters and metabolic enzymes as reported
previously by using the conventional methods. In addition, expression levels of cholesterol transporters and amino acids
transporters were also changed. In the sweetener group, no significant alteration of sugar transporters expression level wasobserved, but there were alterations of expression level of transporters or enzymes that are in charge of amino acids and peptides
absorption and metabolism. Taken all together, over all alterations of membrane protein expression by nutrients were revealed by
the proteomics method. This method can be use not only for nutrient metabolism but also use for many biological systems whichcontain membrane proteins.

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

OthersPWe-190
13:30 － 14:40

Laser Spectroscopic Investigations of 1,3-Dichloro-2-fluorobenzene by
REMPI and MATI Spectroscopy

Sascha Krüger, Juergen Grotemeyer
Kiel University, Kiel, Germany

Keywords:
Ion spectroscopy, vibronic structure, excitation/ionization-energy
Novel aspects:
Geometry distortion during ionization
Abstract:
Introduction Ion spectroscopy is an essential method for investigation of ionic molecular species and their vibronic structure.
Substitution of one or more hydrogen atoms in the benzene ring leads to significant change in electron density distribution.
Previous research showed that certain halogen substitution pattern of benzene lead to strong activity of formally forbidden modes
due to vibronic coupling［ 1 ］. The aim of this study was to assess the impact of replacing the doubly vicinal chlorine atom in
1,2,3-trichlorbenzene by flourine. The vibronic structure in the first excited state（S 1 ）was investigated by means of Resonce
Enhanced Multi Photon Ionization（REMPI）spectroscopy, the ionic ground state（D0 ）by Mass Analyzed Threshold Ionization（MATI）spectroscopy. The experimental findings are supported by quantum chemical calculations.
Experimental The experiment consists of a time-of-flight mass spectrometer equipped with single-stage ion source and reflectron
and two tunable dye lasers. The fundamental（1064 nm）of a Nd：YAG pump laser is frequency-doubled to 532 nm. Then, the 532
nm wavelength is mixed with the fundamental to frequency-tripled 355 nm. One dye laser is pumped by the second harmonic of
the YAG, the other by the third harmonic. The dye wavelength of the 355 nm pumped laser is frequency doubled in a BBO-I. The
second laser was modified to sumfrequency mix the dye wavelength and the 533 nm pump wavelength in a BBO-I. A supersonicmolecular beam of sample molecules and seed gas（Argon）is expanded via a pulsed jet valve into the ion source. Excitation or
ionization is accomplished by multi photon absorption under field-free conditions. In MATI modus the prompt ions are
discriminated against Rydberg neutrals by application of a weak electrical retarding field subsequent to two-photon excitation.
Finally, a high voltage puls ionizes the Rydberg neutrals and accelerates them into the TOF. In REMPI modus the retarding field isomitted and the generated ions are accelerated directly into the TOF. A MCP detector is used to detect the ions. The ion signal isreadout by a digital oscilloscope.
Results and Discussion The findings from the research provided very accurate values for the electronic excitation（IE）and
ionization energy（AE）of 1,3-dichloro- 2 -fluorobenzene. Furthermore a detailed vibrational analysis could be conducted for the
first time. This analysis advances the understanding of vibrational modes active during excitation/ionization. During excitation/
ionization molecules are able to undergo geometric distortion along the eigenvector of certain vibrational modes. The REMPI
spectra show unusual high activity of a symmetry forbidden out-of-plane mode（17b 2 ）at low frequencies. Several cases ofviolation of v = 0 propensity rule［ 2 ］were observed in the MATI spectra. For instance, a short, three membered progression of
the 17b mode with a shift of the Franck-Condon maxmimum to lower energies was observed in the MATI spectrum via 17b 2 .
While the calculations suggest a planar molecular structure in the electronic and ionic ground state, they suggest a non-planar
structure in the first excited state. The experimental findings and calculation indicate a geometrical distortion during excitationand ionization.
Literature［ 1 ］F. Witte, M. Riese, F. Gunzer, J. Grotemeyer, Int. J. Mass Spectrom. 2010, 306 , 129；［ 2 ］M. Child, Theory of Molecular Rydberg States, Cambridge
Molecular Science, Cambridge University Press, 2011.

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

OthersPWe-191
11:10 － 12:20

Selective Extraction and Absolute Quantification of Glutathione using
Nanoengineered Micro Gold Shells and LDI-TOF MS

Jeongwook Lee, Woon-Seok Yeo
Konkuk University, Korea

Keywords:
glutathione, internal standard, LDI-TOF, quantification
Novel aspects:
Selective extraction and absolute quantification of GSH using organic matrix free LDI-TOF MS with nanostructure embedded gold
micro shells.
Abstract:
Glutathione（GSH）is the most abundant biothiol which plays key roles in a variety of biological processes including maintenanceof intracellular redox activities, xenobiotic metabolism, intracellular signal transduction, gene regulation, and protecting against
oxidative stress. Therefore, the various detection techniques, such as gas chromatography, high performance liquid
chromatography, capillary electrophoresis, and optical detection with fluorescence, have been developed for detection of GSH in
biological samples.
In this poster, we describe a new method for selective extraction and absolute quantification of GSH using organic matrix free
LDI-TOF MS with nanostructure embedded gold micro shells（μAuSs）. Our strategy utilizes the Au-thiol interaction which allows
the selective extraction of GSH in samples to μAuSs. After the extraction step, the μAuS-bound GSH is analyzed directly by LDITOF MS without a use of organic matrix. In addition, GSH in the sample was quantitated by using an internal standard（IS）which
was prepared with μAuSs and isotope-labeled GSH. The IS, thus, has the same structure of the GSH but heavy molecular weight
and therefore, allowed absolute quantification of GSH by comparing the mass intensities between the GSH and the isotopelabeled GSH. We believe this strategy for absolute quantification of GSH would be useful by providing the quantitative information
for diagnostic purposes.

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

OthersPWe-192
13:30 － 14:40

Coulomb-Interaction-Induced Effects on Xe Isotopic Ion FT-ICR Mass
Spectra: A Many-Particle Simulation Using GRAPE

Makoto Fujiwara, Naohisa Happo, Koichi Tanaka
Hiroshima City University, Hiroshima, Japan

Keywords:
FT-ICR, Many-Particle Simulation, Coulomb Interaction
Novel aspects:
Coulomb-Interaction-Induced Effects on Xe Isotopic Ion FT-ICR Mass Spectra were simulated in detail using a more powerful
special-purpose computer for many-body problems, GRAPE
Abstract:
In Fourier transform ion cyclotron resonance mass spectrometry（FT-ICR MS）, ion motion trapped in the FT-ICR cell is essentiallya superposition of three modes： cyclotron motion, magnetron motion and z motion. However, in practice, 103 to 106 different m/zions are simultaneously trapped and excited in the cell and the ion cloud distribution consisting of each m/z ion ensemble and its
temporal variation are quite complicated. Moreover at high ion density, the Coulomb interactions between ions considerably
influence the ion cloud distribution. In a previous work［ 1 ］, a special-purpose computer board（MD-One/E, Gazogiken）for
many-body problems was combined with a single processor PC. The computer board makes it possible to calculate thesummation routine of the ion-ion Coulomb interaction force components in high speed. Thereby in the case of 1,000 simulation
particles the computation speed became 10 times as fast as a single processor only. The extended computing configuration wasapplied to the simulation for the influence of ion-ion Coulomb interactions on FT-ICR mass spectra measured especially at a highmagnetic field. Further, the influence of ion-ion Coulomb interactions on Xe isotopic ion mass spectra was preliminarily simulated［ 2 ］.
In the present work, a special-purpose computer GRAPE- 6 （for“GRAvity PipE“）that is a more powerful accelerator board for
many-body problems was employed and the computing configuration was further accelerated. In the case of 10,000 simulation
particles the calculation speed has been 11 times as fast as MD-One/E. On the accelerated configuration Coulomb-interactioninduced effects on Xe isotopic ion mass spectra were in more detail investigated using up to 10,000 simulation particles. The
simulation particle number was divided and assigned to each of seven Xe isotopic ions： 128Xe+, 129Xe+, 130Xe+, 131Xe+, 132Xe+, 134Xe+, and 136Xe+ in proportion to the respective isotopic ratios. Ion excitation was performed by frequency sweep at a magnetic fieldof 5 T and the Xe isotopic ion spectra were simulated as a function of total ion population initially trapped in the FT-ICR cubic
cell. From the simulation results, in the range up to an initial ion population of 10,000 ions, the ratios of Xe isotopic ion peakintensities appear to be consistent with respective Xe isotopic ion abundances initially trapped. However, at high ion density theratios are not maintained. Further from analysis of temporal variations for the ion cloud distributions during detection period it has
been found that Coulomb-interaction-induced dephasing of the coherent ion cyclotron motion excited significantly progresses
with initial ion population in the range of more than 10,000 ions.

References
1 ）M. Fujiwara, etal., J. Mass Specrom. Soc. Jpn., 58, 169-173（2010）.
2 ）M. Fujiwara, et al., Abstr. of the 58th Annual Conference on Mass Spectrometry, Tsukuba, Japan & 1 st Asian and Oceanic
Mass Spectrometry Conference, 17P-024, 188（2010）.

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

OthersPWe-193
11:10 － 12:20

Analysis of flavonoids by Graphene-Based SALDI-TOF MS

Min-Wei Chien1, Ching-Yuan Su2, Lain-Jong Li3, Chien-Chen Lai1

1
Institute of Molecular Biology,National Chung Hsing University, Taiwan, 2Department of Electronic Engineering, Chang
Gung University, Taiwan, 3Institute of Atomic and Molecular Sciences, Academia Sinica, Taiwan

Keywords:
flavonoids, SALDI, graphene,
Novel aspects:
Flavonoids were spotted onto a matrix of graphene-based nanoparticles and analyzed by LDI-TOF-MS
Abstract:
Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry（MALDI-TOF-MS）is a simple and fast technique forthe analysis of large biomolecules but is not suitable for the detection of low molecular weight molecules and compounds, suchas flavonoids and phenylpropanoids, mainly due to the lack of an appropriate matrix. Flavonoids and phenylpropanoids, such as
coumarin and its derivatives, have attracted much attention recently because of their pharmacological activities and putative
therapeutic benefits. In this study, we developed a quick and simple LDI-TOF-MS method for detection of flavonoids andderivatives of coumarin. Analytes were spotted onto a matrix of graphene-based nanoparticles and then analyzed by LDI-TOF-MS
in the negative ion mode. Analysis of the sensitivity and effect of different graphene-based nanoparticles including graphene,graphene oxide, and reduced graphene oxide on desorption/ionizat ion of analytes showed that graphene oxide was the mostsuitable matrix. Moreover, we found that graphene oxide sheets of larger lateral size resulted in better desorption/ionization
efficiency. Overall, we show that graphene oxide is a useful matrix for analysis of flavonoids and the derivatives of coumarin by
LDI-TOF-MS in the negative ion mode.

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

OthersPWe-194
13:30 － 14:40

Quantification of eight catechins and caffeine in black, white and green
tea samples using UHPLC coupled to tandem mass spectrometry

Karolina Skraskova, Michaela Mudrova, Hana Vlckova, Lucie Novakova

Charles University in Prague, Faculty of Pharmacy in Hradec Kralove, Hradec Kralove, Czech Republic

Keywords:
LC-ESI-MS/MS, UHPLC, tea, catechins, caffeine
Novel aspects:
New UHPLC-MS/MS method using mixed-mode sorbent for determination of eight catechins and caffeine in white, green and black
tea samples.
Abstract:
Tea infusions are rich in polyphenolic compounds catechins, which are responsible for the antioxidant activity and the overall
positive effect of tea on human health. Furthermore the tea’s energizing effect caused by the presence of caffeine is well known.
Actual content of catechins and caffeine is recognized as one of the tea quality indicator. According to the treatment of the tea
leaves during processing, three fundamental types are distinguished： green tea（non-fermented）, black tea（fully fermented）andwhite tea（partially fermented）. The fermentation process significantly decreases the content of catechins.
In this project a method for quantitative determination of eight catechins：（+）-catechin（C）,（-）-epicatechin（EC）,（-）-catechin gallate（CG）,（-）-epicatechin gallate（ECG）,（-）-gallocatechin（GC）,（-）-epigallocatechin（EGC）,（-）-gallocatechin gallate（GCG）,（-）-epigallocatechin gallate（EGCG）and caffeine（Caff）in tea
samples has been developed. The analytes were separated on Acquity CSH C18（2.1 x 100 mm, 1.7 µm）column. The separation
was completed within 3.5 minutes. The mass spectrometer was equipped with electrospray ionization source and triple quadruple
analyzer. The polarity switching mode was employed. The quantification was based on selected reaction monitoring.
To prepare the tea infusions, approximately 1 g of accurately weighed dry tea sample was extracted with 100 mL of hot（80 °C）
water for 30 minutes. The cooled down infusion was filtrated through 0.22 µm PTFE filter. Afterwards the samples were diluted 10and 1000-times and used for determination of the analytes.
System suitability test（SST）, method linearity range, limits of quantification and detection, accuracy and precision were assessed.
The method was linear within the range 0.1 ng/mL and 500 ng/mL, with the linear coefficients being typically 0.995. The limit ofquantification was set as the lowest point of the above given range. Accuracy was evaluated by spiking diluted tea extracts withstandard solution mixture. The precision was determined as intra- and inter-day repeatability. All the validation parameters were
evaluated individually for black, white and green teas. The matrix effects were assessed by comparison of the slopes of matrix
matched calibration curves and standard calibration curve. The method was used to monitor the content of catechins and caffeine
in various tea samples.
The authors acknowledge the financial support of the project SVV 265 002.

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

OthersPWe-195
11:10 － 12:20

Low-energy ion scattering (LEIS) study of Cs-sputtering effects on III-V
semiconductor hetero-structures during SIMS analysis

Helena Tellez1, David M McPhail1, Jose M Vadillo2
1Imperial College, London, UK, 2University of Malaga, Malaga, Spain

Keywords:
Low-energy ion scattering, ToF-SIMS, semiconductor, Cs implantation, ion-solid interaction
Novel aspects:
This work presents a novel approach to study Cs surface retention and ion-sputtering effects in ToF-SIMS analysis ofsemiconductor hetero-structures by low energy ion scattering（LEIS）.
Abstract:
Secondary ion mass spectrometry（SIMS）has been widely applied in materials science for surface and interface characterization.
Furthermore, materials properties are strongly affected by their surface and interface morphology and chemistry. In this sense,
SIMS has been successfully applied in the semiconductor industry for the analysis of the surface and interface chemistry, providing
outstanding depth resolution and sensitivity. In this work, we show the analysis of III-V semiconductor hetero-structures by ToFSIMS to study the diffusion and oxidation phenomena that might take place during the multi-layered growth.
Nevertheless, additional ion-sputtering effects during SIMS analysis alter the surface and interface chemistry by atomic mixing andion implantation. These effects are especially relevant when using chemically-active primary species, such as Cs+, which leads tomodifications of ionization probabilities and sputter yields due to the implantation of primary ions on the surface and near-surface
regions and the subsequent material work function reduction（ 1 ）. Since Cs is very volatile and reactive, Cs retention is also
dependent on oxygen availability in the ultra-high vacuum chamber and/or material surface（ 2 ）. Therefore, in order tounderstand the Cs retention on the surface, low-energy ion scattering（LEIS）was used to probe the outermost surface during ToFSIMS analysis. In addition to the first mono-atomic layer specificity, quantitative LEIS analysis of Cs coverage can be performed
when correcting for work function modifications（ 3 ）. In this case, the possibility of performing the LEIS analysis in the same
instrument without exposing the surface to the atmosphere, allows us to avoid any further oxidation/contamination surface toaccurately estimate the surface coverage as Cs dose is building up during ToF-SIMS analysis.
（ 1 ）H. Gnaser, Phys. Rev. B., 1996, 54, 17141.
（ 2 ）B. Berghmans, J. Rip, W. Vandervorst. Surf. Interf. Anal., 2011, 43, 225-227.
（ 3 ）R. Cortnraad, A.W. Denier van der Gon, H.H. Brongersma, S.N. Ermolov, V.G. Glebovsky, Surf. Interface Anal., 2001, 31, 200-205.

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

OthersPWe-196
13:30 － 14:40

Quality control of multilayered/nanoheterostructured solar cells by
secondary ion mass spectrometry and ion beam etching techniques

Jose M Vadillo1, Diana J Padilla1, Helena Tellez2, John W Druce2, David S McPhail2, Jose J Laserna1
1

University of Málaga, 2Imperial College

Keywords:
SIMS, FIB, solar cells analysis
Novel aspects:
The use of SIMS（quadrupole and ToF）and ToF-SIMS imaging over FIB sections to perform depth-resolved analysis withnanometric resolution of multilayered/nanostructured solar cells.
Abstract:
High efficiency solar cells represent the preferred photovoltaic component on spacecrafts and will be used at long term for manyindustrial applications. Its technology requires a continuous development of new materials and fabrication processes. In theparticular case of III-V solar cells, the alloy composition determines its energy band gap and many III-V alloys can be grown
epitaxially on each other, generating a stack of multiple layers（some of the as small as 5 nm）of different band-gap that must
manufactured under strict control of composition, properties and thickness. Secondary ion mass spectroscopy（SIMS）represents
an excellent technique for a complete characterization of a multilayered solar cell device due to its depth-profiling capabilities andsensitivity. The communication will show examples of quality control of III-V solar cells using quadrupole SIMS, and ToF-SIMS
where defects associated to the manufacturing process have been identified. As a complementary technique, Focussed Ion Beam
（FIB）sections of the structure were obtained and subsequently mass-selectively imaged by ToF-SIMS.

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

OthersPWe-197
11:10 － 12:20

Ultra high pressure comprehensive two-dimensional liquid
chromatography combined with hybrid mass spectrometry for the
elucidation of carotenoids in chilired peppers

Marcus Mreyen1, Francesco Cacciola2,3, Paola Donato3,4, Daniele Giuffrida5, Germana Torre3,
Paola Dugo3,4, Luigi Mondello3,4

1
Shimadzu Europa GmbH, Duisburg, Germany, 2Chromaleont S.r.l., Messina, Italy, 3Dipartimento Farmaco-chimico,
Messina, Italy, 4Centro Integrato di Ricerca (C.I.R.), Roma, Italy, 5Dipartimento di Scienze degli Alimenti e dell’Ambi,
Messina, Italy

Keywords:
comprehensive LC, Ion trap, TOF, accurate mass,
Novel aspects:
Combination of high sophisticated LCxLC techniques with advanced hybrid type mass spectrometry for the structural elucidationof natural compounds
Abstract:
Introduction
Some of the important ingredients in paprika extracts are carotenoids formed in the fruit during ripening. These compounds are
not only used in the food industry but also in pharmaceutical and cosmetic products. A total of 33
different compounds have been separated and identified in red chilli peppers.
A comprehensive normal-phase × reversed-phase（NP-LC×RP-LC）liquid chromatographic system was developed, and applied foranalysis of the intact carotenoid composition of chili red peppers, with PDA（photodiode array）and MS（mass spectrometry）. The combination of ion trap technology coupled with accurate massmeasurements was used for the identification and classification of the different compounds.
Methods
A micro-bore cyano column（250x1.0 mm, 5 mm d.p.）was chosen for the first dimension separation, interfaced to a secondary（ 2 D）C18 column（30x4.6 mm, 2.7 mm d.p.）packed with fused-core particles. Subsequently, two columns of the same stationary
phase were coupled serially for second dimension separation, and operated under UHPLC（ultra high pressure LC）conditions,
within a cycle time of 90 or 60 sec. The advantages of the latter set-up over the single-column one are demonstrated by the higher
peak capacity values（nc 836 or 1325 vs. 571）； especially for the separation of the more polar chemical classes（mono-esters andfree carotenoids）, better focusing at the head of the 2 D column resulted in improved
separation.

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

OthersPWe-198
13:30 － 14:40

Identification of Organic Contaminants on Silicon Surface by Mass
Spectrometry

Yoon Mi Lee, Sang Yoon Shin, Jung Dae Park, Pil Kwon Jun, Jong Soo Kim
Semiconductor Business, Samsung Electronics / Gyeonggi-Do, Korea

Keywords:
Semiconductor, Organic Contaminants, TD-GC/MS, TOF-SIMS, LDI-TOF
Novel aspects:
This work shows the differences in detection sensitivity of various mass spectrometry and new application using LDI-ToF MS on
semiconductor.
Abstract:
Increasing yield loss due to non-visual defects and process variations requires new approaches in methodologies, diagnostics andcontrol. Especially, Organic contaminants such as organophosphates, antioxidants, plasticizers, caprolactam, phthalates orsiloxanes on wafers can cause unwanted doping, poor gate oxide reliability, delamination, contact resistance, solder/copper
electroplating failure, mis-align, optical defects, wetting problems, silicon nitride nucleation delays, ellipsometry errors and manyother thin film issues that can reduce yield. For optimum process control, organic contaminants have to be under control.
This research investigated organic contaminants on silicon wafers for semiconductor. During the semiconductor process, trends ofdefect were increased at certain steps. We matched the system of defect trends and then discovered thinner chemical that isadhesion promoter for photo process caused defects. For simulation, thinner was coated on the clean wafer. The defect like thewhirlwind was formed on clean wafers after treating the thinner. C, O, S, Fe and Al elements were characterized by Energy
Dispersive X-ray analysis（EDX）. However, we couldn’t observe any differences between normal and abnormal thinner chemical
about metal impurities. Thermal Desorption GC-MS（TD-GC/MS）analysis was performed that is typical analysis for identifying theorganic contaminants and matching to materials outgassing results to determine the root source of the organic contaminants.
However, any special chromatogram was not detected. Thinner contaminated wafers were examined by using Laser Desorption/
Ionization Time of Flight Mass Spectrometry（LDI-ToF MS）and Time of Flight Secondary Ion Mass Spectrometry（ToF-SIMS）. ToFSIMS result shows the NH4 +, CH4N+, C2H6N+, C4H12N+ ions and LDI-ToF MS result shows 73.9Da in abnormal chemical. According tothese results, tetramethylammonium hydroxide（TMAH）is contaminated in thinner chemical. To convince these results, 2.4 ppm
TMAH coated wafers were investigated and 73.9Da was detected by using LDI-ToF Method. These wafers were also investigatedby using TD-GC/MS（Gerstel TDS 2 thermodesorption system）. However, sufficient signal was detected after concentrating 100
times. TMAH is usually used as an anisotropic etchant of silicon. It is also used as a basic solvent in the development of acidic
photoresist in the photolithography process. In this case, when mixing the chemicals TMAH was not a component of thinner. We
discovered the cleaness of the environment was not controled about organic materials for semiconductor grade.

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

OthersPWe-199
11:10 － 12:20

Comprehensive Targeted Compound Identification Using PseudoPrecursor Ion Scan and Neutral Loss Scan for Degradation Analysis ofElectrolytes

Kaoru Karasawa, Toshiyuki Yamazaki, Sumie Ando
ABSCIEX

Keywords:
QqTOF, comparative study, structure elucidation, pseudo-precursor ion scan and neutral loss scan, impurity and degradation
analysis
Novel aspects:
Various pseudo-precursor ion scan and neutral loss scan data are available easily and comprehensively from acquired high speed
and comprehensive IDA data.
Abstract:
Introduction:
Li-ion batteries and capacitors are being developed for use in mobile phones, electric vehicles, as well as energy storage
especially from natural resources. For development of Li-ion batteries and capacitors, it is important to evaluate their componentsof starting materials, products, and ageing effects. In addition, analysis of electrolyte started gaining attention； however the
method is not fully established because of sample stability, complexity, strong acidity and others. To establish the analyticalmethods, we accomplished comparative studies of electrolytes in Li-ion batteries and structural elucidation of characteristic
electrolyte additives and products in cycle test（reported JSMS and Battery Symposium in Japan last year）. In this report, we
perform further degradation analysis and comprehensive targeted compound identification using pseudo-precursor ion scan andneutral loss scan technique on acquired high speed and comprehensive IDA data.
Material and Methods:
Samples： 35 electrolytes and blank acetonitrile was used as samples. Each electrolyte was extracted with acetonitrile from 35
different cells from six Li ion batteries（# 1 to # 5 ）after discharge and charge cycle tests（0, 100, 500 cycles）and diluted ten times
with acetonitrile before injection.
HPLC Condition: System： Shimadzu UFLC LC System（Shimadzu Corp, Japan）, Column： Acquity HSS T 3 2.1X100mm, 1.8mm（Waters）, Mobile Phase： gradient elution with A： 10mM ammonium acetate in water and B： 20 % THF in acetonitrile, Flow
Rate： 0.500mL/min, Injection Volume： 2 µL, Column Temperature： 40°C, Total Runtime： 19.5min
MS Condition: System： AB SCIEX TripleTOFTM 5600 LC/MS/MS System（AB SCIEX, Canada）, Ionization： ESI, Polarity： Positive and
Negative, Method： IDA（Information dependent acquisition, Survey Scan 100msec and 5 or 10 or 25 Product Ion Scan 50, 25,10msec each, DBS（dynamic background subtraction）ON）, Cycle Time： 0.4sec, CE： +/-35+/-15V
Software: Acquisition： Analyst (R) TF1.5.1 Software, Analysis： PeakView (R) Software（AB SCIEX, Canada）for structure elucidationand pseudo-precursor ion scan and neutral loss scan, MarkerViewTM 1.2.1.1 Software（AB SCIEX, Canada）for multivariable analysis（PCA-DA, t-Test）, MetabolitePilotTM 1.5 Software（AB SCIEX, Canada）for analogue identification and sample comparison
Results and Discussion:* The compounds related to degradation and life time of batteries were identified using PCA-DA of MS data with the results ofdischarge and charge cycle tests and MS/MS data.
* A large number of analogues of these compounds were identified individually, using pseudo-precursor ion scan and neutral loss
scan from one high speed and comprehensive IDA data.
* The intensities of these compounds were corresponded to the results of cycle tests. It suggests that these analogues affect theperformance of batteries.
# With high speed IDA with DBS, MS/MS is acquired for almost all components in one run, and additional MS/MS run is not
required. Various pseudo-precursor ion scan and neutral loss scan data are available easily and comprehensively from theacquired data.
# This technique is applicable to and powerful tool for impurity and degradation analysis, and other applications.

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

OthersPWe-200
13:30 － 14:40

Analysis of degradation products in electrolyte for rechargeable lithiumion battery through high mass accuracy MSn and multivariate statisticaltechniqueHiroki Nakajima, Yamaki Satoshi, Nishine Tsutomu, Furuta Masaru
Shimadzu Co., Kyoto, Japan

Keywords:
LCMS, high mass accuracy MSn, lithium-ion batteries, electrolyte, multivariate statistical technique
Novel aspects:
Formulae and structure of degradation products in the electrolyte were identified using high mass accuracy MSn and multivariate
statistical technique.
Abstract:
Rechargeable lithium-ion batteries are one of the major power sources for portable electronic devices and electric vehicles
because of their high voltage and high energy density. The electrolyte of a lithium-ion battery is consisting of a lithium salt in an
aprotic organic solvent. The typical operational potential of a lithium-ion battery is between 0 and 5 V. Therefore, solvent can be
reduced or oxidizedat the negative and positive electrodes during the battery charging process. As a result, various degradation
products are generated in electrolyte, and cause some problems such as a decrease in the capacitance of battery. Here, we
present the analysis method of degradation products generated in electrolyte using high mass accuracy MSn and multivariate
statistical technique.
The electrolyte was a mixture of ethylene carbonate（EC）and dimethyl carbonate（DMC）（EC ： DMC = 1 ： 2 vol%）containing
1 M lithium hexafluorophosphate（LiPF 6 ）.The electrolyte A taken from unused lithium-ion battery and the electrolyte B takenfrom lithium-ion battery repeated charge and discharge cycles（200 times）were used as samples. Those samples were prepared
1 /10 dilution with methanol for LCMS-IT-TOF（Shimadzu Corporation）measurement. Principal component analysis（PCA）was
performed using data acquired by LCMS-IT-TOF measurement of electrolyte A and electrolyte B（n= 3 ）to find the compounds
generated in electrolyte B. Then, those compounds were identified chemical formula using software“FormulaPredictor“（Shimadzu Corporation）. SIMCA-P+（Umetrics）and Profiling Solution（Shimadzu Corporation）were used for PCA.
On the score plot of PCA, the group of electrolyte A and electrolyte B were located at left side and right side, respectively. 13
unique ions of electrolyte B were observed at right side on the loading plot. And, those ions were not detected on the extracted
ion chromatogram（EIC）of electrolyte A. These results suggested that those ions were degradation products generated in theelectrolyte of lithium-ion battery repeated charge and discharge 200 cycles. The formula of the ion（m/z 284.0982）being one of 13
species；［C9H14O9+NH4］+）using high mass accuracy MS1 data andunique ions of electrolyte B was predicted as C9H14O（ionic9
formula predictor. Indeed, the structure of C9H 14O 9   was predicted as H3C-（OCO2-C2H 4）2-OCOCH3 referring to some articles on
degradation products in electrolyte. We also measured MSn of the ion（m/z284.0982 ）to determine the validity of the predictedchemical structure. The result of MS3 data and neutral loss ions showed that the predicted structure of C9H14O9   was correct. By
the same method, formulae and structures of other 12 ions also were identified as C 4 H 11 O 5 P,C 8 H 18 O 5 , C 3 H 9 O 4 P,C 10 H 22 O 6 ,C 7 H 15 O 8 P,C 9 H 12 O 9 , C 12 H 26 O 7 ,C 6 H 10 O 6 , C 6 H 13 O 7 P,C 16 H 34 O 9 , C 16 H 34 O 9 ,and C 12 H 18 O 12 . It was cleared that the chemical species ofdegradation products generated in the electrolyte with increasing charge and discharge cycles were carbonate and phosphatefrom result of this study.

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

OthersPWe-201
11:10 － 12:20

GC/MS Determination of N-Nitrosamines in Rubber Teats

Peng Gao, Jun Fan, Li X Deng, Hong T Huang
SHIMADZU(CHINA)CO.,LTD,ShangHai,China

Keywords:
N-Nitrosamines； Rubber Teats； Solid Phase extraction
Novel aspects:
A new GC/MS method for the determination of N-Nitrosamines in rubber teats, comparing with traditional GC/TEA method.
Abstract:
A method of GC/MS was developed for the determination of 12 N-nitrosamines（N-nitroso dimethylamine, N-nitroso
methylethylamine, N-nitroso diethylamine, N-nitroso dipropylamine, N-nitroso dibutylamine, N-nitroso-N-methylaniline, N-nitroso
N-ethylaniline, N-nitroso piperidine, N-nitroso pyrrolidine, N-nitroso morpholine, N-nitroso diphenylamine, N-nitroso dibenzylamine）
in rubber teats. The release of N-nitrosamines from rubber teats were accomplished by ultrasonic extraction and then purified by
C18 SPE cartridges. The analyses were separated on Stabilwax capillary column and detected by EI ion source with selected ion
mode. Good linearity of calibration curve was obtained in the concentration range from 50 to 1000 μg/L for all compounds
examined. Recoveries ranged from 70 % to 110 % , with RSD（n= 5 ）less than 10 % . The limits of detection（ 3 S/N）of 12
N-nitrosamines examined ranged from 0.15 to 0.61μg/Kg.

Poster Session
Wedensday, 19th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

OthersPWe-202
13:30 － 14:40

Analysis of black tea by GC-MS and high-resolution UPLC-MS

Christian Grün, Boudewijn Hollebrands, Julien Boelhouwer
Unilever R&D, Vlaardingen, The Netherlands

Keywords:
Black tea, Camellia sinensis, UPLC-TOF-MS, GC-MS, phytochemicals
Novel aspects:
The method described allows the differentiation between different black teas, which is particularly challenging due to theircomplexity. By combining GC-MS with UPLC-TOF-MS, several compound classes can be analyzed simultaneously.
Abstract:
After water, tea（Camellia sinensis）is the most consumed beverage in the world. Of all tea produced, most is consumed as fully
fermented（black）tea. Still, the diversity in between different black teas is enormous, which is on the one hand based on
geographic regions of tea plant cultivation and on the other hand on the procedures used for withering and fermenting of tea
leaves.
Here, we use gas chromatography coupled to mass spectrometry（GC-MS）and liquid chromatography coupled to time-of-flightmass spectrometry（UPLC-TOF-MS）to determine the phytochemical diversity between three common black teas. The teas wereselected on their geographic origins（Yunnan - China, Assam - India or a tea blend）and on differences in the fermentation
processes（orthodox versus CTC）. GC-MS was used in electron ionization mode to identify small phytochemicals such as organic
acids, amino acids, and monosaccharides. UPLC-TOF-MS was used in positive and negative electrospray mode and enabled thedetection of polyphenols such as catechins, chlorogenic acids and flavonol glycosides and other classes of phytochemicals suchas carbohydrates and peptides.
By combining GC-MS with UPLC-TOF-MS, a large range of components was covered, enabling the detailed differentiation between
the three black teas. Both GC and UPLC exhibit impressive chromatographic resolving power required for the separation ofcomplex mixtures such as black tea. Identification was accomplished by comparison with libraries and by making use of accuratemass data.
The data indicate that despite the differences in origin and processing between the black teas, remarkable similarities inphytochemical composition exists. Detailed analysis shows however, that the levels of individual phytochemicals present in black
tea can vary tremendously. Together, the data indicate that the combination of GC-MS and UPLC-TOF-MS forms a strong tool in
differentiating between different black teas.

Plenary Lecture
Thursday, 20th September

Main Hall

08:00 － 08:45

Plenary Lecture 4: Hisayoshi Yurimoto
Chair:

Robert J Cotter（Johns Hopkins University School of Medicine, USA）

PL4-0800
08:00 － 08:45

Science of asteroid sample return mission“HAYABUSA“

Hisayoshi Yurimoto

Hokkaido University, Sapporo, Japan

Keywords:
SIMS, isotope, Asteroid
Novel aspects:
We measured the first asteroidal sample obtained by a planetary exploration HAYABUSA, demonstrating that the most abundantmeteorite species fallen into the Earth come from S-type asteroids.
Abstract:
Of the ~40,000 meteorites we know of, only 14 have had their pre-impact orbits ascertained. The aphelia of these 14 orbits arelocated within the Main Asteroid Belt between Martian and Jovian orbits, which suggests that meteorites are an asteroidal origin.
However, telescopic spectroscopy of asteroidal surfaces had raised serious doubts on the hypothesis. One of the primary scientificpurposes of Hayabusa mission was demonstration of this hypothesis.
The Hayabusa spacecraft made two touchdowns on the surface of asteroid 25143 Itokawa on November 20th and 26th, 2005. After
recoveries from serious accidents on the way home from Itokawa, the spacecraft made the reentry into the terrestrial atmosphereon June 12th, 2010 and the sample capsule was successfully recovered in Australia on June 13th, 2010.
More than 1,500 grains were found in the sample capsule and identified as rocky particles. Although their sizes are mostly less than
10 micrometers, some larger grains of about 100 micrometers or larger were also included. About 50 particles among the larger
grains were subjected to one set of preliminary examinations. The preliminary examinations started from January 21st, 2011. The
examinations include X-ray CT analysis, X-ray diffraction analysis, petrology, mineral chemistry, oxygen isotope analysis, traceelement analysis and noble gas analysis.
In order to determine extraterrestrial origin of the particles in the capsule, isotope analysis of oxygen for individual particles isessential because extraterrestrial materials usually show different isotope ratios from terrestrial materials. However, the degree ofdifference is less than 1 %. A precise analysis technique by SIMS was newly developed and applied.
The SIMS characterization of Itokawa particles certified that Itokawa was composed of similar materials of ordinary chondrites.
This is the first direct link showing that S-type asteroids is one of the sources of the ordinary chondrites, which are the mostabundant meteorite species fallen into the Earth. From this citification, we confirm that insights based on meteorite research are
the right way to study origin of our solar system. New insights for the early solar system are expected by further detailmeasurements of Itokawa particles.

Oral Session
Thursday, 20th September

Main Hall

09:00 － 11:00

Session 31: Native Mass Spectrometry and Structural Biology
Chair:

Satoko Akashi（Yokohama City University, Japan）

S31-0900
09:00 － 09:40[Keynote Lecture] Integrating Native Mass Spectrometry and Top-Down
MS for Defining Protein Interactions Important in Biology and Medicine

Joseph A Loo

University of California-Los Angeles, USA

Keywords:
native MS, top-down MS, ESI
Novel aspects:
Protein binding dynamics and topology can be provided by native mass spectrometry.
Abstract:
The application of mass spectrometry（MS）and ion mobility for studying proteins and protein complexes has utility in structural
biology and biomedical research. The assessment of protein interactions can address the functional role of proteins and protein
complexes. Measuring the molecular masses of protein complexes reveals information on stoichiometry of binding andidentification of interacting partners. MS and ion mobility measurements can supply information on the structural aspects of gasphase/solution-phase protein conformations and supramolecular assemblies in excess of 1 MDa. However, MS can advance anadditional layer of information that is critical to structural biology： location（or topology）and dynamics.
Developments in protein MS and tandem MS（MS/MS, or top-down MS）to define the structures of native protein complexes,
including the sites of ligand bonding, will be discussed. Collisionally activated dissociation（CAD）of gas-phase stable noncovalent
complexes resulting from electrostatic interactions can be used to probe ligand-binding sites（e.g., nucleotides, metals）. Electrontransfer dissociation（ETD）and electron capture dissociation（ECD）can probe the binding sites of weakly bound ligands and the
topology of protein-protein complexes（e.g., hemoglobin）. We are using ECD-Fourier transform ion cyclotron resonance（FTICR）
MS to investigate the molecular action of compounds that prevent amyloid fibril formation in neurodegenerative diseases such as
Alzheimer’s and Parkinson’s disease.
Native proteins and complexes are not necessarily static entities, as they are in constant motion and perhaps changing theirpartners during the“dance“ that defines their roles in biological processes. We are using native MS and top-down MS to
decipher the dynamics of protein interactions that govern iron-capture by pathogens. Bacteria need iron from the host to establish
infection. The blood infection from Staphylococcus aureus affects a large fraction of the human population and its pathologicalmechanism has not been fully elucidated. It has been postulated that a receptor on the surface of S. aureus, IsdH, is involved in
the capture of heme from hemoglobin（Hb）and this is the source of iron required for infection. Native ESI-MS and MS/MS areused to study the dynamics of noncovalent interactions between Hb and the IsdH protein receptor. The complex processes ofsite-specific protein recognition and heme transfer, including the kinetics of multiple binding equilibria, can be monitored by ESIMS to provide important clues to the mechanism of S. aureus infection. This information could be useful for designing newcompounds and strategies for fighting and preventing potentially lethal S. aureus infections. Moreover, it demonstrates the abilityof native ESI-MS for providing a view of the dynamics of protein-ligand assembly-disassembly that would be difficult to measure byother biophysical methods.
Acknowledgments:Support from the US National Institutes of Health（R01 RR20004, S10 RR023045, S10 RR028893）is acknowledged.

Oral Session
Thursday, 20th September

Main Hall

09:00 － 11:00

Session 31: Native Mass Spectrometry and Structural Biology
Chair:

Satoko Akashi（Yokohama City University, Japan）

S31-0940
09:40 － 10:00

Ion mobility-mass spectrometry for the study of conformational space andtopology of proteins and protein-DNA complexesFrank Sobott

Antwerp University, Antwerp/Belgium

Keywords:
ion mobility, native mass spectrometry, protein conformation, protein complexes, protein-DNA complexes
Novel aspects:
Use of native MS and ion mobility to determine protein conformational space and topology of protein-protein and protein-DNA
complexes
Abstract:
While established techniques in structural biology such as x-ray crystallography can reveal biomolecular structure in unsurpassed
detail, they only highlight certain aspects of the different conformational and association states of a protein. Other methods suchas NMR are either not well suited to large, dynamic structures, or they lack the detail necessary（e.g. EM）to resolve individual
species in heterogeneous conformational or association equilibriae.
Native MS as a’single molecule technique’can cope well with such heterogeneity, and in combination with ion mobility
spectrometry, different conformational states can now also be resolved reasonably well. But what does a measured collision crosssection really tell us about the conformational space of a protein or the subunit arrangement of a complex? How important is theabsence of bulk solvent, and how much do the measured size and the flexibility depend on the internal energy（temperature）of
the ions?
We are using native nano-ESI MS coupled with T-wave ion mobility spectrometry（Waters Synapt G 1 and G 2 ）to assess the
structure and conformational space of a couple of well understood proteins and complexes, and correlate this data with resultsfrom other biophysical methods such as size-exclusion chromatography, surface plasmon resonance and small-angle x-ray andneutron scattering（SAXS and SANS）. In side-by-side comparison of closely related protein sequences（e.g. with and without
mutations, truncations or post-translational modifications）, such subtle changes are frequently reflected in the position and widthof the drift time profile（i.e. ’size’ and ’flexibility’ of the ion）. We will correlate these data with candidate structures of the proteinsand complexes.
The binding of ligands or counter-ions however can cause a considerable compaction of the gas-phase structure, as will bedemonstrated with an example. For multiprotein complexes it is an often reported phenomenon that a systematic variation of
instrumental parameters which affect the internal energy of the ions（such as voltage offsets and background gas pressures）leads
to an intial gas-phase collapse of hollow structures such as in the hexameric ring discussed here before further increase of theinternal energy leads to extended, but still oligomeric conformations of the complex. In this way, we can for example determine
the threshold of unfolding or the 50 % unfolding point as an indirect measure of the gas-phase stability of native biomolecular
structures.
Taken together these data highlight the importance of controlling the internal energy of ions carefully in the experiment, and raisesome critical points regarding the measurement of absolute（rather than relative）sizes by IM-MS. They also demonstrate the levelof information we can extract from careful, systematic measurements of collision cross sections of ions of biochemical andpharmaceutical interest.

Oral Session
Thursday, 20th September

Main Hall

09:00 － 11:00

Session 31: Native Mass Spectrometry and Structural Biology
Chair:

Satoko Akashi（Yokohama City University, Japan）

S31-1000
10:00 － 10:20

Structure of a Beta-Crystallin Heterodimer by Ion Mobility and Radical
Probe Mass Spectrometry

Kevin Downard1, Yuichi Kokabu2, Mitsunori Ikeguchi2, Satoko Akashi2
1

University of Sydney, 2Yokohama City Unversity

Keywords:
crystallin, protein complex, ion mobility, radical probe, homology modeling
Novel aspects:
First application of protein footprinting by RP-MS in concert with ion mobility mass spectrometry and homology modeling tocharacterize a protein complex
Abstract:
Beta-crystallins constitute the major proteins of the lens of the eye in mammals. Their association with one another and othercrystallins is critical for lens clarity and vision. The dimerization of beta-crystallins is first step in the formation of many higher ordercomplexes, yet remains poorly understood at the molecular level. We report on the application of Ion Mobility and Radical Probe
Mass Spectrometry, in conjunction with homology modeling, to characterize the structure of the βB 2 B 3 -heterodimer.
NanoESI-TOF and IM-MS were performed on a Waters SYNAPT-G 2 mass spectrometer to identify protein dimers in a solution ofbeta-crystallin. The collision cross sections（CCS）of the B 2 monomer, and B 2 B 2 and B 2 B 3 dimers were measured and foundto be similar in value at 3121Å 2 and 3165Å 2 respectively. The exclusive presence of beta-B 3 -crystallin within the B 2 B 3 heterodimer prompted its analysis by RP-MS. The level of oxidative modification at amino acid residue side chains across the B 2
and B 3 crystallin proteins was established by LC-MS. This data established a solvent accessibility profile of B 3 in complex with
B2. Oxidation levels ranging from 0 -100 % were observed across the reactive residues of the βB 3 subunit. Several homologymodelled structures for the heterodimer were then constructed and assessed in terms of their calculated CCS and whether thesolvent accessibilities were in accord with measured oxidation levels. Two homodimeric structures derived from the βB 2 tetramer structure（PDB 2 BB 2 ）are possible containing two of the βB 2 subunits along the PQ plane in closest proximity（AB）
and along the RQ plane（AD）. Due to high sequence identity among the B 2 and B 3 subunits, these structures were used tomodel both a AB and AD βB 2 B 3 -heterodimer. The calculated CCS’s for the lowest energy structures of the βB 2 B 3 AB and AD
heterodimers had average values of 2888 and 3140 Å 2 respectively. Two additional homology models were constructed. One“half“model used two N and two C-terminal domains extracted from four separate chains（A-D）positioned side-by-side in one half ofthe βB 2 -tetramer. The connecting loops were generated from the structure of the βB 3 - homodimer structure extracted from thehuman βB 3 -hexamer（PDB 3 QK 3 ）. In the final“twisted“ model, a βB 2 monomer（A chain）extracted from the tetramer was
coupled to the N-terminal domain extracted from chain B and C-terminal domain from chain D. These two βB 2 domains wereconnected by elongation of the linker region and the resultant subunit replaced with the βB 3 subunit. The homology modelled
structure for the AD βB 2 B 3 -heterodimer was found to be most consistent with the IM-MS and RP-MS protein footprinting data.

Oral Session
Thursday, 20th September

Main Hall

09:00 － 11:00

Session 31: Native Mass Spectrometry and Structural Biology
Chair:

Satoko Akashi（Yokohama City University, Japan）

S31-1020
10:20 － 10:40

Application of Backbone Amide Hydrogen/Deuterium Exchange-Mass
Spectrometry (HDX-MS) to Characterize Protein-Ligand Interactions andProtein-Protein Interactions

Yoshitomo Hamuro

ExSAR, Monmouth Junction, NJ, USA

Keywords:
hydrogen/deuterium exchange, protein-ligand interactions, epitope
Novel aspects:
Backbone amide hydrogen/deuterium exchange-mass spectrometry（HDX-MS）was used to investigate kinase-ligand interactions
and to map conformational epitopes of antigen-antibody interactions
Abstract:
This presentation will discuss the application of backbone amide hydrogen/deuterium exchange coupled with proteolysis, liquid
chromatography, and mass spectrometry（HDX-MS）to investigate protein-ligand interactions and protein-protein interactions at
the sub-molecular level. HDX-MS is an increasingly popular, widely-applicable, medium-resolution, and medium-throughput
technology for protein dynamic characterization. The presentation consists of three parts： introduction to HDX-MS, theapplication of HDX-MS for protein-ligand interactions, and the application of HDX-MS for protein-protein interactions.
The first part explains the physicochemical background of HDX-MS methodology and an overview of how the method works. In
the HDX-MS work flow, an analyte protein is first mixed with deuterated buffer and the reaction mixture is incubated for apredetermined duration. Next, the addition of acidic buffer quenches the exchange reaction and then exchanged and quenched
protein is flowed over a protease column. The peptic fragments will be desalted and separated by a reverse phase column.
Finally, mass analysis detects the change in mass upon the exchange reaction. When a protein-protein interaction is studied by
HDX-MS, at least two sets of experiments are carried out： the HDX-MS of the unbound protein and the HDX-MS of the protein incomplex with its binding partner. The HDX-MS behavior of the protein in the absence or presence of its binding partner can
describe the dynamic properties of the protein in each condition at the sub-molecular level. The difference of the HDX-MS
patterns can shed light on how the protein dynamics change upon complex formation.
The second part discusses HDX-MS of p38 MAP kinase, a validated cancer target, in the presence and absence of various ligands.
HDX-MS results showed that all tested ligands interact with two regions of the kinase. The two regions are approximately thirty
residues in total and cover both the ATP binding site and the DFG region. The presence of ATP analogs protected HDX reactionsof the ATP binding site without perturbing the HDX reactions near the DFG region, while a known DFG binder retarded the HDX
reactions of only the DFG region without interfere with HDX reactions at the ATP binding site. Moreover, a chemical conjugate ofan ATP binder and the DFG binder protected HDX reactions at both the ATP binding site and the DFG region. The results
exemplify the potential of fragment-based drug discovery by HDX-MS.
The third part illustrates how HDX-MS can be used for protein-protein interactions, particularly for epitope mapping of various
antigen-antibody interactions. HDX-MS successfully mapped challenging discontinuous conformational epitopes. Understanding
antigen-antibody interactions at the sub-molecular level is of particular interest for scientific, regulatory and intellectual property
reasons, especially with increasing demand for monoclonal antibody therapeutic agents. While various techniques are available
for the determination of an epitope, there is no widely-applicable and reliable method available, especially for discontinuous
conformational epitopes. HDX-MS was used to map the epitopes of cytochrome c：E8, IL-13：CNTO607, and IL-17A：CAT-2200
interactions. All three epitopes are discontinuous conformational epitopes and the epitopes identified by HDX-MS are in good
agreement with those identified using high-resolution X-ray crystallography. Other epitope mappings for antigens with therapeuticvalue are also discussed.

Oral Session
Thursday, 20th September

Main Hall

09:00 － 11:00

Session 31: Native Mass Spectrometry and Structural Biology
Chair:

Satoko Akashi（Yokohama City University, Japan）

S31-1040
10:40 － 11:00

An integrated workflow for structural mass spectrometry: combining Xlinking technology with ionmobility for the structural characterisation ofmacromolecular assemblies

Florian Stengel1, Argyris Politis2, Zoe Hall2, Eri Sakata3, Helena Hernandez2, Alexander Leitner1,
Thomas Walzthoeni1, Carol V Robinson2, Ruedi Aebersold1,4

Department of Biology, Institute of Molecular Systems Biology, ETH Zurich, 2Department of Chemistry, University of
Oxford, Chemistry Research Laboratory, Mansfield Road, Oxford, UK, 3Laboratory of Protein Metabolism, Tokyo
Metropolitan Institute of Medical Science, Setagaya-ku, Tokyo, 156-8506, Japan, 4Faculty of Science, University of
Zurich, Switzerland
1

Keywords:
Structural Proteomics Ionmobility Cross Linking
Novel aspects:
Novel method for studying protein complexes that combines cross linking, ionmobility and native mass spectrometry withmodelling approaches in a single workflow
Abstract:
Classical methods for the determination of the structure of protein complexes are increasingly complemented by emerging
experimental techniques. This is especially true if no high-resolution structure can be attained due to the transient nature of themacromolecular assembly or in cases where no pure purification of sufficiently high concentration for classical structural methods
can be achieved.
Here emerging methods in structural mass spectrometry（MS）can pose an exciting alternative, due to their low sample
requirements and comparatively high measuring speed.
MS of intact assemblies is able to assess the absolute stoichiometry of the intact complex and intermediate sub complexes.
Chemical cross-linking coupled with mass spectrometry（CXMS）on the other hand is capable of providing multiple high resolution
interactions for a single protein-protein interaction and at peptide resolution. Additionally both ion mobility mass spectrometry（IMMS）and chemical cross-linking coupled with mass spectrometry（CXMS）provide information on shape and topology.
Combining the different datasets can generate complete interaction maps for subsequent merging with topological information
gained from IM-MS and CXMS. Using those synergies can help to understand the stoichiometry and complete subunit interaction
maps of intact complexes and when combined with computational tools, even generate 3 dimensional ab initio models.
Here, we incorporate for the first time MS of intact assemblies, IMMS and CXMS datasets with modelling approaches in one single
workflow to generate restraints for subsequent structure determination of multi protein complexes. We trained our approach ona set of protein complexes with known high resolution structures before validating it on a protein assembly of high biologicalinterest, the proteasome from Saccharomyces cerevisiae, for which still no atomic-resolution structure does exist.
Our approach presented here converts IMMS measurements into global restraints and combines them with additional local
restraints imposed by CX-MS and the MS of intact complexes. Candidate models are then selected by a search for good scoring
configurations that satisfy all restraints.
Such a hybrid approach can not only generate more complete interaction maps of investigated complexes and provide new
insights into the structure of transient complex macromolecular machines. It should also greatly enhance the interpretation of EM
densities and may become a generic pairing in its own right for high throughput structural biology.
The PPROSPECTS GrantHEALTH-F 4 -2008-201648 under the EU 7 th Framework program and the Wellcome Trust（Sir Henry
Wellcome Fellowship）are gratefully acknowledged for funding.

Oral Session
Thursday, 20th SeptemberRoom A

09:00 － 11:00

Session 32: Formation and Dissociation of Peptide Radical Ions
Chair:

Dominic T W Chan（The Chinese University of Hong Kong, Hong Kong SAR）

S32-0900
09:00 － 09:40[Keynote Lecture] Formation and Dissociation of Peptide Radical Ions

Roman A Zubarev

Karolinska Institutet, Stockholm, Sweden

Keywords:
hydrogen-abundant radicals, nonergodic fragmentation, peptide sequencing, fragment stability, posttranslational modifications.
Novel aspects:
An overview of the recent efforts to decipher the ECD/ETD mechanism, and an outlook for new fragmentation mechods
Abstract:
Electron capture dissociation（ECD）and electron transfer dissociation（ETD）are sister fragmentation techniques where multiply
protonated molecular species capture an electron（which is free, as in ECD, or bound to an anion, as in ETD）and form unstablehydrogen-abundant radical cations. After 15 years of mechanistic studies, many questions remain unanswered. An overview will begiven on the status of mechanistic investigations in ECD/ETD, with emphasis on the following questions：
1. Site of the electron attachment and its effect on the fragmentation pattern. Which process dominates： direct charge
recombination or indirect process（capture, backbone cleavage, charge recombination）?
2. What exactly is the role of fragment stability in determining the fragment abundances?
3. What is the role of secondary structure, charged and neutral hydrogen bondings in determining the fragment abundances?
4. What role does internal energy of the precursor ion play in ECD cross section?
5. What is the role of the kinetic energy of electrons in determining the fragment abundances? What determines the branchingratio between electron capture, electronic excitation and ionization?
6. What is the N-Ca fragmentation time scale upon electroncapture?
7. What is the origin of less conventional backbonecleavages, e.g. leading to radical a-ions?

Oral Session
Thursday, 20th SeptemberRoom A

09:00 － 11:00

Session 32: Formation and Dissociation of Peptide Radical Ions
Chair:

Dominic T W Chan（The Chinese University of Hong Kong, Hong Kong SAR）

S32-0940
09:40 － 10:00

Ab initio MO Study on the Fragmentation Mechanisms of ProtonatedPhosphopeptides in“On-Resonance“ and“Off-Resonance“ Pulsed Gas
Introduction Collision-Induced Dissociations

Takae Takeuchi1,2, Ayaka Takahashi1, Erika Sugawara1, Tomoko Kimura1, Yuka Kurosaki1,
Shigeki Kajihara3, Hiroko Morinaga4, Shinichi Iwamoto3, Koichi Tanaka3

Department of Chemistry, Faculty of Science, Nara Women’s University, Nara, Japan, 2National Institute of Advanced
Industrial Science and Technology (AIST), Ikeda, Osaka, Japan, 3Koichi Tanaka Laboratory of Advanced Science andTechnology, Shimadzu Corporation, Kyoto, Japan, 4Technical Research Laboratory, Shimadzu Corporation, Kyoto, Japan
1

Keywords:“On-Resonance“ Pulsed Gas Introduction CID, Fragmentation Mechanism, Ab initio Calculation, Phosphopeptide, MALDI
Novel aspects:
A new strategy of predicting fragmentation processes of protonated phosphopeptides was developed based on ab initio MO
calculations.
Abstract:
Introduction
The occurrence of facile neutral losses of H3PO4 and/or HPO3 from protonated phosphopeptides makes it difficult to determine
both the site of phosphorylation and the amino acid sequence. In ECD and ETD, fragments retain post-translational modificationsuch as phosphorylation［ 1 - 3 ］, while a conventional low energy CAD/CID of phosphopeptides typically leads to dissociation ofa phosphorylation modification during fragmentation. However, the“On-Resonance“ CID method was developed mainly to
reduce neutral loss and to improve sequence coverage［ 4 ］. In this study, the fragmentation of two different lengths ofprotonated phosphopeptides（FQpSEEQQQTEDELQDK and pYYRK）in both“On-Resonance“ and“Off-Resonance“ pulsed gas
introduction CID were investigated using ab initio MO calculations.
Methods
Optimized geometries and energies of all possible conformers of proton adducts of phosphoptides of FQpSEEQQQTEDELQDKand pYYRK, reaction intermediates, transition states and fragments were calculated using ab initio MO methods.“On-Resonance“
and“Off-Resonance“ CID of the protonated phosphopeptides（FQpSEEQQQTEDELQDK and pYYRK）were observed using MALDI
Quadrupole Ion Trap Time-of-Flight Mass Spectrometer（AXIMA-Resonance, Shimadzu/Kratos, UK）. Protonated pYYRK was
produced using the fragmentation of Insulin receptor（1142-1153）TRDIYETDpYYRK.
Preliminary Data
In“On-Resonance“ pulsed gas introduction CID of protonated FQpSEEQQQTEDELQDK, the ion intensity ratios of［b15-HPO3］/
b 15,［y 14-HPO 3］/y 14 and［y 14-H 3PO 4］/y 14 were much smaller than those in“Off-Resonance“ pulsed gas introduction CID. Thissuggests that the kinetic energy of the precursor ions in“On-Resonance“ CID increased more rapidly compared with“OffResonance“ CID. As a result, the precursor ions excited by“On-Resonance“ CID promptly received enough energy to cleave thepeptide bond（amide bond）. On the other hand, in“Off-Resonance“ CID , vibrational excitation of the precursor ions occurred
gradually through multiple collisions. In this case the excitation energy was less than the energy barrier of an amide bondcleavage.
The b 2  , b 3 , y 2 , a 2 and a 3 ions,［b 3 -HPO 3 ］+ ,［pYYR（H + ）K-HPO 3 ］+ and［pYYR（H + ）K-98］+ appeared in the MS/MS spectrum ofProtonated pYYRK   using“Off-Resonance“ pulsed gas introduction CID. On the other hand, in“On-Resonance“ pulsed gas
introduction CID, the［b3-HPO3］+ peak at m/z 483 became much smaller or even unobservable. The b2, y2, a2 and a3 peaks also
became much smaller in“On-Resonance“ pulsed gas introduction CID.
In order to elucidate the fragmentation mechanism of protonated pYYRK in“On/Off-Resonance“ CID, potential energy surfaces
for their possible fragmentation processes were calculated at the B 3 LYP/ 6 -31+G（d, p）//B 3 LYP/ 6 -31G（d）level［ 5 ］. From
the results of ab initio calculations, it was found that pYYR（H+）K is the most stable isomer of protonated pYYRK and that the（m/z 581）has the lowest energy barrier. An elimination of K from pYYR（H+）K accompanied withproduction of pYYR（H+）（OH）
a six-membered ring formation in the R side chain results in the production of the b3 ion（pYYR（H+））（m/z 563）.
The larger peptide has the greater number of vibrational and rotational modes than the smaller peptide. Therefore, it takes moretime for the larger peptide to gain enough energy for the neutral loss of HPO3 or H3PO4.
References［ 1 ］Creese & Cooper, JASMS 19：1263-1274, 2008.
［ 2 ］Shi et al., Anal. Chem., 73：19-22, 2001.

［ 3 ］Woodling et al., JASMS 18：2137-2145, 2007.
［ 4 ］Takana et al., 58th Annual Conference of MSSJ, 2010.
［ 5 ］Takeuchi et al., 59th Annual Conference of MSSJ, 2011； Takeuchi et al., 60th ASMS Conference, 2012.

Oral Session
Thursday, 20th SeptemberRoom A

09:00 － 11:00

Session 32: Formation and Dissociation of Peptide Radical Ions
Chair:

Dominic T W Chan（The Chinese University of Hong Kong, Hong Kong SAR）

S32-1000
10:00 － 10:20

Electron transfer dissociation of protonated disulfide linked peptides andanalogs without S-S bondShigeo Hayakawa1, Shinya Matsumoto1, Michisato Toyoda2, Yasushi Shigeri3, Michiko Tajiri4,
Yoshinao Wada4

1
Osaka Prefecture Uiniversity,Sakai,Japan, 2Osaka University, Toyonaka, Japan, 3AIST, Ikeda, Japan, 4Osaka MCHRI,
Izumi, Japan

Keywords:
High energy electron transfer dissociation（HE-ETD）, Disulfide linked peptide, N-Ca backbone cleavage, Peptide sequence.
Novel aspects:
HE-ETD using alkali metal targets provides the sequence of disulfide linked peptides regardless of the ring structure by S-S bond.
Abstract:
Introduction: Disulfide linkages in polypeptides are post-translational modifications that play a pivotal role in maintaining higher
order structure and biological functions. Preferential cleavage of the disulfide bond has been reported by electron capture
dissociation（ECD）and low-energy electron transfer dissociation（LE-ETD）. The determination of the position of phosphorylation
and amino acid sequence of phosphopeptides by high-energy electron transfer dissociation（HE-ETD）using an alkali metal target
has been reported. In the present work, HE-ETD using K and Cs targets was applied to doubly protonated disulfide linked
polypeptides. The difference of the peptides with S-S bond and without S-S bond is presented, and the comparison between the
spectra with HE-ETD and LE-ETD is also discussed.
Method: High energy ETD spectra were measure using a sector type tandem mass spectrometer（MS/MS）. Doubly protonatedpeptide ions were generated by an electrospray ionization source. The doubly-protonated ions were accelerated to a kinetic
energy of 10 keV by applying an accelerating voltage of 5 kV. Mono-isotopic ions were mass selected by a JEOL JMS-HX110
double focusing mass spectrometer（MS-I）. The mass-selected precursor ions were led to a 3.7 cm long collision cell in whichalkali metal vapor were filled. The singly- and doubly- charge positive ions were mass-analyzed by a spherical electrostatic
analyzer of a central radius of 216 mm（MS-II）. Low-energy ETD spectra were measured by Finnigan LTQ XL（ThermoFisher
Scientific）using fluoranthene anion. Vasopressin（1）, vasotocin（2）, and oxytocin（3）were used as received.［Ala 1,6 ］-vasopressin（4）in which cysteine residues were substituted by alanine residues was synthesized by use of a Shimadzu PSSM- 8
automated peptide synthesizer.
Results and Discussion： The HE-ETD and LE-ETD spectra of the doubly-protonated peptides（≡［M+ 2 H］2+）of 1, 2, 3, and 4 were
measured with K and Cs targets. HE-ETD spectra of［Ala 1,6 ］-vasopressin（4）which has not the SS ring structure, provided allz-type ions due to NCa bond cleavage as main fragments except z 3 whose position corresponds to the proline residue. Charge
reduced ions with loss of side chains, immonium ions, and some b- and y-type ions were also observed. LE-ETD spectrum also
provided similar fragment ions to the HE-ETD spectrum except lack of z- 1 and the immonium ions. In HE-ETD spectrum ofvasopressin（1）, all the z-type ions due to both NCa and SS or CS bond cleavage except z 3 were observed regardless of theexistence of the ring structure by SS bond. Most of the y-type ions were also observed in the spectrum. HE-ETD spectrum ofvasotocin（2）provided all the y-type ions. While HE-ETD spectra of oxytocin（3）did not provide all y-type ions clearly, all b-typeions were observed. This difference of the observed type of the ions can be attributed to the existence of arginine residue in 1
and 2, and lack of the arginine residue in 3, as the arginine residue can keep positively charge in its side chain. The dependence
on the target alkali metal between Cs and K did not provide the observed peaks but slight difference only for relative intensity ofthe observed fragments. These observed peaks in these spectra indicated that HE-ETD using an alkali metal target can provide thecomplete peptide sequence of the disulfide linked peptides. In the HE-ETD spectrum of 1 and 2, z 4 -S ion formed by N-Ca and C-Sbond cleavage was much larger than z 4 ion which was formed by N-Ca and SS bond cleavage. Since its position correspond to
next of the cysteine residue, it is presumed that hydrogen radical induced the C-S bond cleavage preferentially.

Oral Session
Thursday, 20th SeptemberRoom A

09:00 － 11:00

Session 32: Formation and Dissociation of Peptide Radical Ions
Chair:

Dominic T W Chan（The Chinese University of Hong Kong, Hong Kong SAR）

S32-1020
10:20 － 10:40

Ionization energy of gas phase proteins and its dependence on charge
state and structure

Alexandre Giuliani1, Alexksandar R Milosavljevic2, Konrad Hinsen1, Francis Canon1, Laurent Nahon1,
Matthieu Refregiers1

1
Synchrotron SOLEIL, Gif-sur-Yvette, France / INRA, CEPIA, Nantes, France, 2University of Belgrade, Institute of Physics,
Belgrade, Serbia

Keywords:
photon activation, tandem mass spectrometry, gas phase structure, EID, photoionization
Novel aspects:
We report for the first time the relationship between the ionization energy of a protein and its charge state and structure.
Abstract:
Mass spectrometry（MS）offers the unique ability to manipulate ions in the gas phase. In particular, the potential of mass
spectrometry to perform mass-selected spectroscopy on biomolecules isolated in the gas phase is very appreciable. First,
electrospray ionization is a unique method to bring into the gas phase large and fragile biological species intact. Furthermore, MS
is the only technique to provide additional control on the charge state of the targeted molecule. Indeed, Hirsch et al.［ 1 ］have
reported spectroscopic studies on mass selected clusters of ions, while Thissen et al.［ 2 ］took profit from the storage of the ionsto produce relaxed targets in their electronic ground states We report on a systemic study of the dependence of the ionizationenergy as a function of the protein charge states for cytochrome C, bovine pancreatic trypsin inhibitor（BPTI）and ubiquitin.
A linear ion trap（Thermo Scientific LTQ XL）has been coupled to an ultra-violet beam line at the SOLEIL synchrotron radiation
facility［ 3 ］.Water acetonitrile solutions of 5 µM cytochrome C, BPTI and ubiquitin were electrosprayed at 3 µL/min. Each charge
state has been selected and submitted to 100 ms irradiation between 8 and 16 eV photon energy. At each photon energy, theabundance of the photoionization product has been measured and normalized to the total ionic current and to the photon flux.
From these ion yields, the ionization energy threshold has been determined.
The ionization energies for cytochrome C, BPTI and ubiquitin have been measured for charge states ranging from 4 to 15, 4 to 8
and 4 to 9, respectively. For BPTI a linear dependency of the ionization energy with the charge state is observed. This result is inline with the outcome of previous work, in which the ionization energy of peptide smaller than 3.5 kDa had been measured byelectron impact［ 4 ］. The linear dependence of the ionization energies with the charge state was interpreted as the result of theincrease of the attractive Coulomb potential of the ion. For ubiquitin and cytochrome C, this model is not adequate anymore,since the dependency ceased to be monotonic and he IEs appeared to plateau for particular range of charge states. The
correspondence between these features in the IE vs charge state appear similar to those observed in the collision cross section
obtained from ion mobility experiments.
Our molecular dynamic simulation supports this interpretation. The ionization energy of protein is thus dependent on the gas
phase conformation. We propose a model which links the ionization energy, the charge state and the radius of gyration of theprotein.
［ 1 ］K. Hirsch et al. J. Phys. B 42,154029（2009）［ 2 ］R.Thissen et al. Phys. Rev. Lett. 100,223001（2008）［ 3 ］A.R. Milosavljevic et al. J. Synchrotron Rad., 19, 174（2012）.
［ 4 ］B.A. Budnik et al. J Mass Spectrom 37, 1141（2002）.

Oral Session
Thursday, 20th SeptemberRoom A

09:00 － 11:00

Session 32: Formation and Dissociation of Peptide Radical Ions
Chair:

Dominic T W Chan（The Chinese University of Hong Kong, Hong Kong SAR）

S32-1040
10:40 － 11:00

Direct ECD and CID sequencing intra disulfide C-terminal loops of nontryptic natural peptides

Tatiana Y Samgina, Yegor A Vorontsov, Konstantin V Karandashev, Albert T Lebedev
M.V.Lomonosov Moscow State University

Keywords:
natural peptides, disulfide cycle, CID, ECD, direct sequencing
Novel aspects:
New fragmentation pathways of protonated molecules of natural peptides allowing for the elucidation of the sequence inside
C-terminal disulfide cycle were observed in CID and ECD modes without preliminary derivatization
Abstract:
Usually mass spectrometric sequencing of peptides with an intra molecular disulfide cycle involves preliminary modification of S-S
bond. The proposed earlier methods of direct sequencing of such peptides have some restrictions dealing with length andstructure as well as position of S-S bond in the backbone of the molecules. The present study demonstrates ability of CID and
ECD to carry on sequencing inside disulfide C-terminal loops of non-tryptic natural peptides from the skin secretion of ranid frogs
without any derivatization.
The skin secretions of Rana temporaria, Rana ridibunda, Rana esculenta and Rana arvalis were obtained by mild electric
stimulation. The skin secretion was filtrated（PTF filter, 0,45μ ）, concentrated, liophylized, and analyzed with ESI LC-MS/MS.
NanoHPLC-FTICR-MS system（Thermo LTQ-FT Ultra with Agilent 1100 equipped with homepacked nanocolumn）was used torecord CID and ECD spectra.
CID and ECD mass spectra of 11 non-tryptic peptides containing from 15 to 35 amino acids, with C-terminal cysteine cycle were
processed manually. ECD spectra of all these peptides demonstrate a peak of an ion formed due to the loss of a sulfur atom fromthe protonated molecules. Another group of ions represents N-C α cleavages inside S-S cycle. C-terminal fragmentation issignificantly enhanced when one or better two C-terminal Lys are present. In this case 5 outof 6 possible cleavages inside
7 -member“Rana box“ cycle take place. An increase of the precursor ion charge state does not influence on c-z-ion series,
although favors formation of y and b ions, i.e. promoting additional fragmentation pathways. Sequence coverage in ECD spectra isquite high, reaching 80 % for brevinin 1 and 88 % for brevinin 1 Vb. It is worth mentioning that the cleavage at N-terminus of
C-terminal Cys was not detected for any of the studied peptides.
Despite of great number of publications on ESI CID fragmentation of polypeptides a fragmentation pathway inside C-terminal
cycle of natural peptides not reported earlier was observed in the present work. The exact mass of the neutral loss in conditionsof low energy CID corresponds to the cleavage of amide bond X-Cys-OH, where X is the second amino acid residue from the
C-terminus. The ions peaks due to the cleavages of amide bonds at the C-terminus are present in CID spectra. This series of ionsbreaks with the elimination of S-sulfanylcystein（- 152.92 amu）and formation of dehydroalanine moiety（69.02 amu）at the
C-terminus of the corresponding fragment ion. Thus amide bond cleavage inside the cycle rather than S-S bond cleavage takes
place in conditions of ESI CID. For the Pro-containing peptides similar opening of the cycle is observed as part of secondary
fragmentation processes involving primary ions forming due to the cleavage at the N-terminus of Pro. This type of fragmentation
characterizes 9 peptides out of 11 studied. This direction is not a dominant one being strongly dependent on the composition of
C-terminal cycle and charge state of the molecular ion. Thus the presence of basic Lys（better 2 Lys）in the loop favors thispathway significantly. For three natural peptides with 17 amino acid residues CID spectra allowed their complete sequencing.
The observed feature of ECD and CID spectra dealing with C-terminal disulfide cycles gives a chance to increase efficiency of
direct sequencing of natural peptides of this type.

Oral Session
Thursday, 20th September
09:00 － 11:00

Session 33: JMS Award Symposium
Chair:

Richard M Caprioli（Vanderbilt University,Nashville,TN,USA / Editor-in-Chief, Journal of Mass Spectrometry）

S33-0900
09:00 － 09:20

JMS Award -Overview and Award Ceremony-

Richard M Caprioli1, 2
1Vanderbilt University, Nashville, TN, USA, 2Editor-in-Chief, Journal of Mass Spectrometry

Room B-1

Oral Session
Thursday, 20th SeptemberRoom B-1

09:00 － 11:00

Session 33: JMS Award Symposium
Chair:

Richard M Caprioli（Vanderbilt University,Nashville,TN,USA / Editor-in-Chief, Journal of Mass Spectrometry）

S33-0920
09:20 － 09:40

Integral Membrane Proteins by High-mass MALDI-MS: Direct Access to the
Stoichiometry of their Complexes and to Posttranslational Modifications

FAN CHEN1, Sabina Gerber2, Katrin Heuser2, Volodymyr Kokhov2, Christian Lizak2, Kaspar Locher2,
Renato Zenobi1

1
Department of Chemistry and Applied Biosciences, ETH Zurich, Switzerland, 2Institute of Molecular Biology andBiophysics, ETH Zurich, Swizterland

Keywords:
Integral Membrane Proteins（Complexes）, MALDI
Novel aspects:
High-mass MALDI-MS provides a robust, detergent-friendly and straightforward access to integral membrane proteins, including
the stoichiometry and posttranslational modifications, with high mass accuracy.
Abstract:
A precise understanding of the chemistry of integral membrane proteins is important for optimizing crystallization conditions andfor subsequent structure determination. Mass spectrometry（MS）is a powerful and versatile tool that provides deep insight into
the state of integral membrane proteins, e.g., with respect to posttranslational modification. There are, however, only very few
examples where electrospray ionization（ESI）- after quite a lot of efforts for optimizing buffer and instrument conditions - ormatrix-assisted laser desorption/ionization（MALDI）-MS have been used to study integral membrane proteins or their complexes.
In all of these cases, the experimental conditions were highly specific and could not be easily transferred to other membraneproteins. In other words, it is still challenging for soft ionization MS to analyze integral membrane proteins and their complexes.

In this work, we succeeded in using the traditional dried droplet method, sinapinic acid as the matrix, and a commercial high-mass
detector（HM2, CovalX）retrofitted to our MALDI-MS instrument to explore integral membrane proteins in detergent micelles
directly. Oligosaccharyltransferase（PglB）was measured with a mass error around 0.1 % . By protecting the membrane protein
complexes via chemical cross-linking with glutaraldehyde, we unambiguously determined the subunit stoichiometry of a series ofATP-binding cassette（ABC）transporter complexes, the homomeric Campylobacter jejuni-encoded ABC transporter PglK, and theheteromeric vitamin B12 importers BtuCD and BtuCDF. Moreover, the precise molecular weight determined by high-mass MALDIMS allowed determining the site of N-linked glycosylation in Candida aibicans drug resistance protein 1 （Cdr 1 p）, which assistsin circumventing problems due to heterogeneous glycosylation in future crystallization studies. All the above information would
not be accessible at all with SDS-PAGE or gel filtration chromatography, the commonly used tools in laboratories studying
membrane proteins.

Oral Session
Thursday, 20th SeptemberRoom B-1

09:00 － 11:00

Session 33: JMS Award Symposium
Chair:

Richard M Caprioli（Vanderbilt University,Nashville,TN,USA / Editor-in-Chief, Journal of Mass Spectrometry）

S33-0940
09:40 － 10:00

Great Insights from a Small System: Structure and reactivity of [VPO4]+ incomparison with [V2O4]+.

Nicolas Dietl, Maria Schlangen, Helmut Schwarz
Technical University Berlin, Berlin, Germany

Keywords:
oxo-clusters, DFT-calculations, IRPD-spectroscopy, gas-phase reactions
Novel aspects:
Improved mechanistic understanding of structure-reactivity relationships in thermal reactions of oxo-clusters with smallhydrocarbons in the gas phase.
Abstract:
The selective oxidation of hydrocarbons still constitutes one of the major challenges in contemporary chemistry to solve global
problems, such as the environmentally benign and economically feasible conversion of natural gas into value-added products.[1]Today, there exist numerous effective homogeneous and heterogeneous catalysts which cover a broad spectrum of oxidation
reactivity； however, it is not exaggerated to note the lack of substantiated knowledge about the intrinsic properties of many of
the catalysts which, after all, control the selectivity of the various oxidation processes. A well-known example for such a poorly
understood catalytic process represents the chemical transformation of n-butane to maleic anhydride by the so-called VPOcatalysts； this highly complex and selective reaction involves the abstraction of eight hydrogen atoms from, as well as the transferof three oxygen atoms to C4H10. One approach in the elucidation of mechanistic aspects at a molecular level employs reactivity
studies which are conducted under near single-collision conditions in a mass spectrometer in conjunction with computational
studies.[2]Our recent results report on the electrospray-ionization（ESI）generation of the small, mixed-oxo cluster［VPO4］+, its electronic
structure in the gas phase as well as its reactivity towards small hydrocarbons, thus permitting a comparison to the long-time
known and extensively investigated vanadium-oxide cation［V2 O 4 ］+ . [3] As described in previous studies, the latter exhibits noreactivity towards small hydrocarbons, such as CH4, C2H6, C3H8, C4H10, and C2H4, while the substitution of one vanadium atom bya phosphorous atom yields the reactive［VPO4］+ ion which brings about oxidative dehydrogenation from saturated hydrocarbons,i.e. propane and butane, as well as oxygen-atom transfer to unsaturated hydrocarbons, i.e. ethene, at thermal conditions. Further,
the structure of［VPO4］+ was characterized in the gas phase by advanced IR photodissociation spectroscopy, and a comparison ismade to the also structurally characterized gaseous［V 2O4］+ system, including structure-reactivity relationships. Finally, reaction
mechanisms are elucidated by DFT calculations； the results underline the key role of phosphorous in terms of C-H bondactivation of hydrocarbons by mixed VPO-clusters.
［ 1 ］J. R. Webb, T. Bolaño, T. B. Gunnoe, ChemSusChem 2011, 4, 37.
［ 2 ］H. Schwarz, Angew. Chem. Int. Ed. 2011, 50, 10096.
［ 3 ］a）R. C. Bell, K. A. Zemski, K. P. Kerns, H. T. Deng, A. W. Castleman, J. Phys. Chem. A 1998, 102, 1733； b）K. R. Asmis, J.
Sauer, Mass Spectrom. Rev. 2007, 26, 542.

Oral Session
Thursday, 20th SeptemberRoom B-1

09:00 － 11:00

Session 33: JMS Award Symposium
Chair:

Richard M Caprioli（Vanderbilt University,Nashville,TN,USA / Editor-in-Chief, Journal of Mass Spectrometry）

S33-1000
10:00 － 10:20

Charge-State Dependent Compaction and Dissociation of Protein
Complexes: Insights from Ion Mobility and Molecular Dynamics

Zoe Hall1, Argyris Politis1, Matthew F Bush1,2, Lorna J Smith1, Carol V Robinson1
1

University of Oxford, Oxford, UK, 2University of Washington, Seattle, USA

Keywords:
ion mobility CID protein complex
Novel aspects:
The charge-state dependece of protein complex CID pathways is demonstrated by IM-MS, in combination with charge
manipulation in solution, and is supported by solvent-free molecular dynamics simulations.
Abstract:
Combining computational methods with gas-phase experiments can provide powerful insights into the structure and interactionsof biomolecular ions. Here we combine ion mobility-mass spectrometry（IM-MS）with molecular dynamics（MD）simulations toinvestigate the structural changes occurring in four multimeric protein complexes during gas-phase activation1. Importantly, using
IM-MS, we find that all four complexes retain their native-like topologies at low energy. Upon increasing the collision energy, twoof the four complexes adopt a more compact state. This collapse was most noticeable for pentameric serum amyloid P（SAP）
which contains a large central cavity. The extent of collapse was found to be highly correlated with charge state, with thesurprising observation that the lowest charge states were those which experience the greatest degree of compaction.
Intermediate charge states unfolded without prior collapse, releasing highly charged, extended monomers. The highest charge
states ejected compact monomers and dimers. We compared our experimental results with in vacuo MD simulations of SAP,
which were carried out over increasing temperature, in order to impart increasing amounts of energy to the system, to mimic IMMS activation experiments. Simulations showed that low charge states of SAP exhibited compact states, corresponding to collapseof the ring, whilst intermediate and high charge states unfolded to more extended structures, maintaining their ring-like topology,
as observed experimentally. To simulate the collision-induced dissociation（CID）of different charge states of SAP, we used MS tomeasure the charge state of the ejected monomer and assigned this charge to one subunit, distributing the residual charges
evenly among the remaining four subunits. Under these conditions, intermediate charge states of SAP dissociated via the unfolding
and ejection of a single subunit. The highest charge states dissociated to compact monomers and dimers with similar collisioncross sections to those released in CID experiments for supercharged SAP. This strong correlation between theory and experimenthas implications for further studies as well as for understanding the process of CID and for applications to gas-phase structural
biology more generally.
References
1. Hall Z., Politis A., Bush MF, Smith LJ, Robinson CV. J. Am. Chem. Soc.（2012）134, 3429

Oral Session
Thursday, 20th SeptemberRoom B-1

09:00 － 11:00

Session 33: JMS Award Symposium
Chair:

Richard M Caprioli（Vanderbilt University,Nashville,TN,USA / Editor-in-Chief, Journal of Mass Spectrometry）

S33-1020
10:20 － 10:40

Odd products from even-electron ions: Exploiting charge-remote bondhomolysis to compare bond dissociation energies

David L Marshall1, Martin Paine1, Philip J Barker2, Stephen J Blanksby1
1

University of Wollongong, Wollongong, Australia, 2BlueScope Steel Research, Port Kembla, Australia

Keywords:
Nitroxyl radical, alkoxyamine, even-electron rule
Novel aspects:
CID of alkoxyamines produces structure-dependant competitive bond homolysis, violating the even-electron rule. Relative BDEs
are derived in agreement with theoretical calculations.
Abstract:
Collision induced dissociation（CID）of even-electron precursor ions typically yield even-electron fragment ions： a phenomenon
known as the“even-electron rule“. There are, however, exceptions where even-electron ions fragment via bond homolysis,
particularly where the result is a stable radical ion. When independent of the charged moiety, the efficacy of homolysis isindicative of the bond dissociation energy（BDE）. In this research we derive the relative BDEs of N-O and O-C bonds inalkoxyamines to provide new insight into the mechanistic action of these anti-oxidants.
Hindered amine light stabilisers are commonly added to polymer coatings. The key to their anti-oxidant action involves nitroxides（R1R2NO.）scavenging macroradicals to form alkoxyamines（R1R2NOR3）, thus inhibiting radical induced degradation. There exists
significant debate about the fate of the alkoxyamine and the mechanism（s）by which the nitroxyl radical is recycled. Mass
spectrometric analyses of stabilised polyesters exposed to high temperatures identified both secondary amine（R 1 R 2 NH）andalkoxyamine products.［ 1 ］One plausible explanation is that N-O homolysis competes with O-C homolysis, the former resulting inan aminyl radical and ultimately a secondary amine.
To test this hypothesis, a suite of alkoxyamines were synthesised, each incorporating a remote acidic moiety, but differing in thenature of the R3 functional group. The［M-H］- ions formed from electrospray ionisation of these alkoxyamines were subsequently
activated by collision induced dissociation in a triple quadrupole mass spectrometer.
The fragmentation of alkoxyamine precursor ions to nitroxides by O-C homolysis is shown to be another violation of the“evenelectron rule.“ This is not surprising, given the inherent stability of nitroxides. Transient aminyl radicals are also identified, from acompetitive N-O homolysis channel, which for certain R 3 functionalities dominates O-C homolysis. The relative efficacy of theseprocesses can be attributed to the relative stability of the alkyl and alkoxyl radicals. Remarkably, no significant even-electron
product channels are observed upon CID. The selective formation of nitroxyl and aminyl radicals, together with the variableenergy imparted in the triple quadrupole, has been used to probe the structure-dependant relative thresholds of N-O and O-Cbond homolysis. Whilst this technique does not provide the precision of guided ion beam methods, the qualitative trends aresupported by ab initio quantum chemical calculations.［ 2 ］
Understanding the structure-dependant relationship between these competing processes will assist in the selection of systems
that selectively promote one homolysis pathway. Furthermore, this distonic ion approach may prove useful for the further
characterisation of aminyl radicals in the gas phase, or the evaluation of polymer stabilisation mechanisms by alkoxyamines.
［ 1 ］M. R. L. Paine, P. J. Barker, S. J. Blanksby, Analyst, 2011, 136, 904.
［ 2 ］J. L. Hodgson, L. B. Roskop, M. S. Gordon, C. Y. Lin, M. L. Coote, J. Phys. Chem. A, 2010, 114, 10458.

Oral Session
Thursday, 20th SeptemberRoom D

09:00 － 11:00

Session 34: MS Informatics for Identification and Characterization
Chair:

Shigeki Kajihara（Koichi Tanaka Laboratory of Advanced Science and Technology, Shimadzu Co , Japan）

S34-0900
09:00 － 09:40[Keynote Lecture] The value of different types of information in MS based
identification

David Fenyö

New York University, New York, NY, USA

Keywords:
MS Informatics
Novel aspects:
The value of different types of information in MS based identification
Abstract:
Mass spectrometry-based protein identification has become an invaluable tool for elucidating protein function, and several
methods have been developed for protein identification, including sequence collection searching with masses of peptides andtheir fragments, spectral library searching, and de novo sequencing. In all mass spectrometry-based identification methods, ascore is calculated to quantify the match between the observed mass spectrum and the collection of possible sequences. Thesescores are highly dependent on the details of the algorithm used, and they are not always easy to interpret because theinterpretation of the score depends on properties of the data and the search results. Therefore, it is desirable to convert the score
to a measure that is easy to interpret, such as the probability that the result is random and false. It is also important to choose anappropriate collection of sequences to search, because if the search space is expanded to include a lot of irrelevant sequences,
the sensitivity decreases, but if a sequence is not included it will not be found. The current development of next-generation
sequencing technologies has made it possible to within the budget of many proteomics projects to also generate RNA-Seq data,
i.e. sequence the transcriptome, and use it to construct sample-specific sequence collections for searching. Here, we will give an
overview of mass spectrometry-based protein identification and discuss strengths and weaknesses of different strategies.

Oral Session
Thursday, 20th SeptemberRoom D

09:00 － 11:00

Session 34: MS Informatics for Identification and Characterization
Chair:

Shigeki Kajihara（Koichi Tanaka Laboratory of Advanced Science and Technology, Shimadzu Co , Japan）

S34-0940
09:40 － 10:00

Improving endogenous peptide sequence characterization using electroncapture dissociation and collision-induced dissociation.

Eisuke Hayakawa1, Gerben Menschaert2, Walter Luyten1, Geert Baggerman3, Liliane Schoofs1
1

K.U. Leuven, Leuven, Belgium., 2Ghent University,Ghent, Belgium, 3VITO, MOL, Belgium

Keywords:
peptide identification, peptidomics, ECD, ETD, MS/MS
Novel aspects:
A combination of two fragmentation methods, CID and ECD, enables highly accurate and confident endogenous peptide
identification. This approach is also effective for peptide identification in large search space.
Abstract:
Tandem mass spectrometry（MS/MS）and bioinformatics tools have enabled fast and systematic peptide identification. Especially,
the combination of LC-MS/MS and MS/MS peptide search software has become the method of choice for the characterization of
peptides in complex biological samples. However, it is still a challenge to achieve accurate characterization of endogenous
peptides, such as neuropeptides, peptide hormones, peptide pheromones and antimicrobial peptides, at high confidence from
MS/MS spectra. Since various types of enzymes are potentially involved in the processing of endogenous peptides, the MS/MS
search has to be done without any cleavage site specification. In addition, many endogenous peptides have various posttranslational modifications（PTMs）and these need to be taken into account in the search. These characteristics of endogenous
peptide lead to a huge search space and result in poor confidence of peptide characterization. As a consequence,characterization of endogenous peptide from MS/MS spectra is still difficult.
We developed a new MS/MS peptide search method to enable highly accurate and confident endogenous peptide
characterization by using a combination of two different fragmentation methods. Collision-induced dissociation（CID）is commonlyused as a peptide fragmentation method in general LC-MS/MS analysis. On the other hand, relatively new methods： electroncapture dissociation（ECD）or electron-transfer dissociation（ETD）, achieve fragmentation that is complementary to CID. We
have employed a new approach to take advantage of the combination of these two independent fragmentation methods forhighly accurate endogenous peptide identification. Peptide-spectrum matching（PSM）is carried out for CID and ECD separately,
and the score of a peptide sequence is given as a combination of the two PSM scores. This approach is quite effective todiscriminate correct peptide characterization from false hits and therefore enables highly accurate and confident peptide
identification. We applied this new approach to CID and ECD spectra of mouse neuropeptides for validation. This method
significantly increased the peptide identification rate and the reliability, as compared to a conventional MS/MS peptide searchusing a single fragmentation method. Especially, it is effective for endogenous peptide identification with various post-translational
modifications against large protein databases.
This new MS/MS search method will contribute to the accurate and confident identification of novel endogenous peptides.
Furthermore, this method will be effective for other MS/MS peptide search applications that require to be done against large
databases, such as proteogenomics, peptidogenomics and metaproteomics.

Oral Session
Thursday, 20th SeptemberRoom D

09:00 － 11:00

Session 34: MS Informatics for Identification and Characterization
Chair:

Shigeki Kajihara（Koichi Tanaka Laboratory of Advanced Science and Technology, Shimadzu Co , Japan）

S34-1000
10:00 － 10:20

LipidXplorer software supports high-throughput shotgun lipidomics at any
mass spectrometer platform and acquisition mode

Ronny Herzog1, Dominik Schwudke2, Kai Schuhmann1, Julio Sampaio1, Andrej Shevchenko1

1
Max Planck Institute of Cell Biology and Genetics, Dresden, Germany, 2National Centre for Biological Science,
Bangalore, India

Keywords:
Lipidomics, Computational, Mass Spectrometry, High Throughput
Novel aspects:
LipidXplorer software supports high-throughput shotgun lipidomics at any mass spectrometer platform and acquisition mode.
Abstract:
Lipidomics aims at quantifying the full lipid complement in cells, tissues or organisms. Shotgun lipidomics relies on the direct
analysis of total lipid extracts, in which lipids are recognized by either accurately determined m/zin survey MS spectra or by
structure-specific“signature ions“ in MS/MS spectra of prospective precursors. The shotgun approach has been widely applied inbiological research, but a major bottleneck still exists in the accurate interpretation of spectra. The available software is typicallytuned to a particular type of mass spectrometer, spectra acquisition and lipid identification method.
Here we report on LipidXplorer that supports shotgun lipidomics by any common acquisition method at any instrumentation
platform. Importantly, it offers resolution-dependent interpretation of MS and MS/MS spectra and supports high throughput
lipidomics screens. The interpretation of shotgun MS and MS/MS spectra relies upon the Molecular Fragmentation Query
Language（MFQL）［ 1 ］and is independent from reference spectra libraries or lipidomics databases. Using MFQL, any lipid
identification routine is described in simple and intuitive terms and can be adjusted to particular instrumentation features（massaccuracy and mass resolution）and instrument-dependent fragmentation pathways. In particular, common MFQLs support
identification and quantification of lipids on triple quadrupole, ion trap, hybrid quadrupole time-of-flight and linear ion trap
Orbitrap instruments by multiple reaction monitoring, data-dependent MS/MS, precursor and neutral loss scans and accurate
mapping of intact masses［ 2 ］. We report on benchmarking LipidXplorer lipid identification performance and validated its crossplatform capabilities by comparative analysis of total lipid extracts on different mass spectrometric platforms.
LipidXplorer has its own wiki page that features MFQL queries library linked to more than 14 application papers. It also provides afull documentation, step-by-step tutorial and test example queries and datasets.
Since tandem mass spectrometers are now commonly available, we argue that LipidXplorer eliminates one of the major hurdles in
adopting lipidomics methodologies by a broad scientific community in the fields of cell biology and molecular medicine.

References：［ 1 ］R. Herzog, D. Schwudke, K. Schuhmann, S. Bornstein, M. Schroeder and A. Shevchenko. A Novel Informatics Concept for HighThroughput Shotgun Lipidomics Based on the Molecular Fragmentation Query Language［ 2 ］R. Herzog, K. Schuhmann, D. Schwudke, J. L. Sampaio, S. R. Bornstein, M. Schroeder, A. Shevchenko. LipidXplorer： A Software
for Consensual Cross-Platform Lipidomics PLoS ONE 7 （ 1 ）： e29851. doi：10.1371/journal.pone.0029851.

Oral Session
Thursday, 20th SeptemberRoom D

09:00 － 11:00

Session 34: MS Informatics for Identification and Characterization
Chair:

Shigeki Kajihara（Koichi Tanaka Laboratory of Advanced Science and Technology, Shimadzu Co , Japan）

S34-1020
10:20 － 10:40

MassBank: Public Mass Spectral Database

Takaaki Nishioka, Yoshito Nihei, Yuya Ojima, Tasuku Ikeda
Nara Institute of Science and Technology, Nara, Japan

Keywords:
peak annotation, structure elucidation, public repository
Novel aspects:
Chemical annotation of ESI-MS 2 data of high mass accuracy are useful for the analysis of peak-chemical structure relationships.
These were applied to the chemical structure elucidation of unknown ESI-MS 2 data.
Abstract:［Summary］Mass spectral data, as the experimental data supporting research conclusions, should be open to the public forallowing other researchers to verify the experiments. Additionally they are valuable research products that should be shared
among the researchers using mass spectrometry for chemical identification and other applications. MassBank（http：//www.
massbank.jp）has been internationally accepted as a public repository of mass spectra of small chemical compounds. ESI-MS 2
data of high mass accuracy have steadily increased in MassBank. Their product ions are so accurately analyzed that we havesuccessfully annotated with the molecular formula. MassBank provides users with these data that are searchable by molecular
formulae as well as conventional peak m/z values. We report novel applications of the chemically annotated ESI-MS 2 data in
MassBank to the structure elucidation from LC-MS2 data.
［Current Data Statistics］Researchers have made their data open to the public from MassBank at their own expenses. That is,they have to prepare their records in the MassBank Record Format and provide their MassBank data servers. In spite of such
expenses, MassBank contributors and their data increase steadily. Currently（April 2012）, 21 research groups（15 Japan, 3 USA, 2
Germany, 1 China）deposit a total of 29,644 high-quality MS data of 13,534 small molecules, which include ESI-MS, MS2 16,440 dataof 2,204 compounds from nine data servers. Among them, the number of ESI-MS2 data of relative mass accuracy within 50 ppm is6,146. Researchers timely share the data analyzed on up-to-date instrument models. Accesses to MassBank in the period of
October, 2011 were 10,063 unique IP addresses per month.
［Tools for Sharing Mass Spectral Data］Mass++（http：//www.first-ms 3 d.jp/english/achievement/software）and MassBankprojects have collaborated to relieve the data contributors and users of tedious manual tasks in data preparations and retrieves.
For example, in metabolomics study, a single LC-MS2 analysis of a biological sample outputs hundreds of MS2 data. With these
outputs as queries, spectral search of the MassBank data for the metabolite identification was a tedious manual work previously.
The two projects developed the batch spectral search, by which users are able to submit a set of the query data at once.
［Chemical Annotation of MassBank ESI-MS2 Data］We manually annotated the molecular formula to product ions observed in
the MassBank ESI-MS2 data that were analyzed on ESI-QTOF and ESI-QFT mass spectrometers. The annotated spectral data aresearchable with the molecular formula of peaks and peak differences on MassBank. In combination with the substructure search,peak search with the molecular formula is helpful to analyze the relationships between product ions and chemical substructures
and to evaluate the specificity of the relationships. These relationships are accumulated as Fragmentation Library on a wiki system（http：//metabolomics.jp/wiki/Index：MassBank）.
One of the applications of the relationships is the chemical structure elucidation of unknown metabolites from their ESI-MS2 data.
The relationships and KNApSAcK database（http：//kanaya.aist-nara.ac.jp/KNApSAcK/）are integrated in a MassBank tool thatpredicts the chemical substructure of unknown metabolites. Currently the tool works to predict unknowns that are derivatives ofprimary metabolites. To predict secondary metabolites more effectively, the relationships should cover broader substructures that
secondary metabolites integrate. Identification or structure elucidation of unknown metabolites is a bottleneck of metabolomics
study. Sharing mass spectral data of secondary metabolites is a steady and effective solution to remove the bottleneck.
［Acknowledgements］The authors thank to all the researchers who contribute MassBank with their data at their own expenses.
MassBank is the official database of the Mass Spectrometry Society of Japan. MassBank project is financially supported by“Program for Coordination Toward Integration of Related Databases“, the National Bioscience Database Center in Japan.

Oral Session
Thursday, 20th SeptemberRoom D

09:00 － 11:00

Session 34: MS Informatics for Identification and Characterization
Chair:

Shigeki Kajihara（Koichi Tanaka Laboratory of Advanced Science and Technology, Shimadzu Co , Japan）

S34-1040
10:40 － 11:00

MSPTM-DB: a known PTM database for high-speed and accurate searchavailable on the“ProteoAnalysis“ web site

Akiyasu C Yoshizawa1, Tsuyoshi Tabata2, Takayuki Kimura2, Ken Aoshima2, Yoshiya Oda2,
Shigeki Kajihara1, Koichi Tanaka1

Koichi Tanaka Laboratory of Advanced Science and Technology, Shimadzu Corporation, Kyoto, Japan, 2Eisai ProductCreation Systems, Eisai Corporation, Ltd., Tsukuba, Japan

1

Keywords:
database, post-translational modification（PTM）, sequence, literature, annotation
Novel aspects:
We constructed two databases： One contains variety of peptide sequences with known PTM sites for high-speed and accurate
search. The other contains literature information about these PTMs for rich annotation.
Abstract:
Introduction
The analysis of mass spectrometry-derived data is the mainstream of proteomic research, although software for this purpose isstill evolving. We are constructing a web site for proteomic research called“ProteoAnalysis,“ and implementing several new
functions for the analysis of mass spectrometric data. Using functions on this site, users can extract peak lists from raw data, andidentify the proteins corresponding to the MS peaks： During this process, users can utilize our original databases,“MSPTM-DB“
and“PTM annotation DB.“ MSPTM-DB is an attempt at high-speed and accurate search for sequences with known（databaserecorded）post-translational modifications（PTMs）, whereas the conventional variable modification method calculates all possible
PTMs. PTM annotation DB is a database of detailed information of known PTMs. In addition to this, the sequence databases ofisoforms and mutated proteins are also available on ProteoAnalysis. Using these databases, users can obtain more accurate andrich information of PTMs in a shorter time.
Methods and Materials
For the construction of MSPTM-DB, we extracted data from the“Modified Residue“ lines of the“Feature“ section in the SwissProt database（Release 2012_04）. At this time, we focus on the phosphorylation and acetylation data； we adopted thephosphorylation information of Serine（S）, Threonine（T）, and Tyrosine（Y）, and the acetylation information of Lysine（K）,
Alanine（A）, and Methionine（M）. These data correspond to 98.8 % and 90.2 % of data in each category, respectively, andcollectively cover 85.3% of all PTM data in Swiss-Prot.
We produced trypsin-digested peptides of all extracted sequences mentioned above； we made a variety of peptide sequenceswith one or more known modified sites, whose known（potential）PTM sites are either modified or not modified. In the databasesearch, characters not assigned to any amino acid are treated as“new amino acids“ by temporally assigning them to themodified amino acid residues（We call this technique“Stand-in method“）. If“ProteoAnalysis“ users just specify this databaseas the search database, all processes including the Stand-in method are automatically executed.
“PTM annotation DB“ at this time contains the literature information of all PTMs recorded in Swiss-Prot. Users can look up
literature information by inputting the protein name, the position of the modified amino acid, and the kind of PTM as the searchkeys.
To assess the usability, we performed the database search of HeLa cell extract, which was reduced, alkylated, and digested by
Lys-C and Trypsin, and phosphopeptides were enriched by immobilized metal affinity chromatography（IMAC）. The search engine
was X! Tandem Version：2010.01.01.4. The search results against our MSPTM-DB, isoform/mutation database, and Swiss-Prot werecompared with the results obtained by the conventional search against our isoform/mutation database and Swiss-Prot. For thisevaluation test, we chose targeted PTM data according to the irreliabilities； we used experimentally confirmed data only.
Results
Using MSPTM-DB for the phosphorylation analysis, we could identify 367 peptides （both phosphorylated and nonphosphorylated）in contrast with that 321 peptides were identified by the conventional method and 249 peptides were identified
by the normal search method. Although theMSPTM-DB method cannot detect unrecorded PTMs, it can however detect both
unmodified peptides and peptides with known PTMs； surprisingly, it managed to detect peptides that were not detected by theconventional method. In the case of acetylated peptides, the effect of MSPTM-DB is larger； more than 100 additional peptides
were detected compared with the conventional method. The search speed using the MSPTM-DB method is at least 20 times
faster.

We are expanding the data contents and resources of both databases； and the“ProteoAnalysis“ web site will be freely
accessible on the web in the near future.

Oral Session
Thursday, 20th SeptemberRoom E

09:00 － 11:00

Session 35: Environment I
Chair:

Peter Haglund（Umeå University, Sweden）

S35-0900
09:00 － 09:40[Keynote Lecture] Chiral Chemicals as Tracers of Sources and Fate
Processes in a World of Changing Climate

Terry F Bidleman1,2,3, Liisa M Jantunen2, Perihan B Kurt-Karakus4, Fiona Wong5

1
Chemistry Department, Umeå University, Umeå, Sweden, 2Centre for Atmospheric Research Experiments, Environment
Canada, Egbert, ON, Canada, 3Department of Chemistry, University of Toronto, Toronto, ON, Canada, 4Department ofEnvironmental Engineering, Bahcesehir University, Istanbul, Turkey, 5Department of Applied Environmental Science(ITM), Stockholm University, Stockholm, Sweden

Keywords:
persistent organic pollutants, pesticides, chiral, climate, arctic
Novel aspects:
Chiral compounds offer special advantage for following POPs pathways because of their ability to distinguish emissions fromracemic（newly released or protected from microbial attack）and nonracemic（microbially weathered）sources.
Abstract:
Elimination of persistent organic pollutants（POPs）under national and international regulations reduces“primary“ emissions, but“secondary“ emissions continue from residues deposited in soil, water, ice and vegetation during former years of high usage. In
a future, secondary source controlled world, POPs follow the cycle of organic carbon and biogeochemical processes determine
their transport, accumulation and fate（ 1 ）. Climate change is likely to affect mobilisation of POPs through e.g., increased
temperature, loss of ice cover in polar regions, melting glaciers and changes in soil microbiology which affect degradation andtransformation（ 2 ）. Chiral compounds offer special advantage for following transport and fate pathways because of their ability
to distinguish racemic（newly released or protected from microbial attack）and nonracemic（microbially weathered）sources. Useof GC or LC with chiral stationary phases and MS makes enantiomer-specific analysis possible for chiral compounds of many
chemical classes. This paper discusses the rationale for this approach and suggests applications where chiral POPs（ 3 ）and othercompounds could aid investigation of climate-mediated exchange and degradation processes. Examples include distinguishingagricultural vs. non-agricultural and recently used vs. residual pesticides, degradation and sequestration processes in soil, historicalvs. recent atmospheric deposition, sources in arctic air and influence of ice cover on volatilisation.
1.Nizzetto, L., MacLeod, M., Borgå, K., Cabrerizo, A., Dachs, J., et al., 2010. Past, present, and future controls on levels of persistentorganic pollutants in the global environment. Environ. Sci. Technol. 44, 6526-6531.
2.UNEP/AMAP 2011. Climate Change and POPs: Predicting the Impacts. Report of the UNEP/AMAP Expert Group, Secretariat of theStockholm Convention, Geneva, 62 pp.
3.Bidleman, T.F., Jantunen, L.M., Kurt-Karakus, P.B., Wong, F. 2012. Chiral compounds as tracers of sources and fate： review andprospects for investigating climate change influences. Atmos. Pollut. Res., submitted.

Oral Session
Thursday, 20th SeptemberRoom E

09:00 － 11:00

Session 35: Environment I
Chair:

Peter Haglund（Umeå University, Sweden）

S35-0940
09:40 － 10:00

POLLUTION OF MOSCOW AIR: GC/MS STUDY OF SNOW SAMPLESOlga V Polyakova1, Viatcheslav V Artaev2, Dmitry M Mazur1, Albert T Lebedev1
1

M.V.Lomonosov Moscow State University, 2LECO Corporation, 3000 Lakeview Avenue, St. Joseph, MI,USA

Keywords:organic pollutants, GC/MS, ICP/MS, high resolution mass spectrometry, snow
Novel aspects:
A representative list of organic pollutants in the atmosphere of Moscow was created for the first time using high resolution mass
spectrometry.
Abstract:
Moscow is the largest European city with population about 15 millions and hundreds of enterprises. More than 4 millions cars areregistered in Moscow. However just the most common atmospheric pollutants are monitored in the city at the regular basis. To
propose a list of priority pollutants for the atmosphere of Moscow 16 snow samples were collected along the perimeter（109 km）
of the Moscow belt road at the end of March 2011 and 2012. Snow is an excellent preserving matrix allowing keeping the majorityof chemical compounds including not very stable ones（e.g. phenols）. In the countries with cold climate analysis of snow gives achance to estimate long term atmospheric pollution（several months）.
Mass spectrometry was used as an analytical tool to identify individual organic compounds（gas chromatography/mass
spectrometry, GC/MS）and the most environmentally relevant chemical elements（inductively coupled plasma with mass
spectrometric detection, ICP-MS）. Sample preparation was carried out according to US EPA 8270 and 200.8 Methods, while LECO
Pegasus IVD and Agilent7500c instruments were used correspondingly. Both target and non-target approaches were used. As aresult more than 500 organic compounds belonging to various classes were identified in each sample. Besides classic pollutants
like PAH, PCB, phthalates several classes of other anthropogenic contaminants including, organophosphates, esters of N,Ndiethylcarbamodithioic acid, various nitrogen and sulphur containing compounds, antioxidants, and some others were represented
by a number of compounds. Quite surprising was the detection of several compounds with dichloromethyl group in 2012 samples,
including dichloronitromethane as the major ingredient. The confirmation of the identification was obtained by parallel analysis of
all the samples with LECO Pegasus GC-HRT instrument with the resolving power exceeding 60000. Several valuable issues
concerning reliability and new possibilities for the identification of new compounds were discovered when dealing with a highresolution instrument.
The levels of organic compounds using internal standards as well as the levels of chemical elements were quantified. The dataobtained allow estimating atmospheric pollution in Moscow in the period between December and March and proposing a draft
list of priority pollutants for the atmosphere of Moscow.

Oral Session
Thursday, 20th SeptemberRoom E

09:00 － 11:00

Session 35: Environment I
Chair:

Peter Haglund（Umeå University, Sweden）

S35-1000
10:00 － 10:20

AQUEOUS-PHASE REACTIONS OF ATMOSPHERICALLY RELEVANT
VOLATILE ORGANIC COMPOUNDS THROUGH TANDEM MASS
SPECTROMETRY: AN INTRIGUING STORY OF AEROSOL FORMATION

Rafal Szmigielski, Krzysztof Jan Rudzinski, Inna Kuznietsova

Institute of Physical Chemistry Polish Academy of Sciences, Warsaw, Poland

Keywords:
volatile organic compounds, isoprenoids and their oxygenated metabolites, atmospheric aerosols, tandem mass spectrometry
Novel aspects:
The presented study falls into a hot topic research on the formation, transformation and environmental implications of ambient
aerosols（SOA）. Our research revealed a number of novel wet aerosol components.
Abstract:
Volatile organic compounds（VOC）are a broad group of species that play important roles in the environment. They come toexistence in the air through different pathways, ranging from the vital activity of plant vegetation（a prevailing source）toanthropogenic emissions（a minor source）. Regardless of the emission source, they are engaged in the formation of ambient
secondary organic aerosols（SOA）in the atmosphere through a complex photo-oxidation chains followed by a gas-to-particle
conversion. In addition, they affect human health, a quality of life, and in the larger scale the Earth’s climate. A poorly understoodchemical transformations of VOCs contribute to a significant fraction of organic matter in the atmosphere（~35% to ~90%）which is“frozen“ in the form of aerosol particles.
Isoprene（ 2 -methyl-buta-1,3-diene）and its first oxidation products（i.e., methacrolein, methyl vinyl ketone and methacrylic acid）
are relevant volatile precursors of ambient SOA in the atmosphere. Isoprene is the most abundant non-methane hydrocarbon
emitted to the atmosphere as a result of living vegetation. According to the recent data, the isoprene emission rate is estimatedto be at the level of 700 TgC per year. While heterogeneous transformations of isoprene and its first oxidation products have been
well documented, aqueous-phase reactions of these species with radical intermediates leading to the production of new class of
wet SOA components such as polyols and their sulfate esters（organosulfates）, are still poorly recognized. The chain reactions ofisoprene with sulfoxy radical-anions（SRA）are one of the recently researched routes leading to the formation of organosulfates in
the aqueous phase. The latter radical species originate from the auto-oxidation of SO2 in the aqueous phase and are behind thephenomenon of atmospheric acid rain formation. This is a complicated chain reaction that is catalyzed by transition metal ions,such as manganese（II）, iron（III）and propagated by sulfoxy radical anions.
The presented work addresses the chemical interaction of selected VOCs such as isoprene, methacrolein, methyl-vinyl ketone,
with sulfoxy radical-anions in the aqueous solution under both dark and solar conditions. We showed that triple quadrupole mass
spectrometry is a powerful technique to follow the chemical changes of monitored processes. The use of collision-induced
dissociations revealed the formation of novel components of wet SOA, including oxygenated polar species with C- 5 skeleton
bearing SO3H（MW 182, 180）and SO2H（MW 166, 164）moieties on the hydroxyl group. The structures of these products were firmly
confirmed by comparison of their liquid chromatography and mass spectrometry behaviors with that corresponding to thesynthesized model compounds. It is believed that newly discovered highly polar low molecular weight compounds may contributeto the growth of wet aerosol particles by the formation of higher molecular weight species.

Oral Session
Thursday, 20th SeptemberRoom E

09:00 － 11:00

Session 35: Environment I
Chair:

Peter Haglund（Umeå University, Sweden）

S35-1020
10:20 － 10:40

HRAM Screening and Quantitative Analysis of Pesticides in Environmental& Food Matrices using a bench top LCMS Orbitrap system

Dipankar Ghosh, Charles Yang, Leo Wang, Jonathan Beck
Thermo Fisher Scientific

Keywords:
Pesticides, Orbitrap, LCMS, UHPLCNovel aspects:
Usage of a novel Second Generation Orbitrap platform to screen, quantify and confirm a large number of pesticides at the low
ppb level.
Abstract:
Introduction:
Current methodologies for the quantitation of pesticides in food revolve around using triple quadrupole platforms and long run
times. The method described here utilizes UHPLC- with a second generation Exactive TM mass spectrometer（MS）using highresolution accurate mass. The second generation Exactive MS is capable of resolving power settings of up to 140,000（FWHM）atm/z 200, providing the ability to resolve matrix from analyte in full MS mode even in most complex matrices. This work describes amethod to do screening and quantitation of a 100 pesticides mixture together with high level confirmation.
Method:A Hypersil Gold aQ C18 50x2.1mm 1.9u column was utilized with a run time of less than 8 mins with all analytes eluting within 5
mins. A standard curve containing 110 compounds was spiked in orange solution ranging from 50 pg/mL levels to 250 ng/mL levels
was injected in triplicate and screening of different food matrix（green bell pepper and hot peppers）samples was analyzed fortargeted list of 100 compounds and also screened for possible other unknown pesticides. The spectrometer was set to a resolving
power of 70,000（FWHM）at m/z 200 in full MS mode to minimize matrix interference, and all-ion-fragmentation（AIF）spectra werecollected to qualify. The data was then compared to a current MS/MS library for confirmation, as well as calibration curves weregenerated for the individual compounds.
Preliminary Data:
Calibration lines were generated for the compounds analyzed with r2 better than 0.9940, the limits of detection（LOD）varied from50pg/mL to 500pg/ml based on the individual compounds. For confirmation of each compound the exact mass of the compound,its isotopes and as well as the AIF produced were collected and compared to limit the amount of“false positives“in the results.
One of the main challenges using a high resolution accurate mass system is data mining, but with novel ExactFinderTM software
the data processing becomes straight forward. Using the novel second generation Exactive the workflow to screen and confirm ina single run increases throughput for repeat runs.

Oral Session
Thursday, 20th SeptemberRoom E

09:00 － 11:00

Session 35: Environment I
Chair:

Peter Haglund（Umeå University, Sweden）

S35-1040
10:40 － 11:00

Development and validation of methodology using liquid
chromatography-tandem mass spectrometry (LC-MS/MS) for monitoringfor use of anabolic steroids in animals

Simon J Hird, George Stubbings

The Food and Environment Research Agency

Keywords:
anabolic steroids, urine, serum, LC-MS/MS
Novel aspects:
High sensitivity, validated methodology suitable for official control purposes, chromatographic and mass spectrometric selectivity,
overcoming matrix effects
Abstract:
A wide range of anabolic steroids has been used in animal fattening because of their capacity to increase weight gain and theimprovements in feed conversion efficiency. The use of anabolic steroids in animal fattening is prohibited in the European
Community and monitoring for use of anabolic steroids is carried out through the National Plans of the individual Member States.
For controls at retail level and for products imported in the EU, it is necessary to have analytical methods applicable to meat
samples, whereas at farms and abattoirs, misuse of anabolic steroids in living animals is monitored by analyses of the animal’surine and/or serum. Because of the complexity of these matrices and the low“Recommended Concentrations“ established, it isnecessary to have sensitive, selective and specific methods for the detection of the anabolic steroids.
Methodology has been developed for the determination of 15 anabolic steroids in bovine, ovine and porcine urine. The procedure
involved enzymatic hydrolysis prior to extraction and SPE prior to analysis by ultra high performance liquid chromatography
coupled to a tandem mass spectrometer operating in electrospray, switching between positive and negative ion modes. Dataacquisition was performed in selected reaction monitoring（SRM）mode. The method was successfully validated according to theCommission Decision 2002/657/EC for the detection and confirmation of residues in products of animal origin. The methodology
developed is suitable for the detection, quantification and confirmation of identity of these anabolic steroids in bovine, ovine andporcine urine and can be used for residue control programs.

Oral Session
Thursday, 20th September

Main Hall

15:00 － 17:00

Session 36: Advances in Ion Mobility Mass Spectrometry
Chair:

Joseph A Loo（University of California-Los Angeles, USA）

S36-1500
15:00 － 15:40[Keynote Lecture] Peptide and protein aggregation: Mechanisms,
inhibition and disease

Michael T Bowers

University of California at Santa Barbara, Santa Barbara CA USA

Keywords:
ion mobility, amyloid, Alzhimer’s, aggregation
Novel aspects:
IMS-MS can not only determine oligomer distributions and structures for aggregating systems but can also effectively screen small
molecule inhibitors to this aggregation.
Abstract:
One of the outstanding problems in understanding amyloid diseases at the molecular level is an inability to determine oligomer
distributions and oligomer structures for rapidly aggregating systems. This kind of aggregation characterizes a wide variety ofimportant diseases including Alzheimer’s disease, Type 2 Diabetes, Parkinson’s disease and many others. The problem istraditional spectroscopic structural methods simply don’t work since they measure average solution properties and individualoligomers cannot be isolated for x-ray analysis. In the past several years we have shown that ion mobility based mass
spectrometry（IMS-MS）is ideally suited to attack this important problem. Here we will present our latest results on peptide
assembly mechanisms and results on inhibition of this assembly for important disease systems.

Oral Session
Thursday, 20th September

Main Hall

15:00 － 17:00

Session 36: Advances in Ion Mobility Mass Spectrometry
Chair:

Joseph A Loo（University of California-Los Angeles, USA）

S36-1540
15:40 － 16:00

Behavior of intrinsically disordered regions within a protein complex of
Swi5-Sfr1 characterized by IM-MS and SAXSKazumi Saikusa1, Naoyuki Kuwabara2, Yuichi Kokabu1, Yu Inoue1, Mamoru Sato1, Hiroshi Iwasaki3,
Toshiyuki Shimizu2, Mitsunori Ikeguchi1, Satoko Akashi1
1
Yokohama City University, Yokoham, Japan, 2University of Tokyo, Tokyo, Japan, 3Tokyo Institute of Technology,
Yokohama, Japan

Keywords:
small-angle X-ray scattering, dummy residue model, intrinsically disordered protein, ion mobility mass spectrometry
Novel aspects:
First application to evaluate the experimental CCS values of IDPs using SAXS dummy residue models.
Abstract:
It has now been recognized that intrinsically disordered proteins（IDPs）play important roles as hubs in intracellular networks, andtheir structural characterization is of significance. Due to their highly dynamic features, it is not easy to investigate their structures
only by conventional methods, such as X-ray crystallography. In the present study, we tried to characterize IDP complexes using
ion mobility mass spectrometry（IM-MS）in combination with small-angle X-ray scattering（SAXS）. This method enables structuralcharacterization even of proteins that have difficulties with crystallization. With this method, we have characterized the
Schizosaccharomyces pombe Swi 5 -Sfr 1 complex, which is expected to have a long disordered region at the N-terminal portionof Sfr1. Here we contrasted the structural characteristics of the intrinsically disordered（ID）region in the gas phase with those in
the solution phase proved by SAXS.
The collision cross sections（CCSs）and low-resolution structures were analyzed for the Swi 5 -Sfr 1 and Swi 5 -Sfr 1 C complexes by
traveling-wave IM-MS and SAXS. Sfr 1 C is a mutant of Sfr 1 lacking the ID region of the N-terminal 177 amino acid residues, and theatomic-level structure of the Swi 5 -Sfr 1 C complex has already been determined by X-ray crystallography.
NanoESI-MS of Swi 5 -Sfr 1 and Swi 5 -Sfr 1 C revealed that Swi 5 （9.7 kDa）formed 1 ： 1 complexes with Sfr 1 （33.5 kDa）and Sfr 1 C（14.1 kDa）, respectively. The SAXS experiments suggested that these complexes exhibited the elongated shapes, especially for
the Swi 5 -Sfr 1 complex. The CCS values of multiply-charged ions of the complexes were obtained by converting their arrival times
in the ion mobility region. The CCS distribution of the Swi 5 -Sfr 1 C complex indicated that there were four conformers exist withthe centroid CCSs of 2100, 2310, 2870 and 3720 A2. As for the Swi 5 -Sfr 1 complex, three conformers with the centroid CCSs of 3160,
6380, and 7940 A 2 were found. The most dominant ions observed in the ESI mass spectra of the Swi 5 -Sfr 1 and Swi 5 -Sfr 1 C
complexes were with the most compact shapes.
To evaluate the experimental CCS values, low-resolution dummy residue models for the Swi 5 -Sfr 1 C and Swi 5 -Sfr 1 complexes
were constructed with the SAXS data and their theoretical CCS values were calculated. Upon CCS calculation, a dummy residue
radius of 5.8 A was adopted, which was obtained by comparing the X-ray and dummy-residue model structures constructed basedon the PDB atomic coordinates of 10 model proteins.
The experimental CCS value of the most dominant ion observed in the ESI mass spectrum of the Swi 5 -Sfr 1 complex was
extremely smaller than the theoretical value. In the case of Swi 5 -Sfr 1 C, the compaction level was not as high as Swi 5 -Sfr1. Thisimplies that a considerable compaction occurred for the ID region of the complex during IM-MS measurement. Furthermore, thisstudy indicates that the experimental CCS values of proteins containing long ID regions can be evaluated with the theoretical CCS
values calculated for their SAXS low-resolution solution structures.

Oral Session
Thursday, 20th September

Main Hall

15:00 － 17:00

Session 36: Advances in Ion Mobility Mass Spectrometry
Chair:

Joseph A Loo（University of California-Los Angeles, USA）

S36-1600
16:00 － 16:20

Probing Protein Quaternary Structures by Surface Induced Dissociation
and Ion Mobility - Mass Spectrometry

Mowei Zhou1,2, Shai Dagan1, Vicki H Wysocki1,2
1

University of Arizona, Tucson, United States, 2Ohio Sate University, Columbus, Ohio, United States

Keywords:
surface induced dissociation, ion mobility, protein complexes
Novel aspects:
First comprehensive presentation illustrating the advantages of combining surface induced dissociation with ion mobility to study
large non-covalent protein complexes
Abstract:
Quaternary structures of protein complexes are highly important to their biological functions. The combination of ion mobility（IM）
and mass spectrometry（MS）, in which both conformational and molecular weight information of the assemblies can be obtained,
greatly expanded the application of MS in structural biology. Subunit organization of protein complexes can be studied withtandem mass spectrometry by activating the original assemblies and releasing sub-structures in a controlled manner. The major
challenge of applying this method is to overcome the undesired unfolding of protein subunits in the commonly used gas collision
activation（collision induced dissociation, CID）which usually results in the loss of information for the native conformation.
We present here the incorporation of a custom surface induced dissociation（SID）device in a modified quadrupole/IM/time-offlight mass spectrometer. The SID device is positioned either before IM cell（SID-IM）which provides conformational information
on product ions, or after IM cell（IM-SID）for shape selected activation. With collisional cross sections（CCSs）of remaining
precursor ions after activation measured using the SID-IM setup, C-reactive protein（CRP）pentamer is shown to undergo
significant structural rearrangement and unfolding upon CID. Major monomer product ions in CID are partially unfolded. Instead,
CRP pentamer dissociates rapidly into compact monomers in SID without remarkable change in collisional cross section of the
precursors. This illustrates the advantage of using SID to activate protein complexes with minimum disruption in their conformation
so that the product ions are informative to their native architecture. Additionally, SID-IM simplifies interpretation of tandem MS
spectra because of the separation of oligomeric subunit products which could overlap in mass-to-charge, elucidated by SID
spectra of several protein complexes. With the IM-SID setup, SID of selected conformations of a protein complex can be
performed. As an example, two conformational ensembles of serum amyloid P（SAP）decamers are separated and activated by
SID, which revealed differences in subunit packing between the two structures.

Oral Session
Thursday, 20th September

Main Hall

15:00 － 17:00

Session 36: Advances in Ion Mobility Mass Spectrometry
Chair:

Joseph A Loo（University of California-Los Angeles, USA）

S36-1620
16:20 － 16:40

Energy-resolved ion mobility tandem mass spectrometry: A new approachfor probing fragmentation pathways involving ion isomerization

Yayoi Hongo1, Takemichi Nakamura1, Takae Takeuchi2, Shunya Takahashi1, Hiroyuki Koshino1
1

RIKEN, Wako, Japan, 2Nara Women’s University

Keywords:
energy-resolved ion-mobility tandem mass spectrometry（ER-IMS/MS）, collision cross section, small molecular characterization
Novel aspects:
Energy-resolved ion mobility tandem mass spectrometry was applied to a macrocyclic system and found to be a useful tool for
probing the fragmentation involving acyclic intermediate.
Abstract:
In tandem mass spectrometry（MS/MS）, activated gas-phase ions undergo a variety of reactions that can be categorized as
fragmentation and isomerization. Among various reaction pathways from activated even-electron ions, isomerization may be noless common than fragmentation. Structure specific isomerization（or rearrangement）should give us rich information on ionic
structure. However, ion isomerization in MS/MS has not been well documented or studied in the past, partly because rather few
direct methods for probing such reactions have readily been available.
While on the other hand, ion mobility spectrometry（IMS）has widely been recognized as a useful tool for studying gas-phase ion
structure. Consequently, recent advancements in hybridization of IMS and MS/MS devices should give us unique opportunity forthe study of isomerization upon activation of gas-phase ions. Here we report a new approach to probe fragmentation pathways
involving ion isomerization using ion mobility analysis combined with energy-resolved tandem mass spectrometry（ER-IMS/MS）.
Ring-opening reactions of cyclic molecules are considered to be one of the distinct examples of ion isomerization. To demonstrate
the proof of concept of ER-IMS/MS, we’ve investigated fragmentation behavior of a compound, cyclo-di-SIP-B（C 50 H 56 O 10 ）, a
derivative of a natural-product Phomopsin B[1] and related compounds. The structure of cyclo-di-SIP-B includes 20-membered-ringcorresponding to dimer of ’SIP-B’ unit. The ER-IMS/MS experiments were conducted by using a Synapt G 2 HDMS, IMS Q-TOF
instrument（Waters）, equipped with an ESI source.
［C 25H28O5 Na］+）corresponding
The sodiated molecule of cyclo-di-SIP-B（［M+Na］+, m/z 839）gave a major fragment ion at m/z 431（
to the mass of ’SIP-B’ unit, generation of which requires cleavages at two sites in the ring structure, i.e. ring-opening to give an
acyclic structure and 2 nd cleavage to give the fragment ion. In the ER-IMS/MS experiment, in which precursor ion was collisionallyactivated prior to entering to the mobility cell, a single peak was detected in the mobiliogram of m/z 839 with lower collisionenergies（CE）. On the other hand, interestingly, a second peak of m/z 839 appeared at a little larger drift time at higher CE. The
second peak is attributable to an isomerized precursor as it showed substantially larger collision cross section（CCS）comparedto that of the original cyclic ion. In other words, the ion with larger CCS may be attributable to the hypothetical ring-opened ’intermediate’ of the fragmentation pathway from m/z 839 to 431. In order to confirm the hypothesis, we’ve designed a double CID
ER-IMS/MS experiment. The mass analyzed m/z 839 precursor were subjected to the first CID to before entering to the mobility
cell. After the mobility separation, the isomers of m/z 839 were fragmented in the 2 nd CID cell. The energy-resolved MS/MS
spectra of two isomers were virtually identical showing dominant fragment ion at m/z 431； the larger CCS isomer was suggested
to be an ’intermediate’ of fragmentation from m/z 839 to 431. There appeared to be a few eV difference in CID onset for theoriginal cyclic structure and the acyclic isomer. The energy difference may be associated with the energy requirement for the ringopening reaction, or isomerization prior to fragmentation.
Combined with theoretical assessment of CCS and energetics of reactions, ER-IMS/MS can be a powerful means to probe the ion
fragmentation pathway which contains ion isomerization in mass analyzer. Potential application of ER-IMS/MS is further explored.
［ 1 ］Izuchi, Y., Koshino, H., Hongo, Y., Kanomata, N., Takahashi. S., 2011, Org. Lett., 13, 3360-3363

Oral Session
Thursday, 20th September

Main Hall

15:00 － 17:00

Session 36: Advances in Ion Mobility Mass Spectrometry
Chair:

Joseph A Loo（University of California-Los Angeles, USA）

S36-1640
16:40 － 17:00

Understanding protein-DNA Interactions and Tumorigenic Mutations in
IDPs: IM-MS rises to the challenge

Ewa Jurneczko1, Faye Cruickshank1, Penka Nikolova2, Ian Campuzano3, Michael Morris3,
Perdita E Barran1
1

The University of Edinburgh, 2King’s College London, London, UK, 3Waters Corporation, Manchester, UK

Keywords:
Ion Mobility-Mass Spectrometry, p53-DNA Interactions
Novel aspects:
First reported use of（ion mobility）mass spectrometry to systematically examine protein-DNA interactions and Tumorigenic
Mutations in IDPs
Abstract:
Introduction
Common approach to understanding protein function is to ’solve’ its structure and subsequently probe interactions between theprotein and its binding partners. The first part of this approach is non-trivial for proteins where localised regions or even their
entire structure fail to fold into a 3 -D structure and yet they possess function. These so called intrinsically or inherently disordered
proteins（IDPs）or intrinsically disordered regions（IDRs）constitute up to 40 % of all expressed proteins, and a much higher
percentage in proteins involved in the proliferation of Cancer. The tumour suppressor p53 has an intimate and multifaceted role inthe cell cycle and that is reflected in its equally complex structure. The full length protein has a modular domain structure,
consisting of DNA-binding and tetramerisation domains, flanked by intrinsically disordered regions at both the amino andcaroboxy termini, which poses a challenge for the structural biologists. Here we use ion mobility mass spectrometry to assess therelative disorder of p53, in a complex with different lengths of DNA and also mutants of the DNA binding domain.
Methods
Wild type p53 and mutants of the core DNA-binding domain（amino acid residues 94-312）were dialysed in 50 mM ammonium
acetate for 2 h at 4 °C. 10% by volume isopropanol was added prior toanalysis by mass spectrometry to aid desolvation. Nonnative samples were sprayed from buffered solutions containing 10% by volume isopropanol and 1 % formic acid. DNA oligomers
contained the following sequences： 12mer： GGAACATGTTCC and 20mer： GAACATGTTCGAACATGTTC which are derived fromthe p53 consensus binding site. All oligomers were annealed by heating them to 95°C for 5 min. Data were obtained on a TW IMMS（SynaptHDMS, Waters, Manchester, UK）in positive ionisation mode and a home built linear DT IM-MS instrument. In the linear
DT IM-MS instrument data was also obtained at different buffer gas temperatures to examine the thermal unfolding of these
proteins.
Preliminary Data
Mass spectrometry studies of WT p53 and mutants of the DNA binding domain following nano-electrospray from bufferedconditions show a wide charge state distribution（ 8 z 18）formonomeric species of the general form［M+zH］z+, which we highlight
as signature behaviour of IDPs in vacuo as reported by mass spectrometry. The collision cross-sections of the monomer are
observed to increase with increasing charge in a stochastic fashion attributed to protein unfolding due principally to Coulombic
repulsion, which can be attributed to the ’plasticity’ of these IDPs. Differences in unfolding transitions between the WT andmutated samples can be characterized. The H115N mutation located at the boundary of the IDR loop I appears to be lessstructured, favoring the more extended conformations when compared with WT p53. The structural ’hot spot’ mutation in the loop
III region R249A shows less population in unfoldomers and the structure appears even tighter, contrary to what expected.
The majority of the mutations occur in the core domain which contains the sequence-specific DNA binding activity of the p53
protein. And they result in a loss of DNA binding. The DNA binding is critical for the biological activity of p53, and provides a frame
work for understanding how mutations inactivate it. In a complex with DNA, p53 core domain binds all available consensus
binding sites in both DNA constructs. WTp53 binds to the 12merDNA as a dimer and as four monomers to the 20mer DNA. Theinteraction is highly cooperative, and relies on the recognition of DNA sequences by the core domain and on the tetramerizationof the protein, which is heavily mediated by the presence of DNA. These results are interpreted in terms of the biological activity
and also in terms of a view of the allosteric regultion in these proteins.

Oral Session
Thursday, 20th SeptemberRoom A

15:00 － 17:00

Session 37: Challenges in High Resolution and High Accuracy Mass Measurement Mass
Spectrometry
Chair:

Evgeny N Nikolaev（The Institute for energy problems of chemical physics Russian Academy of Sciences, Russia）

S37-1500
15:00 － 15:40[Keynote Lecture] Mass Resolution and Mass Accuracy: How Much isEnough?

Alan G Marshall, Greg T Blakney, Nathan K Kaiser, Yuan Mao, Amy M McKenna, Ryan P Rodgers,
Brian M Ruddy, Christopher L Hendrickson
Florida State University,Tallahassee, Florida, U.S.A.

Keywords:
Fourier Transform Mass Spectrometry, FTMS
Novel aspects:
Hardware and software developments leading to higher mass resolving power, mass resolution, and mass accuracy. Minimum
resolving power for unique assignment of elemental composition and amino acid composition.
Abstract:
Accurate mass measurement must be preceded by the highest possible mass resolution, to ensure that only a single elemental
composition contributes to the mass spectral peak in question. Although mass resolution is conventionally defined as the closest
distinguishable separation between two peaks of equal height and width, the required mass resolving power is ~10x higher for
equal width peaks whose peak height ratio is 100：1. Ergo, minimum resolving power requires specification of maximum dynamic
range, and is thus 10-100x higher than the conventional definition.
The highest available broadband mass resolving power and mass accuracy is from Fourier transform ion cyclotron resonancemass spectrometry. I shall describe how, over the past five years, FT-ICR MS mass accuracy has improved by about an order ofmagnitude, based on higher magnetic field strength, conditional co-adding of time-domain transients, better mass calibration（spectral segmentation； inclusion of a space charge term； radially dispersed excitation； phase correction to yield absorptionmode display； and new azimuthal and axial ICR cell segmentation designs. Work supported by the National Science Foundation（DMR-06-54118； CHE-1016942； CHE-1019193 and the State of Florida.
Xian, F.； Hendrickson, C. L.； Blakney, G. T.； Beu, S. C.； Marshall, A. G.“Automated Broadband Phase Correction of Fourier
Transform Ion Cyclotron Resonance Mass Spectra,“Anal. Chem.2010, 82, 8807-8812.
Savory, J. J.； Kaiser, N. K.； McKenna, A. M.； Xian, F.； Blakney, G. T.； Rodgers, R. P.； Hendrickson, C. L.； Marshall, A. G.“PartsPer-Billion Fourier Transform Ion Cyclotron Resonance Mass Measurement Accuracy with a ’Walking’ Calibration Equation,“Anal.
Chem.2011, 83, 1732-1736.
Kaiser, N. K.； Quinn, J. P.； Blakney, G. T.； Hendrickson, C. L.； Marshall, A. G.“A Novel 9.4 Tesla FT-ICR Mass Spectrometer with
Improved Sensitivity, Mass Resolution, and Mass Range“ J. Am. Soc. Mass Spectrom. 2011, 22, 1343-1351.
Kaiser, N. K.； Savory, J. J.； McKenna, A. M.； Quinn, J. P.； Hendrickson, C. L.； Marshall, A. G.“Electrically Compensated FT-ICR
Cell for Complex Mixture Analysis,“Anal. Chem.2011, 83, 6907-6910.
Blakney, G. T.； Hendrickson, C. L.； Marshall, A. G.“Predator Data Station： A Fast Data Acquisition System for Advanced FT-ICR
MS Experiments,“Int. J. Mass Spectrom.2011, 306, 246-252.
Valeja, S. G.； Kaiser, N. K.； Xian, F.； Hendrickson, C. L.； Rouse, J. C.； Marshall, A. G.“Unit Mass Resolution for an Intact 148 kDa
Therapeutic Monoclonal Antibody by FT-ICR Mass Spectrometry,“Anal. Chem.2011, 83, 8391-8395.
Xian, F.； Hendrickson, C. L.； Marshall, A. G.“High Resolution Mass Spectrometry,“Anal. Chem.2012, 84, 708-719.
Vladimirov, G.； Hendrickson, C. L.； Blakney, G. T.； Marshall, A. G.； Heeren, R. M. A.； Nikolaev, E. N.“Fourier Transform Ion
Cyclotron Resonance Mass Resolution and Dynamic Range Limits Calculated by Computer Modeling of Ion Cloud Motion, J. Am.
Soc. Mass Spectrom.2012, 23, 375-384.

Oral Session
Thursday, 20th SeptemberRoom A

15:00 － 17:00

Session 37: Challenges in High Resolution and High Accuracy Mass Measurement Mass
Spectrometry
Chair:

Evgeny N Nikolaev（The Institute for energy problems of chemical physics Russian Academy of Sciences, Russia）

S37-1540
15:40 － 16:00

Enhanced Fourier transform and filter diagonalization method mass
spectrometry for top-down analysis of antibodies and petroleomics

Yury O Tsybin, Ünige A Laskay, Luca Fornelli, Konstantin O Zhurov, Anton N Kozhinov
Ecole Polytechnique Federale de Lausanne, Switzerland

Keywords:
top-down mass spectrometry； Fourier transform mass spectrometry； Orbitrap； filter diagonalization method； petroleomics
Novel aspects:
Enhanced Fourier transform and filter diagonalization method-based signal processing accelerate high resolution and high massaccuracy data acquisition in FT mass spectrometry improving petroleomics and top-down structural characterization of antibodies
Abstract:
Fourier transform mass spectrometry（FTMS）provides superior resolution and mass accuracy among the palette of the MS
instruments. However, comprehensive structure analysis of molecules and macromolecules present in extremely complex
biological and environmental samples and/or performed under time-constrained experimental conditions demands a substantial
increase in the acquisition speed of high-resolution and high mass accuracy MS data. For this reason, absorption mode spectra
representation has been implemented in Orbitrap FTMS as a part of enhanced FT（eFT）signal processing and its development isin progress in FT-ICR MS. On the other hand, filter diagonalization method（FDM）is capable of overcoming the FT resolution
limitation. Recently, we described the first implementation of FDM for FT-ICR MS.［1,2］Here, we demonstrate performance
optimization of hybrid Orbitrap Elite FTMS in the top-down analysis of intact proteins as well as feasibility and limitations ofOrbitrap FTMS compared to FT-ICR MS in routine analysis of petroleum-type samples. In addition, we examine performances of
FT, eFT, and FDM approaches for the transient data treatment.
Proteins were obtained from commercial sources or expressed in-house. Complex molecular mixtures, including crude oil
fractions, were obtained through collaboration. MS and MS/MS experiments were performed using 10 T LTQ ICR and LTQ Orbitrap
Elite FT mass spectrometers（both Thermo Scientific）. Ionization was achieved by atmospheric pressure electrospray orphotoionization, either in direct infusion or in on-line liquid chromatography（LC）-MS/MS approach. The instrumental parameters
and operation were controlled by standard and advanced user interface data acquisition software allowing acquisition of transient
signals of user-defined length. Data treatment using FT and FDM signal processing methods was performed using in-housedeveloped Python-based software running on a multi-core workstation.
First, we examined performance gain of eFT and FDM signal processing for the data treatment compared to the conventional
magnitude mode FT. The results indicate that FDM-based MS presents a number of advantages compared to other signal
processing methods, including FT and eFT.［ 1 ］In contrast to FT-ICR MS performed at 10 T, FDM-based signal processing of thesame data requires transients containing only 1 isotopic beat for multiply charged 10-20 kDa proteins to provide their isotopic
distribution resolution. Importantly, FDM-based MS confirms its applicability in the case of high spectral complexity.［ 1 ］Briefly, incase of extremely complex mixtures the application of FDM for the analysis of time-domain transient signals in FT-ICR MS reduces
the required transient length by a factor of 2 -4. Furthermore, FDM MS provides complementary molecular structure information,such as peptide fine isotopic structures, on a LC time scale.［ 2 ］
We will demonstrate further method and technique development of FDM MS methodology for FT-ICR MS and extent thismethodology to the Orbitrap FTMS. Particularly, we will present the application of eFT and FDM MS to the analysis of intact
proteins, including monoclonal antibodies, by electron transfer dissociation（ETD）-based top-down approach.［ 3 ］In the area of
petroleomics, the new hybrid high-resolution Orbitrap FTMS platforms with eFT bridge the resolution gap with FT-ICR MS in awide m/z range. We will apply FDM MS to push the upper mass limit for separation of closely spaced molecular peaks in Orbitrap
FTMS-based petroleomics. Particular attention will be paid to the mass accuracy challenge in Orbitrap FTMS-based petroleomics
with FT, eFT, and FDM signal processing. We will also discuss the current limitations of the FDM-based MS and some technicaldetails of its current implementation.
1. Kozhinov A. N., Tsybin Y. O. Anal. Chem.（2012）84, 2850-2856
2. Miladinovic S. M., Kozhinov A. N., Gorshkov M. V., Tsybin Y. O. Anal. Chem.（2012）, in print
3. Tsybin Y. O., et al. Anal. Chem.（2011）, 83（23）, 8919-8927

Oral Session
Thursday, 20th SeptemberRoom A

15:00 － 17:00

Session 37: Challenges in High Resolution and High Accuracy Mass Measurement Mass
Spectrometry
Chair:

Evgeny N Nikolaev（The Institute for energy problems of chemical physics Russian Academy of Sciences, Russia）

S37-1600
16:00 － 16:20

Characterization and Selective Classification of Petroleum Crude Oils
using High Resolution Time-of-Flight Mass Spectrometry

Clecio Klitzke2, Marcos Eberlin2, Kevin Siek1, Jeffrey S Patrick1, Joe Binkley1, Yuri Corillo2
1

LECO Corporation, 2UNICAMP, ThoMSon Mass Spectrometry Laboratory, Campinas, São Paulo Brasil

Keywords:
Resolution, Petroleomics, Time-of-flight, FTMS, Heteroatoms
Novel aspects:
This work demonstrates the practical retention of key information from petroleum characterization in translation form FTMS to HR
TOF including heteroatom contributions and the definition of key petroleum attributes.
Abstract:
Crude oil samples from different sources and having different quality chemical attributes are examined by high performance timeof flight mass spectrometry. The data are evaluated using an software specifically designed to process the TOF data an accessmolecular level information of the oil samples through the high resolution data. Crude oil samples are ionized using electrosprayionization by direct infusion and the mass analyzes is performed at a variety of resolving powers on FT-ICR MS（7.2 T）and on ahigh resolution time of flight（HRT）mass spectrometer. Specifically, at 50,000 and ca. 90,000 on the HRT and 100,000, 200,000 and
400,000 on the FT-ICR MS systems. These data are used to classify the crude oil samples based on their most abundant content ofheteroatomic species as well other measurements of significance to petroleum processing. Comparison is made between
samples, resolving power conditions and with real and theoretical data obtained from other measurements. The ability toselectivity access the significant descriptors of crude oil pertinent to source identification and processing quality is demonstrated.
The data were acquired on a time of flight mass spectrometer using acquisition times of less than 10 minutes per sample with asimple sample preparation. The information content of significance to petroleum researchers and engineers that is retained at thevarious resolving powers is compared and contrasted with the information content being retained on the HRT compared with
400,000 resolving power on the FTMS.

Oral Session
Thursday, 20th SeptemberRoom A

15:00 － 17:00

Session 37: Challenges in High Resolution and High Accuracy Mass Measurement Mass
Spectrometry
Chair:

Evgeny N Nikolaev（The Institute for energy problems of chemical physics Russian Academy of Sciences, Russia）

S37-1620
16:20 － 16:40

Detailed structure analysis of fluorinated polymer by means of pyrolysis
comprehensive two-dimensional gas chromatography / high-resolution
time-of-flight mass spectrometry

Yoji Nakajima, Yuko Arinami, Tsuguhide Isemura, Kiyoshi Yamamoto
Asahi Glass Co., Kanagawa, Japan

Keywords:
TOFMS GCxGC polymer high-resolution
Novel aspects:
The usefulness of Py-GCxGC/HRTOFMS for chemical structure analysis of fluorinated polymers was demonstrated. Detailed
structure of the polymer such as sequence and end-group were successfully identified by using accurate mass.
Abstract:
We demonstrate the usefulness of pyrolysis（Py）comprehensive two-dimensional gas chromatography（GCxGC）/ high-resolution
time-of-flight mass spectrometry（HRTOFMS）for chemical structure analysis of fluorinated polymers. New fluorinated polymers are
rapidly emerging in recent years to meet requirements of advanced applications in many fields. Introduction of fluorine intopolymers leads to drastic improvements in several properties, such as chemical stability, electric and optical properties. However,there are peculiar technical difficulties for chromatography and mass spectrometry of fluorinated polymers. For example, in
relation to chromatography, fluorinated polymers are often insoluble in common organic solvent. In this case, Py-GC/MS isgenerally useful for structure analysis of insoluble polymers. However, pyrolysis of fluorinated polymers often generates great
numbers of unidentified peaks which cannot be separated by one-dimensional GC.
Recently, comprehensive GCxGC has been developed and applied to separate complex samples mainly in the fields ofgeochemistry and environmental chemistry. Separation ability of GCxGC is several tens times higher than one-dimensional GC. In
relation to mass spectrometry, most of pyrolysis products of fluorinated polymers are unidentified and not included in commercial
database of mass spectra. In this regard, accurate mass spectrometry by using HRTOFMS appears as a useful technique instructural analysis of such unidentified compounds. In this study, combination of GCxGC and HRTOFMS were used to resolve theproblems of separation and identification of the pyrolysis products. Our Py-GCxGC/HRTOFMS system consists of a microfurnace
pyrolyzer（Frontier Lab PY-2010D）and a gas chromatograph（Agilent GC7890）equipped with a high-resolution time-of-flight mass
spectrometer（Jeol AccuTOF）and a GC×GC jet modulator（Zoex KT2006）. Typical measurement conditions of the system are as
follows. Pyrolysis temperature is 600 °C. GC columns are DB- 5 for the first dimension and DB-17 for the second dimension. GC×
GC modulation is performed with liquid nitrogen cooling during 6 sec. GC oven temperature is programmed as 40 °C（ 5 min）,
ramp to 300 °C at 5 °C /min. Helium is used as a carrier gas at 1.5 ml/min. HRTOFMS is operated at the acquisition rate of 25 Hz.
Mass spectra are collected from m/z 35 to 800.
We have applied the Py-GCxGC/HRTOFMS system to structural analysis of various fluorinated polymers. The advantages ofapplication to structural analysis of fluoroacrylate（FA）/methylmethacrylate（MMA）copolymer are clearly described. GC×GC
chromatogram of FA/MMA copolymer shows several hundred well separated peaks. GC×GC chromatogram pattern had good
reproducibility. Many of those peaks couldn’t have been separated and detected by means of one-dimensional GC. In addition to
the separation ability, sensitivity of GC×GC is also higher than that of one-dimensional GC because the GC×GC modulation
process makes the peaks sharper. The first dimension of GC×GC represents the separation according to boiling points and thesecond dimension represents polarity. FA derived compounds were detected in the lower polarity region of the GC×GC
chromatogram, and fully separated from MMA derived compounds detected in the higher polarity region. The advantages of GC×
GC include enhanced identification due to well ordered and classified detection of the compounds in GC×GC chromatogram.
Accurate mass spectra of the compounds obtained by HRTOFMS can provide us not only molecular weight and fragmentation
information but also elemental composition information. By combining those peaces of information together, many compounds
including detailed structure information of the polymer such as sequence and end-group were successfully identified. In addition,
the Py-GCxGC/HRTOFMS seems to be a powerful tool for detailed structure analysis of not only fluorinated polymers but also
some other advanced synthetic polymers.

Oral Session
Thursday, 20th SeptemberRoom A

15:00 － 17:00

Session 37: Challenges in High Resolution and High Accuracy Mass Measurement Mass
Spectrometry
Chair:

Evgeny N Nikolaev（The Institute for energy problems of chemical physics Russian Academy of Sciences, Russia）

S37-1640
16:40 － 17:00

Dynamically harmonized FTICR cell with specially shaped electrodes forcompensation of inhomogeneity of the magnetic field.

Yury Kostyukevich1, Gleb Vladimirov1, Eugene Nikolaev1,2,3

1
Institute for Energy problems of chemical physics, Russia, 2Emanuel Institute of Biochemical Physics of Russian
Academy of Sciences, Moscow, Russia, 3Institute of Biomedical Chemistry of Russian Academy of Medical Sciences,
Moscow, Russia

Keywords:
FTICR, dynamically harmonized cell, simulation
Novel aspects:
Methods of compensation of the magnetic field inhomogeneity inside a dynamically harmonized FTICR cell by introducing specificelectric field corrections
Abstract:
The recently introduced FTICR ion trap with dynamic harmonization shows the highest resolving power ever achieved both forions with moderate masses 500-1000 Da（peptides）as well as ions with very high masses of up to 200 kDa（proteins）. Time oftransient duration corresponding to the time of synchronous motion of the ion clouds with m/z around 500 excited to cyclotron
motion is more than 5 minutes and seems to be limited only by the vacuum inside FT ICR cell. Such results were obtained forsuperconducting magnets of very high homogeneity of the magnetic field.
In superconducting magnets used in FTICR mass spectrometry the inhomogeneity of the magnetic field in its axial direction
prevails over the inhomogeneity in other directions and should be considered as the main factor influencing the synchronous
motion of the ion cloud［ 1 ］. The inhomogeneity leads to a dependence of the cyclotron frequency on the amplitude of axial
oscillation in the potential well of the ion trap. As a consequence the ions in an ion cloud become dephased, which leads to adecrease in the resolving power.
Ion cyclotron frequency is also affected by the radial component of the electric field. Hence by appropriately adjusting the electricfield one can compensate the inhomogeneity of the magnetic field and align the cyclotron frequency in the whole range of
amplitudes of z-oscillations. Such compensation has recently been experimentally demonstrated for Gabrielse’s type FT ICR trap
［ 2 ］. Here a method of magnetic field inhomogeneity compensation in a dynamically harmonized FT ICR cell is presented, which
is based on the introduction of additional electrodes into the cell shaped in such a way that the averaged electric field created bythese electrodes produces a counterforce to the forces caused by the inhomogeneous magnetic field.
Realistic computer simulation clearly demonstrates the possibility of the proposed cell design to sufficiently（by the factors of
5 -10）increase a time of synchronic ion motion for the magnetic field of the following dependence on z coordinate： B=B 0（ 1 +a*z*z）. Such inhomogeneity has the main influence on cyclotron frequency for magnets used in FTICR mass spectrometry.
The authors acknowledge the support from the Russian Foundation of Basic Research（grant 10-04-13306）, from the RussianFederal Program（state contracts 14.740.11.0755, 16.740.11.0369）, and from the Fundamental Sciences for Medicine Program of theRussian Academy of Sciences.
1) W.A. Anderson, Electrical Current Shims for Correcting Magnetic Fields Rev. Sci. Instrum., 32（1961）241.
2) Nathan K. Kaiser, Joshua J. Savory, Amy M. McKenna, John P. Quinn, Christopher L. Hendrickson, and Alan G. MarshallElectrically Compensated Fourier Transform Ion Cyclotron Resonance Cell for Complex Mixture Mass Analysis Anal. Chem., 2011,
83（17）, pp 69076910

Oral Session
Thursday, 20th SeptemberRoom B-1

15:00 － 17:00

Session 38: Mass Spectrometry for Metabolic Diseases
Chair:

Makoto Yoshino（Kurume University, Japan）
Seiji Yamaguchi（Shimane University, Japan）

S38-1500
15:00 － 15:20

LC/MS/MS analysis of protein-bound uremic toxins in hemodialysis
patients

Toshimitsu Niwa1, Yoshiharu Itoh2, Atsuko Ezawa2, Kaori Kikuchi2, Yoshinari Tsuruta3

Nagoya University, Nagoya, Japan, 2Biomedical Research Laboratories, Kureha Corporation, Tokyo, Japan, 3Meiyo
Clinic, Aichi, Japan

1

Keywords:
uremic toxin, LC/MS/MS, hemodialysis
Novel aspects:
This study describes simultaneous LC/ESI-MS/MS measurement of 12 uremic solutes in hemodialysis patients, and first
demonstrates the protein-binding of indoxylglucuronide, p-cresylglucuronide, phenyl sulfate, phenylglucuronide, and 4 -ethylphenyl
sulfate in uremic serum.
Abstract:
Introduction: Cardiovascular disease（CVD）isprevalent in patients with chronic kidney disease（CKD）. In hemodialysis（HD）
patients, some protein-bound uremic toxins are considered to be associated with CVD. However, it is not yet known which uremic
toxins are important in terms of endothelial toxicity. Methods: Serum samples were obtained from 45 hemodialysis（HD）patients
before and after HD. Total and free serum concentration of indoxyl sulfate, indoxyl glucuronide, indoleacetic acid, p-cresyl sulfate,p-cresyl glucuronide, phenyl sulfate, phenyl glucuronide, phenylacetic acid, phenylacetyl glutamine, hippuric acid, 4 -ethylphenyl
sulfate, and 3 -carboxy- 4 -methyl- 5 -propyl- 2 -furanpropionic acid （CMPF） were simultaneously measured by liquid
chromatography/electrospray ionization-mass spectrometry/mass spectrometry（LC/ESI-MS/MS）. The effects of these solutes attheir pre-HD mean and maximum serum concentrations on reactive oxygen species（ROS）production in human umbilical vein
endothelial cells（HUVEC）were measured with a ROS probe. Results: Serum levels of 11 solutes except 4 -ethylphenyl sulfate
were significantly increased in HD patients compared to healthy subjects. All the 12 solutes showed more or less protein-binding.
Especially, indoxyl sulfate, p-cresyl sulfate, CMPF, and 4 -ethylphenyl sulfate showed high protein-binding ratios（more than 95%）
and low reduction rates by HD（less than 35%）. Indoxyl sulfate at its mean and maximum pre-HD serum concentration even with
4 % albumin stimulated ROS production in HUVEC most intensely, followed by CMPF. Conclusion: Serum levels of 11 proteinbound uremic toxins were increased in HD patients. Indoxyl sulfate, p-cresyl sulfate, and CMPF could not be removed efficiently
by HD due to its high protein-binding ratio. Indoxyl sulfate most intensely induced endothelial ROS production, followed by CMPF.

Oral Session
Thursday, 20th SeptemberRoom B-1

15:00 － 17:00

Session 38: Mass Spectrometry for Metabolic Diseases
Chair:

Makoto Yoshino（Kurume University, Japan）
Seiji Yamaguchi（Shimane University, Japan）

S38-1520
15:20 － 15:40

Early indicator of hepatic fibrosis in patients with chronic hepatitis C:
discovery, assay optimization and clinical validation

Fumio Nomura1, Kazuyuki Sogawa1, Kenta Noda1, Toshihide Miura1, Yoshio Kodera2, Osamu Yokosuka1
1

Chiba University, Chiba, Japan, 2Kitasato University, Kanagawa, Japan

Keywords:
hepatic fibrosis, fibrinogen alpha chain,
Novel aspects:
A novel biomarker candidate to detect hepatic fibrosis related to hepatis C virus at an early stage was discovered by MS, andsimple and high-throughput assay was developed.
Abstract:
When the damage to the liver is chronic, excess fibrous connective tissue accumulates irrespective of the etiology. Withadvancements in the treatment of chronic liver diseases, the accurate assessment of hepatic fibrosis is a pivotal need forsuccessful individual management of patients. Liver biopsy has long been the gold standard for assessing the degree of liver
fibrosis. However, it is invasive and also prone to sampling errors and/or interobserver variation. Therefore, there is a need for
non-invasive biomarkers for hepatic fibrosis. Proteome analyses should be a promising tool to search for novel fibrosis markers.
Although current proteomic technologies permit identification of new biomarkers, diagnostic test development remains
challenging. Indeed, a large number of marker candidates detected and identified by MS-based proteomic methods are left
untested because an appropriate ELISA for a validation study is not available.
We previously identified a 5.9 kDa peptide fragment of fibrinogen α C-chain（FIC 5.9）as a novel biomarker candidate for heavy
drinking. Since our initial identification of FIC5.9, the same peptide has been reported to be of diagnostic value in a variety ofpathological conditions including cancers and inflammatory diseases. To establish diagnostic values of FIC 5.9 in a large scale, it ismandatory to develop simple and reproducible assay system.
Since FIC5.9 was first detected and identified by MALDI-TOF, a stable isotope-labeled internal standard dilution（SID-MS）assay
was the initial option for quantitative determination of FIC5.9 concentrations in serum samples. We constructed a stable isotopelabeled internal standard dilution（SID-MS）assay to determine serum FIC 5.9 levels. Although the SID-MS FIC 5.9 determinations
were reproducible, other methods were desirable in terms of throughput and feasibility.
Since the size of FIC 5.9 was outside the detection limit of MS combined with MRM or SRM, we decided to constructimmunoassays for FIC 5.9. FIC 5.9 is a fragment of the fibrinogen alpha C chain, and it is likely that serum samples contain anumber of fibrinogen fragments of different sizes. It was not an easy task to obtain an antibody that can specifically react with one
particular fragment. For this purpose, two monoclonal antibodies specific to the N and C-termini of the 5.9-kDa peptide were used
to develop a FIC5.9 sandwich ELISA. The assay was evaluated by comparing the results with those obtained by the stable isotopelabeled dilution mass spectrometry（SID-MS）.
The ELISA results correlated with the SID-MS findings（slope=0.795, intercept=-0.011, r（ 2 ）=0.908）and the performance of the
ELISA was satisfactory in terms of recovery（98.5-103.0%）and within-run（1.4-4.7%）and between-day（2.8-8.4%）reproducibility.
We then applied this immmunoassays to serum samples obtained from patients with chronic liver diseases of various etiologies. A
total of 371 serum samples obtained from 65 patients with chronic HBV infection, 126 patients with chronic HCV infection and 180
healthy volunteers were analyzed by the ELISA assay.
Serum levels of FIC 5.9 in healthy subjects were 12.1 + 1.7 mg/mL, whereas those for patients with chronic hepatitis C were 5.1 +2.6 mg/mL（p<0.001）. Interestingly, FIC 5.9 levels were markedly decreased in as early as F 1 （namely very early）stage of chronic
hepatitis C（5.5 + 1.7 mg/mL； p<0.001）, whereas other representative conventional markers to assess hepatic fibrosis（type IV
collagen and hyaluronic acid were not.
Measurement of serum FIC 5.9 levels could be a sensitive indicator to detect hepatic fibrosis at an early stage in chronic hepatitis
C. A larger-scale multicenter study is currently underway to further confirm the results obtained in this preliminary study.

Oral Session
Thursday, 20th SeptemberRoom B-1

15:00 － 17:00

Session 38: Mass Spectrometry for Metabolic Diseases
Chair:

Makoto Yoshino（Kurume University, Japan）
Seiji Yamaguchi（Shimane University, Japan）

S38-1540
15:40 － 16:00

Liquid chromatography-mass spectrometry based analysis of thecerebrospinal fluid metabolome for the study of inborn errors of
metabolism

Maria del Mar Amadore2, Foudil Lamari2,5, Benoit Colsch1, Fanny Mochel2,3, Francois Seguin4,
Frederic Sedel2, Christophe Junot1

CEA, Gif-sur-Yvette, France, 2Pitie-Salpetriere Hospital, AP-HP & University Pierre and Marie Curie, Paris, France,
INSERM UMR S975, Paris, France, 4INSERM U1082, Paris, France, 5Department of Metabolic Biochemistry- PitieSalpetriere Hospital, Paris, France
1
3

Keywords:
Metabolomics, cerebrospinal fluid, liquid chromatography coupled to mass spectrometry, inborn errors of metabolism.
Novel aspects:
LC/MS based metabolomics of CSF provides new insights into the pathogenic mechanisms of known inborn errors of metabolism,
and is also relevant for the stratification of patients with unexplained encephalopathies.
Abstract:
Introduction
Many complex neurological disorders remain of unknown etiology despite extensive biochemical and genetic work-up. These“unexplained encephalopathies“ may be caused by inborn errors of metabolism（IEM）. Diagnosis of IEMs can usually be
accomplished by the biochemical analysis of biofluids（blood and urine in most cases）to highlight the consequences of an
enzymatic or a transport protein defect. However, biochemical analyses available at clinical chemistry laboratories are restricted
to well-known metabolites, and many metabolic pathways remain unexplored. By enabling the concomitant detection of a wide
range of metabolites in biological fluids in a single analysis, metabolomics appears to be a relevant tool for the diagnosis of IEMs
［ 1 - 3 ］. Regarding disorders with predominant nervous system dysfunction, the cerebrospinal fluid（CSF）is the body fluid more
likely to be altered because of its proximity with neuronal and glial cells. In the present pilot study, we aimed at evaluating liquid
chromatography coupled to mass spectrometry（LC/MS）-based CSF metabolomics for the study and characterization of IEMs
involving the nervous system.
Material and methods
Patients. For this study, 79 CSF samples were selected from a biobank collected from adult patients seen in the neurometabolic
unit of the Pitie-Salpetriere Hospital. CSF sample collection was realized after written informed consent, in accordance with french
ethical rules. Selected CSF samples were classified into 4 categories：（i） 7 Negative controls without progressive neurologicaldisease,（ii）12 patients with well-defined neurometabolic disorders,（iii）60 patients having unexplained encephalopathies, and（iv）15 patients with various neurodegenerative diseases.
LC/MS based metabolomics. Hundred µl of CSF samples were deproteinized in microcentrifuge tubes by adding 300 µl of
methanol. Samples were vortexed and centrifugated. Supernatants were evaporated to dryness under nitrogen and resuspended
in 100 µl of water containing 0.1% of formic acid. Five µl of a solution of internal standards were added to all samples. In addition,a quality control CSF sample was injected every 10 samples in order to evaluate the analytical error at the level of eachmetabolite. Samples were analyzed by ultra performance liquid chromatography coupled to an ESI-LTQ-Orbitrap operated inpositive and negative modes. Data processing and treatment were performed as previously described［ 4 ］.
Results
The developed LC/MS and processing methods led to the identification of 104 metabolites in the CSF. CSF analysis revealed a
specific abnormal fingerprint in 4 out of 12 patients with confirmed metabolic diseases, emphasizing that the possibility tomeasure metabolites not previously analyzed may be of help to depict underlying pathophysiological mechanisms. For the others
8 patients, LC/MS analysis of the CSF was normal. This can be explained by incomplete coverage of the whole metabolome.
Furthermore, 22 of the 60 patients with unexplained encephalopathies presented at least one or more metabolites in an
abnormally high concentration. Among these 22 patients, our LC/MS study enabled the grouping of 3 patients, given the highnumber of common abnormalities detected in their fingerprints. Interestingly, two of these patients have already been groupedfrom a previous NMR study in which a significant elevation of free sialic acid was detected exclusively in their CSF［ 5 ］.
In conclusion, The CSF profile of abnormal metabolites might provide new insights into the pathogenic mechanisms of known
diseases. In patients with unexplained encephalopathies, the precise delineation of individual biochemical phenotypes makes LC/
MS based metabolomics of CSF a promising complementary tool to high throughput genetic approaches.

Reference List
［ 1 ］B.A.Binzak et al. Am J Hum. Genet., 68（2001）839.
［ 2 ］M.Oostendorp et al. Clin. Chem, 52（2006）1395.
［ 3 ］S.H.Moolenaar et al. Magn Reson. Med, 46（2001）1014.
［ 4 ］D.Darghouth et al. Blood, 117（2011）e57-e66.
［ 5 ］F.Mochel et al. Brain, 132（2009）801.

Oral Session
Thursday, 20th SeptemberRoom B-1

15:00 － 17:00

Session 38: Mass Spectrometry for Metabolic Diseases
Chair:

Makoto Yoshino（Kurume University, Japan）
Seiji Yamaguchi（Shimane University, Japan）

S38-1600
16:00 － 16:20

Highly sensitive and specific clinical diagnostics of lysosomal storage
diseases using multiple reaction monitoring mass spectrometry,fluorimetry, and affinity-proteomics

Michael Przybylski1, Claudia Cozma1, Marius I Iurascu1, Laura Ion1, Adolf Muehl2, Alina Petre1,3,Michael L Gross3, Stefan Maeser2

University of Konstanz, Konstanz, Germany, 2Centogene GmbH, Freiburg & Rostock, Germany, 3Washington University
St. Louis, St. Louis, MO, USA

1

Keywords:
LSDs； diagnostics； mass spectrometry, fluorimetry；
Novel aspects:
Development of molecular clininical diagnostics for lysosomal storage diseases by MS-MRM and fluorimetry using identicalsubstrates, suitable for multiplex diagnostics on dry blood spots.
Abstract:
Lysosomal storage disorders（LSDs）are a group of approximately 60-70“rare“ metabolic disorders caused by the deficiency of
single lysosomal enzymes in metabolizing biopolymer substrates, or by a defect lysosomal membrane protein. If enzymes as aresult of an inherited genetic defect or loss of function due to biochemical defects are out of function,（LSDs）are manifesting
through a broad, unspecific spectrum of life-threatening clinical symptoms including bone, muscle and organ enlargement（e.g.,
heart, liver and spleen）, differing by disease, age of onset and severity. Most LSDs are inherited in an autosomal recessive
manner； however, Fabry disease and Hunter syndrome（MPSII）are X-linked disorders. For some LSDs, enzyme replacement
therapies（ERT）have been developed and have been shown to be curative； however, ERT is critical to start as soon as possible
and therapy at late-stages is often no longer effective, thus rendering rapid and efficient diagnosis of key importance. Here we
describe highly specific and sensitive diagnostics on dry blood spots（DBS）for（i）, molecular determinations of LSDs, particularly
muco-polysaccaridoses and muco-lipidoses, by simultaneous fluorimetric and mass spectrometric analysis using newly developed,
identical substrates and standard derivatives；（ii）, clinical diagnostics of LSDs by multiplex- MS-MRM analysis using specificfragmentations of substrates and multiply-13C-labelled standard derivatives；（iii）, the clinical validation of the new methods with
DBS samples, as shown for patients with established LSDs（Fabry； MPS-I； MPS-VI）. The new methods enable a substantial
extension and improvement of current diagnostics, and provide access to a spectrum of LSDs not hitherto amenable to clinicaldiagnostics. Moreover, most recently methods have been developed for the direct determination of LSD proteins in vivo by affinityproteomics and -mass spectrometry［ 1 - 3 ］, using epitope- specific antibodies.
［ 1 ］Iuszczyk P. et al.,（2009）J. Med. Chem. 52, 2420-2428.
［ 2 ］Dragusanu, M., et al.（2010）J. Am. Soc. Mass Spectrom.21, 1643-1648.
［ 3 ］Sladewska, A. et al.（2011）J. Mol. Recogn. 24, 687-699.

Oral Session
Thursday, 20th SeptemberRoom B-1

15:00 － 17:00

Session 38: Mass Spectrometry for Metabolic Diseases
Chair:

Makoto Yoshino（Kurume University, Japan）
Seiji Yamaguchi（Shimane University, Japan）

S38-1620
16:20 － 16:40

Mass spectrometry in newborn screening of fatty acid oxidation disorders- diagnostic approach using lymphocytes

Yosuke Shigematsu1, Ikue Hata1, Go Tajima2
1

University of Fukui, Fukui, Japan, 2Hiroshima University Graduate School of Biomedical Sciences, Hiroshima, Japan

Keywords:
fatty acid oxidation, newborn screening
Novel aspects:
Labeled acylcarnitines in lymphocytes loaded with deuterium-labeled palmitate can be measured using tandem mass
spectrometry, and are useful for quick and accurate diagnosis of fatty acid oxidation disorders.
Abstract:［Background］Fatty acid oxidation disorders, as inborn errors of metabolism, are characterized by sudden infant death or acuteencephalopathy due to hypoglycemia and hyperammonemia during fasting events, and are designated as important target
disorders for newborn screening using tandem mass spectrometry（MS/MS）, in which acylcarnitines in dried blood spots aremeasured. However, there are some difficulties in selecting optimum screening markers with adequate cut-off values, partly due tounique nature of disorders or lack of consistency in measurement among MS/MS instruments, which caused a considerable
numbers of cases with false-positive results and even with false-negative results. Thus, enzyme assays and mutation analyses aremandatory for the diagnosis, although these tests are not convenient in clinical settings. Instead, we tried to analyze acylcarnitines
in lymphocytes loaded with stable-isotope labeled fatty acid for quick diagnosis of fatty acid oxidation disorders.
［Methods］Heparinized whole blood samples（ 5 ml）were obtained from patients with very-long-chain acyl-CoA dehydrogenase
deficiency（VLCADD）, carnitine palmitoyltransferase I deficiency（CPT 1 D）, CPT 2 D, mitochondrial tri-functional protein
deficiency（TFPD）, and multiple acyl-CoA dehydrogenase deficiency（MADD）. Lymphocytes, collected using Ficall-Paque
solution, were suspended in 1.0 ml of Dulbecco’s-PBS（D-PBS）. The cell suspension was mixed with D-PBS containing fatty acidfree BSA, l-carnitine, and d31-palmitate（d15-caprylate, if necessary）, and was incubated for 2 hrs. The washed lymphocytes were
homogenized in methanol and centrifuged. Acylcarnitines in the supernatant, which was spiked with stable-isotope-labeled
acylcarnitines as internal standards, were analyzed by flow-injection electrospray-ionization tandem mass spectrometry using amodel API4000 LC/MS/MS system（AB Sciex）. Precursor ion scanning using a product ion of m/z 85 was performed, andquantification was done using the peak heights of the respective acylcarnitines.
［Results& Discussion］In lymphocytes loaded with d31-palmitic acid, a series of labeled acylcarnitines, from d31-palmitoylcarnitine（d31C16）to d1-acetylcarnitine（d1C 2 ）, were observed, and the concentrations were determined. The amount ratios of d1C 2 to
d 31 C16（d 1 C 2 /d 31 C16）in patients with long-chain fatty acid oxidation disorders（VLCADD, CPT-II, TFPD）and MADD weredecreased significantly as compared with those in controls, and were thought to be a useful marker to assess fatty acid oxidation
ability. With regard to diagnosis of these disorders, metabolic blocks were assessed by the ratio of the accumulated labeledacylcarnitine to the decreased one； d31C16/d27C14 was elevated in CPT 2 D, d27C14/d23C12 in VLCADD, d29C16OH/d31C16 in TFPD,
and d 11 C 6 /d 1 C 2 in patients with MADD. In lymphocytes of CPT 1 D, however, the concentrations of d 31 C16 were decreased
because of the deficient activity of CPT1. In the present study, lymphocytes collected from an aliquot of heparinized blood varied
considerably, and the concentrations of labeled acylcarnitine derived from d31 -palmitate themselves were hardly evaluated. In
contrast with palmitate, caprylate can be metabolized in lymphocytes of CPT 1 D by the other transferase than CPT1. We
measured acylcarnitines in lymphocytes loaded with d31-palmitate and d15-caprylate, in addition, and found that, in CPT 1 D, theratios of d 7 C 4 /d 31 C16 value in lymphocytes loaded with d 31 -palmitate and d 15 -caprylate to d 7 C 4 /d 31 C16 value in lymphocytes
loaded with d31-palmitatealone were increased as compared to the ratios in controls.
［Conclusion］Enzyme defects can be detected by the labeled-acylcarnitine ratios designated to the disease. The present test
using lymphocytes loaded with labeled fatty acid is simple and quick, and useful in clinical settings.

Oral Session
Thursday, 20th SeptemberRoom B-1

15:00 － 17:00

Session 38: Mass Spectrometry for Metabolic Diseases
Chair:

Makoto Yoshino（Kurume University, Japan）
Seiji Yamaguchi（Shimane University, Japan）

S38-1640
16:40 － 17:00

Application of in-vitro probe acylcarnitine assay using tandem mass
spectrometry for the evaluation of mitochondrial fatty acid oxidation

Jamiyan Purevsuren, Hironori Kobayashi, Yuki Hasegawa, Kenji Yamada, Tomoo Takahashi,
Seiji Yamaguchi
Shimane University School of Medicine, Shimane, Japan

Keywords:
acylcarnitine, fatty acid oxidation disorder
Novel aspects:
In vitro probe assay（IVP）was developed for diagnosis of primary carnitine deficiency and carnitine palmitoyltransferase- 1
deficiency.IVP assay was applied for evaluation of fatty acid oxidation in various conditions.
Abstract:
Background: Fatty acid oxidation（FAO）disorder is a relatively common type of inherited metabolic disorders, and detected byblood acylcarnitine analysis using tandem mass spectrometry（TMS）. In vitro probe acylcarnitine（IVP）assay with cultured cells
and TMS is used for determination of FAO capacity to confirm diagnosis of FAO disorders. We applied the IVP assay to evaluate
the effects of endogenous and exogenous factors such as heat stress, cytokines, bacterial toxins or some drugs on FAO capacity
in the cells. Furthermore, we developed a strategy to make diagnosis of primary carnitine deficiency（PCD）or carnitine
palmitpoyltransferase- 1 （CPT 1 ）deficiency using IVP assay, for which the IVP assay diagnosis has been difficult.
Materials and Methods: Fibroblasts from healthy controls and patients with medium chain acyl-CoA dehydrogenase（MCAD）,
very long chain acyl-CoA dehydrogenase（VLCAD）, CPT- 1 and CPT- 2 deficiencies, glutaric acidemia type 2 （GA 2 ）and PCD
were examined for acylcarnitine（AC）profiles using in IVP assay. Acylcarnitine profiles were analyzed in various conditions, andintra- and extracellar lysates of fibroblasts. The followings are the modified conditions： a）various fatty acid substrates such asoctanoic（C 8 ）or palmitic acid（C16）loaded； b）various concentration of free L-carnitine（reduced（10 µmol/L）, physiological（50µmol/L）or excessive concentration（400µmol/L）loaded. Effect of endogenous factors, cytokines（IL- 1 β, IL-6, IL-10 and TNF-α ）and/or exogenous bacterial toxin, cereulide, on FAO were analyzed.
Results and Discussions: In healthy control, acetylcarnitine（C 2 ）, which is a final product of fatty acid oxidation, was the only
prominent metabolite when palmitic acid were loaded with excessive free L-carnitine. A substantial accumulation of specific ACs
was observed in the culture medium of cells with MCAD, VLCAD, CPT- 2 deficiencies and GA2. In this condition, however, AC
profile in cell cultured medium was not specific for PCD and CPT- 1 deficiency. It was revealed that the profile in intracellular
lysates with low concentration of free L-carnitine was diagnostic for PCD and CPT- 1 deficiency.
In the experiments with endogenous cytokines, AC profile in the presence of IL- 6 and IL-10 was similar to those without cytokines
in controls and patients with various FAODs. In the presence of IL- 1 β and TNF-α, however, C16 was significantly increased in
CPT- 2 deficiency, while C14 and C16 were elevated in VLCAD deficiency. Wide range ACs（C 6 -C16）were significantly increasedin GA2, whereas ACs were barely affected in MCAD deficiency and controls.
In summary, the current IVP assay is a simple and powerful method to make an accurate diagnosis of FAO disorders, and also agood method to evaluate FAO capacity in various conditions.

Oral Session
Thursday, 20th SeptemberRoom D

15:00 － 17:00

Session 39: MS Informatics for Quantitation
Chair:David Fenyö（New York University, USA）

S39-1500
15:00 － 15:40[Keynote Lecture] Detection of differential expression of splice variants in
the MaxQuant framework for quantitative proteomics

Jürgen Cox1,2, Nagarjuna Nagaraj1,2, Cornelia Schoenbauer1,2, Frank Schnorrer1,2, Matthias Mann1,2
1

Max Planck Institute for Biochemistry, Martinsried, Germany, 2Max Planck Institute for Biochemistry

Keywords:
Quantification, alternative splicing
Novel aspects:
Proteome-scale automated detection of quantitative differences between proteins from differentially spliced transcripts allows
distinguishing functionally redundant from functionally distinct isoforms.
Abstract:
Mass spectrometry based quantitative proteomics is becoming more comprehensive in terms of providing protein expression
values or changes in expression for the majority of proteins expressed in a cell. However, in most cases only a summary
quantification of all the protein isoforms originating from the same gene location is available. Biologists know many cases in which
the specific function of a gene is carried out by a particular splice variant, while other products from the same gene do not havethe same functional capabilities. Therefore is desirable in general to perform protein quantification on peptides derived fromexons or splice junctions that are specific to a certain spliced transcript.
Label-free or labeling-based peptide quantification is carried out with the MaxQuant software. A new MaxQuant module maps theidentified peptides to the ENSEMBL genome including to annotated splice variants. We define and apply a statistical test designed
to detect genes with significant differential regulation between at least two isoforms over two or multiple sample groups. Whilethe basic form of the test requires at least two peptides specific for an isoform, a modified version of the test allows in somecases for detection of splice variants that are distinguished by a single peptide.
Several high-resolution LC-MS data sets obtained with different Thermo Fisher Orbitrap platforms were obtained including the Q
Exactive and Velos platforms. Label-free as well as SILAC labeled data was included. The data originates from multiple different
species, including human, mouse and Drosophila. Raw data was processed with MaxQuant in the standard manner. The
quantitative peptide data was further processed with the Perseus module which is dedicated to the statistical analysis andsystems biology of proteome and other high-throughput data. The resulting normalized quantitative peptide data was further
analyzed with the newly developed splice-variant specific quantification and genome mapping module of MaxQuant. We detected
hundreds of cases where different splice variants have significantly different quantitative profiles over the measured samples,
implying different regulation and different biological functions. Several positive controls were detected where the MaxQuantoutput agrees with prior biological knowledge and many as yet unknown cases of differential splice variant regulation are found.
Comparison with deep sequencing data of the transcriptome reveals many cases where the transcriptome and proteome dataagree. In addition, specific cases of disagreement between transcriptomics and proteomics data were clearly evident. We analyze
data from a quantitative study of 28 different mouse tissues and discuss particular examples of correlating and non- or anticorrelating slice variants. Furthermore we investigate if there are significant differences between isoforms originating from thesame gene and isoforms encoded in different genomic locations. Finally, we applied our framework to functionally distinct muscle
types in Drosophila. Preliminary results indicated striking differences in the structural sarcomeric components, for instance
between leg muscles and flight muscles. These results are important to understand tissue specific transcriptional regulation anddifferential tissue specific splicing. They would have been very difficult to discover with traditional targeted approaches such as
immuno detection.

Oral Session
Thursday, 20th SeptemberRoom D

15:00 － 17:00

Session 39: MS Informatics for Quantitation
Chair:David Fenyö（New York University, USA）

S39-1540
15:40 － 16:00

Peptide Yields Depend on Digestion Conditions

Stein E Stephen, Yuxue Liang, Eric Yan

National Institute of Standards and technology, Gaithersburg, MD, USA

Keywords:
protein digestion, proteomics, variability, qa/qc
Novel aspects:
A quantitative determination of variations in peptide yields in a tryptic digestion for individual proteins is presented using digestsof N15-labeled proteins as internal standards.
Abstract:
Introduction： Quantitative proteomics is based on the assumption that concentrations of proteins are proportional to theirderived peptides. Therefore, changes in relative yields of peptides from a protein will cause apparent changes in concentrations of
the proteins themselves. We examine these changes by measuring relative peptide yields from tryptic digests of two different
plasma proteins under various digestion conditions using spiked-in isotopically-labeled mixtures peptides from digests of thecorresponding N15-labeled proteins.
Method： The proteins examined were human serum albumin and apoliproptein A1. They were each digested in trypsin for 2 and18 hours using up to 12 different digestion conditions. Denaturants included urea, guanidine（at low and high temperature）,
methanol, trifluoroethanol, rapigest and heat alone. Proteomics grade（alkylated）trypsin was generally used, but some runs
employed non-alkylated trypsin and Lys-C. Time course digestion studies were also done over 15 min. to 18 hours. Analysis wasdone with a Velos Orbitrap by monitoring relative MS 1 signals of labeled and unlabeled peptides, as identified by MS 2 analysis
using in-house software. Aliquots of N15-digest were added to digests dpme under varying conditions immediately after quenching
the digestions relative concentrations of the labeled peptides were therefore the same in each digest. Moreover, initial proteinsconcentrations were identical since proteins came from a single protein solution. Relative peptide yields of over one hundred
peptide ions were determined for each protein. Peptides included simple tryptic, missed cleavage, semitryptic and a variety ofmodifications. Measurement accuracy was better than 5 % as determined by comparing different charge states of the same
peptide, which should have the same relative concentrations of N15 peptides.
Results： Significant variations in yields of most peptides from trypsin digestion from the same protein were found with changing
digestion conditions. Some variations clearly result from different different typsin activities or digestion times. But most could not
be explained so simply. Almost all peptides varied at least a factor of two for the different digestion conditions, even whendigested for the same time period many varied far more. At the current stage of analysis, this complex behavior cannot be
expressed in rules, but appears to be a consequence of a variety of hard-to-separate factors including peptide release rates,
solubility and chemical reactivity. One trend did seem clear, variations increased with increasing peptide length. Furthermore,digest-to-digest reproducibility was not high, indicating that a great deal of attention will have to be made to ensure sample-tosample consistency of peptide yields even using the same materials and methods. Other implications of these results forquantitative proteomics are discussed.
Conclusion： Yields of tryptic peptides from proteins are variable and depend on both the method and peptide in a way thatcannot presently be predicted. Without specific validation, concentrations of ’proteotypic’ peptides cannot be assumed to beequal to their precursor proteins.

Oral Session
Thursday, 20th SeptemberRoom D

15:00 － 17:00

Session 39: MS Informatics for Quantitation
Chair:David Fenyö（New York University, USA）

S39-1600
16:00 － 16:20

Quantitative Proteomics of Embryonic Stem Cells Differentiation to
Cardiomyocytes

Ekaterina Mostovenko, André M Deelder, Christine L Mummery, Robert Passier, Magnus Palmblad
Leiden University Medical Center, Leiden, Netherlands

Keywords:
label-free, quantitative proteomics, stem cell differentiation, scientific workflows
Novel aspects:
New, high-throughput quantitative proteomics methodology applied in the investigation of protein expression and localization
during stem cell differentiation
Abstract:
Introduction
The pluripotency of embryonic stem cells（ESC）was demonstrated in mice in 1981 and much work has since been undertaken to
create from them mature（fully differentiated）, specialized somatic cells. In recent years, Mummery et al. successfully
differentiated human ESC（hESC）into cardiomyocytes using different growth factors, including activin A and BMP 4. Resulting cellaggregates beat spontaneously and have a direct electrophysiological comparison with primary human fetal cardiomyocytes inculture. Although we are now able to influence the development of stem cells, many details in the differentiation process are still
unclear. A better understanding of stem cell differentiation may lead to a better control of the differentiation process and tosuccessful myocardium regeneration therapies. The aim of this work was to follow in detail, changes in protein expression andlocation during differentiation of hESCs into cardiomyocytes using label-free quantitative proteomics. Specifically, we relied on a
recently developed and implemented FTICR-ion trap cluster designed for high throughput label-free quantitative proteomics tomeasure protein concentrations in many time points and biological replicates.
Methods
To obtain various stages of differentiating cardiomyocytes, hESCs were resuspended in defined differentiation medium containing
BPEL medium and growth factors BMP4, ActivinA, VEGF and SCF for the first three days in 96-well plates. Cells were collected after
0 （undifferentiated hESC）, 3, 7 and 15 days of incubation. The medium was removed, cells washed twice with warm（37°C）PBS
and transferred to Eppendorf tubes. The cells were then lysed in 1 % SDS in a hot（70 °C）ultrasonic bath（VWR Ultrasonic
Cleaner）for 15 min. Thirty µg of each lysate was loaded on a 10-lane 4 -12% NuPAGE(R) Bis-Tris gel（Invitrogen, Carlsbad, CA,USA）.
From each lane 48 slices were cut out using a custom-made cutter（The Gel Company, San Francisco, CA, USA）and digested ingel and then analysed using LC-MS/MS using splitless parallel NanoLC-Ultra 2 D plus ultra-high pressure liquid chromatographysystems（Eksigent, Dublin, CA）coupled on-line to amaZon ETD high-capacity 3 D ion traps（Bruker Daltonics, Bremen,Germany）.
Each time point and replicate was then analyzed on a 12 tesla solariX FTICR（Bruker）using the same type of column, but without
any preceding gel fractionation. The acquired data was converted to mzXML by compassXport and processed by a scientificworkflow in Taverna workbench, the core of the workflow derived from the Trans-Proteomic Pipeline. In the workflow, first thedata from the ion traps were searched with X!Tandem and SpectraST for peptide identifications which were then combined by
iProphet and assigned with accurate masses and ion intensities from the FTICR.
Results
We obtained a wide range of peptide identifications（~40,000 peptides, 5,200 proteins）for further use in combination with accurate
and quantitative FTICR data to generate protein expression profiles from 15 time point（sampling each day of incubation）andreplicates. Gene ontology analysis revealed that sample preparation is robust and reproducible with respect to the relative yieldsof membrane（22%）, intracellular（42%）, extracellular（10%）and nuclear proteins（26%）, with the number of proteins identified
in different cellular compartments being consistent between samples. The difference in protein expression profiles increased as afunction of differentiation stage. From 2,470 proteins presented in all samples, as expected, proteins involved in motor and channeltransporter activities increased in relative abundance in later time points, when cells become differentiated cardiomyocytes.
All steps of the data handling（from converting raw files to mzXML all the way to graphical visualization）were piped in one
workflow using Taverna. Such automated analysis led to an easier data processing and allowed us to deal with large datasets.

Oral Session
Thursday, 20th SeptemberRoom D

15:00 － 17:00

Session 39: MS Informatics for Quantitation
Chair:David Fenyö（New York University, USA）

S39-1620
16:20 － 16:40

Estimation of relative protein abundance and statistical analysis ofproteomic data from multiple iTRAQ experiments, to assess micronutrient
deficiencies.

Ingo Ruczinski, Keith P West, Robert N Cole, Kerry Schulze, Shelley Herbrich, Parul Christian,Jim D Yager, John D Groopman
Johns Hopkins University, Baltimore MD, USA

Keywords:
iTRAQ, statistical models, micronutrient deficiency
Novel aspects:
Development of novel robust, scalable, and efficient methods for relative protein abundance estimation and statistical inference,
applied to a chronically undernourished child population in South Asia.
Abstract:
Introduction
We investigate strategies to estimate relative protein abundance from multiple iTRAQ experiments. We show that using theavailable biological data instead of masterpools for normalization within and across experiments yields more efficient estimators.
We show how previously proposed linear mixed effects models can be improved and simplified for inference in case-control
studies and studies with quantitative outcome, producing simple, efficient, and scalable approaches to detect proteins associatedwith the outcome of interest. We illustrate our methods using data from replicate iTRAQ experiments on samples of pooled
serum, and from a large project carried out to define the nutriproteome（proteins in circulation that covary with nutrient
concentrations or respond to nutritional exposures）in a young, chronically undernourished child population in South Asia.
Methods
8 -plex stable isotope masstags（iTRAQ）were employed to identify and quantify serum proteins of 400 Nepalese children by highthroughput tandem mass spectrometry. Isotopically resolved masses in MS and MS/MS spectra were extracted with and without
deconvolution using Thermo Scientific Xtract software. Both data sets were searched against the RefSeq 40 database using Mascot（Matrix Science）through Proteome Discoverer software（v1.3, Thermo Scientific）. Peptide identifications from Mascot searches
were processed within the Proteome Discoverer to identify peptides with a confidence threshold 1 % False Discovery Rate（FDR）,
based on a concatenated decoy database search. Peptide identifications with the highest mascot score from the same peptide
matched spectrum before and after deconvolution by the Xtract function were used for quantitation of reporter ion intensities.
Results
A micronutrient deficient population of early school aged children, typical of the Terai of Nepal, has been established using 20
nutrient-indicators（including Vitamins A, B6, B12, copper, iron and zinc）and indicators of inflammation（including CRP）, alongwith their health profile and associated plasma proteome. With a 1 % false discovery rate, over 4600 proteins have been so far
identified in 400 child plasma samples at least once, reflecting the breadth of detection that is possible within the dynamic range
reached in this project. The vast majority of protein identifications, however, are based on single peptides and have been
identified in small numbers of children. Nonetheless, 140 proteins have so far been identified and quantified in every child, and an
additional 160 proteins have been quantified in more than 70% of all children.
Novel methods to estimate protein relative abundances were employed, accounting for dependencies of reporter ion intensities
within peptides, and peptides within protein. Newly devised linear mixed effects models were used to assess protein-nutrient
relationships, accounting for between experiment variability. We demonstrate that these methods provide a robust, scalable, andmore efficient alternative for statistical inference. For each nutrient indicator in our case study we assessed the nutriproteome ofcorrelated proteins（p-values < 0.05 and/or q-values < 0.10）, generating lists in excess of 100 proteins for some of the nutrients,
with very strong relationships emerging for many protein-nutrient combinations（some R^ 2 >0.75, p< 1 e-100）. We also definedprotein“signatures“（linear combinations of relative protein abundances）with predictive power for micronutrient status.

Oral Session
Thursday, 20th SeptemberRoom D

15:00 － 17:00

Session 39: MS Informatics for Quantitation
Chair:David Fenyö（New York University, USA）

S39-1640
16:40 － 17:00

Accurate mass quantitation of in vivo plasma samples using highresolution QTof and MSE data analysis across a global network

McDonald Stephen1, Mark Wrona1, Julie Laterreur2, Nigel Ewing3, Eric Lanlois2, Joanne Mather1,
Debadeep Bhattacharya1, Robert S Plumb1
1

Waters Corporation, Milford, USA, 2Vertex, Laval, Canada, 3Vertex, Cambridge, USA

Keywords:
DMPK, Bioanalysis, Metabolite Identification, Exact Mass, Quan/Qual
Novel aspects:
Unbiased, MSE methodology for obtaining in vivo high resolution quantitative datasets using UPLC-Qtof platforms in acollaborative environment.
Abstract:
The current realm of drug metabolism has been challenged with dealing with ever increasing need for sensitivity, selectivity andresolution. As instrument platforms have evolved to address and often exceed these basic tenets, laboratories are also beginningto look for additional ways to use the data more intelligently. QTof platforms equipped with MSE data analysis offer a balance ofscan speed, sensitivity and resolution have the ability to both provide rich qualitative datasets and put the data into context（thequantitative aspect）. This presentation focuses on the analysis of in vivo plasma PK with multiple lab, company, site, and country
variables on the Quant of platform generating datasets that can be used for both qualitative and more importantly quantitativedecision making.
Pharmaceutical compound（Propranolol）was dosed in rats at 75 mg/kg. Samples were protein precipitated, using a 2 ： 1
acetonitrile（containing IS）followed by centrifugation. Samples were frozen（-80 °C）and shipped to several locations. Samples
were analyzed on four different Acquity -Xevo Qtof UPLC-MS systems across two companies, four different sites in three different
geographies（Canada, USA and the United Kingdom）. PK curves for animals were reported with levels of ~0.5ng/mL to 1000 ng/mL（as measured by traditional QqQ approach）, a difference of ~ 4 orders of dynamic range in the presence of plasma matrix
background. Results presented will show the accuracy and precision（with standard curves and QCs）with which these analyses
were performed and the benefits of such an approach.
All Xevo G 2 -QTofs were operated in positive ESI resolution mode with a minimum measured resolution of 25,000（FWHM at m/z520）with an analysis mass range of m/z 50 to 1200 running at 10Hz. With MS E enabled to facilitate collection of both intact
precursor full scan data（for quantitative analysis and screening）and generate fragmentation data for all precursors（high collisionenergy, ramping from 10 to 50eV）, samples were effectively being scanned at 5 scans per second. The average chromatographic
peak width observed across the sites was 3 seconds, and this typically yielded a minimum of 10 points for both identified intact
precursors and internal standard（used for quantitation）as well as 10 points of fragment ion data（which is then available for
qualitative analysis and confirmation of putative metabolites）. Rat plasma from two rats, collected from 0 -24hrs were pooled（thisallowed more material to provide additional injections for analytical reproducibility, rather than measure biological variability
which is assumed to exist）. Pooled propranolol samples were quenched with acetonitrile containing propranolol-d 7 to provide
reliable and accurate quantitation of the dataset. Samples analysis showed extremely consistent injection reproducibility,
obtaining R2 values of >0.95 across several orders of dynamic range in the presence of complex matrix.
The methodology demonstrates the ability to capture a broadly applicable data set well suited for quick analyses where bothquantitative and qualitative data find utility in the drug metabolism environment.
This need for maximizing knowledge obtained from each analysis, management of the data itself, the ability to share this data with
scientific departments and leveraging expertise distributed, across sites, across companies and indeed across oceans is acontinuing theme across the field of drug discovery. This presentation will show new ways to conduct experiments that increase
the capabilities of modern DMPK laboratories.

Oral Session
Thursday, 20th SeptemberRoom E

15:00 － 17:00

Session 40: Environment II
Chair:

Takeshi Nakano（Osaka University, Japan）

S40-1500
15:00 － 15:40[Keynote Lecture] Powerful GC-ToF-MS Techniques for Quantification ofLegacy Pollutants and Screening and Identification of Emerging Pollutants

Peter S Haglund, Conny Danielsson, Mikael Harju, Patricia Moreira-Bastos, Ulrika OlofssonUmeå University, Umeå, Sweden

Keywords:
GC×GC, multi-reflectron ToF, PCBs, dioxins, emerging pollutants
Novel aspects:
Two-dimensional gas chromatography and ultra high-resolution mass spectrometry provide exceptional peak capacity, mass
resolution and mass precision and open new possibilities in environmental analyses.
Abstract:
Environmental samples often present a challenging task to the analytical chemists. The target compounds are generally present attrace levels in a complex cocktail of natural and anthropogenic compounds. In order to accurately quantify such trace compounds
it is essential to reduce the background, but also to efficiently separate the target compounds from each other and residual
matrix. Although some advances have been made in the streamlining of sample preparation, e.g. using simultaneous extraction
and clean-up, the focus on this talk will be on powerful GC-MS methods for quantitative and qualitative analysis of legacy andemerging pollutants.
Over the last decades, comprehensive two-dimensional GC（GC×GC）has evolved into a robust and exceptionally powerful
technique for group-type as well as within-group（isomer）separations. Group-type separations of petroleum hydrocarbons were
one of the first and most important areas of application. In the case of isomer separations, one of the more demanding tasks isthe complete separation of the 209 PCBs or 210 polychlorinated dioxins and furans（PCDD/Fs）. Although the quest still continues,
it has been possible to greatly enhance the separation. It is now possible to separate all dioxin-like PCBs from each other andfrom other PCBs, and separate all 2,3,7,8-PCDD/Fs from each other and from most other PCDD/Fs. It is also possible to perform
enantioselective analysis of PCB atropisomers（asymmetric tri- and tetra-ortho PCBs）. However, careful selection and optimizationof the column sets are essential. For PCB and PCDD/F analysis the coupling of a long efficient non-polar first and a shape-selective
liquid crystal second dimension column proved to be most successful； while for the atropisomers, the first column had to bechanged to a permethylated cyclodextrin column. In these separations, the target compounds were more and less retained,
respectively, by the liquid crystal phase than the potential interferences.
The high peak capacity of GC×GC ToF MS can also be utilized to perform comprehensive characterization of complex samples,e.g. extracts of soil from contaminated sites or water from sewage treatment plants（STPs）. However, the number of components
detected and tentatively identified in such non-target analyses are overwhelming and logical and efficient prioritization tools are
required to make such studies feasible. Recently, two different approaches were tested： a risk-based prioritization and aproperty-based classification. In the first, quantitative structure-activity relationships were applied to all tentatively identified
compounds in contaminated soil extracts and the measured concentrations were compared to the estimated no-effect levels to
obtain a risk-ratio. All compounds with high risk ratios were prioritized for confirmatory analysis and, if confirmed, further analyzed
in biota potentially affected by soil contaminants. The property based classification was also based on ration calculations, i.e. theconcentration ratio of STP effluent and influent water. Compounds with ratios much lower than 1 are removed to a great extend,
those with a ratio close to 1 are poorly removed, and those with a ratio above may actually be formed in the treatment process.
In this way, compounds that are classified as poorly removed or metabolites may be identified, which will aid in the improvementof existing and design of new STP processes.
Although ultimate confirmation requires authentic standards novel GC-MS techniques such as ultra-high resolution ToF-MS may be
used to provide valuable information. Such techniques deliver full-scan spectra at a resolution up to 50,000 and a mass precision of
1 ppm, which is sufficient to unambiguously determine elemental compositions of ions up to ca 150 amu（may be extended to ca.
500 amu using isotope abundance analysis）. This is generally sufficient to confirm or reject tentative structures and greatly
enhances the possibility to perform a correct manual interpretation.

Oral Session
Thursday, 20th SeptemberRoom E

15:00 － 17:00

Session 40: Environment II
Chair:

Takeshi Nakano（Osaka University, Japan）

S40-1540
15:40 － 16:00

Dioxin Food Crises and New POPs: Challenges in Analyses

Jef Focant

University of Liege, Liege, Belgium

Keywords:
Food； Screening； SectorHRMS； HRTOFMS； GCxGC
Novel aspects:
Comprehensive emerging POP analyses
Abstract:
When more than a million of broiler chickens suddenly and unexpectedly died in the eastern and midwestern parts of the UnitedStates in late 1957, the first dioxin crisis record was set. The 1999 Belgian dioxin chicken gate affair ultimately demonstrated theeconomic damage such a contamination episode could yield to and pushed the European Union（EU）to set an efficient and proactive monitoring program to ensure proper quality of the European food and feed web and try to maintain most of the
population below tolerable weekly intake. The current European Commission（EC）strategy relies on the implementation of
maximum（and action）residue levels（MRLs）for selected polychlorinated dibenzo-p-dioxins（PCDDs）, polychlorinateddibenzofurans（PCDFs）（both families actually called ’dioxins’）, and dioxin-like polychlorinated biphenyls（DL-PCBs）.
To ensure the adequate production of comprehensive and reliable data on the presence of PCDD/Fs dioxins and DL-PCBs in foodand feed, a screening-confirmatory approach was thus early adopted for the official control of the PCDD/F dioxins and DL-PCBs. In
practice, screening is most of the time performed using chemical activated luciferase gene expression（CALUX）bioassays,
response-binding assays（RBAs）based on the aryl hydrocarbon receptor（AhR）, although the sole confirmatory method is gas［ 1 ］. More thanchromatography coupled to 13C-labeled isotope dilution sector high resolution mass spectrometry（GC-IDHRMS）
10 years after the implementation of the screening-confirmatory approach, the analytical situation has drastically evolved becauseof advances in automation and hyphenation of parallel sample preparation techniques that allowed both cost and result delivery
time to be significantly reduced（down to 350 EUR and 24h, respectively）for the confirmatory GC-IDHRMS method.
Nevertheless, RBAs could and should now be used with non-restrictive sample preparation techniques that would allow most
toxicants present in the sample to interact with the AhR to give a general persistent organic pollutant（POP）toxicity information
rather than an estimation limited to dioxin regulation compliance. This would be much more biologically relevant and would allow
to enlarge current food safety practices to other known and unknown POPs. Sample showing high response for the biologicalscreening should be further analyzed in the hope of identifying new compounds. The extension of the list of target compounds tomore ’exotic’（un）suspected persistent molecules present in our food requires both chromatographic resolution and instrumental
limits of detection（iLODs）to be improved.
Using comprehensive two-dimensional GC（GCxGC）［ 2 ］or cryogenic zone compression（CZC）［ 3 ］GC coupled to HR time-offlight MS（HRTOFMS）operating in full scan（FS）mode［ 4 ］could nicely complement the classical GC-sector IDHRMS performing
in selected ion monitoring（SIM）used for target analyses. This would open the possibility to screen for other compounds（organochlorine pesticides, halogenated flame retardants, GC-amenable perfluorinated compounds, ）than the one under current
regulation without compromising sensitivity. Recent advances in coupling between GCxGC and HRTOFMS nicely put this approach
one step further as it allows to produce elemental composition data for unknown compounds present in complex mixtures.
Furthermore, when considering halogenated compounds, such a system can be granted by a tremendous improvement insensitivity（back to low fg iLODs）by favoring resonance electron capture（REC）when operating in negative chemical ionization（NCI）rather than electron impact（EI）. Major recent analytical advances in the GC-MS of emerging GC-amenable POPs in thecontext of prioritization of new targets for food control will be highlighted.
［ 1 ］Eppe G et al.（2005）, Encyclopedia of Mass Spectrometry, vol. 8 ： Elsevier, Amsterdam（Ch. VI- 3 ）, pp 531-541［ 2 ］Focant J-F et al.（2005）J Chromatogr A 1067： 265［ 3 ］Patterson Jr DG et al.（2011）J Chromatogr A 1218： 3274
［ 4 ］Shunji H et al.（2008）J Chromatogr A 1178： 187

Oral Session
Thursday, 20th SeptemberRoom E

15:00 － 17:00

Session 40: Environment II
Chair:

Takeshi Nakano（Osaka University, Japan）

S40-1600
16:00 － 16:20

New Perspectives in the Mass Spectroscopy Determination of Dioxin-like
Substances in Environmental and Food Samples

Angel Garcia-Bermejo2, Manuela Abalos1, Lisa Mattioli1, Maria Jose Gonzalez2, Belen Gomara2,
Esteban Abad1
1Lab. of Dioxins, IDAEA-CSIC, 2Department of Instrumental Analysis and Environmental Chemistry, IQOG-CSIC

Keywords:
dioxin-like substances, triple-quadrupole, GC-QqQ（MS/MS）, HRGC-HRMS
Novel aspects:
A novel approach for the analysis of dioxin-like substances in environmental and food samples based on the use of GC-QqQ（MS/MS）is compared to reference techniques based on HRMS.
Abstract:
Due to an increased concern of the dietary exposure of dioxin-like compounds to the general public, the accurate determinationof these compounds has become of great interest. These compounds are found in the environment and food and feed matrices atvery low levels therefore analytical techniques and instrumentation providing precision, sensitivity and adequate limits of detection
are necessary.
In the past dioxin-like compounds have been analyzed with gas chromatography coupled to single quadrupole and ion trap mass
spectrometry, two-dimensional gas chromatography coupled to micro electron capture detection, and time-of-flight mass
spectrometry. These methods are alternatives to the current reference instrumentation of high resolution gas chromatographyhigh resolution mass spectrometry（HRGC-HRMS）for the analysis of many persistent organic pollutants, mainly dioxin-likecompounds. In general, dioxins are analyzed at 10,000 resolution, ionized with a positive electron impact（EI+）source andquantified based on the isotope-dilution method. Magnetic sector remains the choice instrument for the analysis of dioxins as itallows for the necessary sensitivity at very low levels and the high resolution to separate the peaks. Although HRGC-HRMS iscommonly used to analyze for dioxin-like compounds, this instrumentation is costly and high maintenance therefore alternative
instrumentation are continuously under development1. Gas chromatography coupled to tandem mass spectrometry with a triple
quadrupole analyzer（GC-QqQ（MS/MS））seems to potentially have the sensitivity and selectivity compared to HRGC-HRMS 2,3 .
Transitions from different precursors to product ions have been evaluated at several collision energies in order to determine theoptimal conditions for the analysis of PCDD/Fs and DL-PCBs by GC-QqQ（MS/MS）. Results are compared to those obtained by
GC-HRMS in order to assess the applicability of this technique for the analysis of dioxins in environmental and food samples.
Comparative analyses were performed on a TRACE GC Ultra gas chromatograph（Thermo Fisher Scientific, Milan, Italy）equippedwith a triple quadrupole（TSQ Quantum XLS, Thermo Fisher Scientific, Bremen, Germany）and on a Trace GC Ultra gas
chromatograph（Thermo Fisher Scientific, Milan, Italy）coupled to a DFS high resolution mass spectrometer（Thermo Fisher
Scientific, Bremen, Germany）controlled by an Xcalibur data system.
For optimization of the multiple reaction monitoring（MRM）method, different transitions were studied in order to select the mostintense and, if possible, to achieve the highest number of identification points. For PCDD/Fs, the loss of the COCl group was themost abundant when collision induced dissociation（CID）voltages around 25 and 30 V were applied. Collision energies werealways higher for PCDFs. In addition, other transitions were observed and studied when CID was set to higher values（40-50 V）.
Similar behaviour was observed for DL-PCBs, with the most intense transitions from the molecular cluster to the loss of two
chlorine atoms for the congeners investigated.
Acknowledgements
This study was supported by MICINN（project AGL2009-09733）and the Comunidad Autónoma of Madrid（Spain）and European
funding from FEDER programme（project S2009/AGR-1464, ANALISYC-II）.
References
1. Hernández F, Sancho J.V., Ibáñez M, Abad E, Portolés, Mattioli L（2012）Anal Bioanal Chem
2. Commision Regulation（EC）1883/2006； Off. J. Eur. Union L 364：32-43
3. Commision Regulation（EC）152/2009； Off. J. Eur. Union L 54： 1 -130

Oral Session
Thursday, 20th SeptemberRoom E

15:00 － 17:00

Session 40: Environment II
Chair:

Takeshi Nakano（Osaka University, Japan）

S40-1620
16:20 － 16:40

GC-MS studies on POPs in Brazilian Ecosystems

Joao Paulo M Torres1, Jose Lailson-Brito2, Paulo R Dorneles1, Rodrigo O Meire1, Larissa S Cunha1,
Daniele Botaro1, Giselle C Saldanha3, Claudio E Azevedo e Silva1, Dayse Aline M Rocha1, Olaf Malm1,
Karl W Schramm4, Begona Jimenez5

Universidade Federal do Rio de Janeiro, Brazil, 2Universidade do Estado do Rio de Janeiro - MAQUA, Brazil, 3Instituto
Federal de Educacao Tecnologica - Rondonia, Brazil, 4Helmholtz Centrum, Munich, Germany, 5Instituto de Quimica
Organica General - CSIC, Spain

1

Keywords:
GC-MS, POPs, PCBs, OCPs, PCDD/PCDFs
Novel aspects:
Using GC-MS, Hot Spots of POPs of Pollution were found in the Amazon, on fishes from tropical rivers and in Coastal Zones ofBrazilAbstract:
Environmental contamination by POPs where investigated in the biota of different Brazilian ecosystems, including the Amazon
equatorial forest in the northern part of the country, tropical river basins, coastal zones and some enclosed bays located near
densely populated areas. In the Amazon and on tropical river basins, we have focused our work on different kinds of fishes that
represent the main protein source for the local population. In costal zones, we collected samples of several species of marine
mammals（e.g.： dolphins and porpoises）as well as one species of tuna（Thunnus albacares）, one piscivorous bird species（Sulaleucogaster）sampled from 3 （three）different archipelagos and two species of bivalves（Perna perna and Nodipecten nodosus）
obtained from aquaculture established inside coastal bays and lagoons. Most of this work was done in cooperation with foreignlaboratories under academic cooperation and joint research projects where the fat extracts obtained with soxhlet apparatus and/or pressurized automatic extraction devices（e.g.： Dionex or FMS）. After clean-up using acidic silica gel and florisil columns, themain equipment used in these laboratories was the high resolution gas chromatography, operating in NCI（Negative Chemical
Ionization）mode and coupled to low resolution mass spectrometer（e.g.： Quadrupole）. Among several OCPs, PCBs and thepesticide DDT and its main metabolite DDE were found in all of the edible fish tissues（e.g.：Prochilodus sp.）samples analyzed in
the Amazon and in the tropical rivers may represent a health risk to the local population. Levels of POPs pesticides and PCBs arealso present in hair samples taken from this population. Regarding marine mammals, we could identify in coastal dolphins thepresence of high levels of POPs on the blubber of the animals, including a whole bunch of organochlorinated pesticides,
brominated flame retardants（e.g.：PBDEs）and also moderate levels of dioxins and furans, and in this case, a high resolution mass
spectrometer（“magnetic sector“）equipment was used. The overall pattern of contamination is so clear and separate quite well
3 （three）different bays that we can even say the place where the animal lives, only by looking to it´s POPs results. A pelagic
animal tends to have less contamination than coastal animals. Taking in account the work we did on the avian predatory specie,
their eggs are also very contaminated near Rio de Janeiro city, with reducing concentrations in birds eggs sampled far from themain land. On the other hand, ocean pelagic fishes like tuna as well as the cultivated bivalves seems not to concentrated highlevels of pollutants in their edible tissues, even if they grown in highly impacted ecosystems. This work represents part of theeffort and the commitment of different scientists from different Universities in Brazil to keep tuned with the utmost developmentof this field of work and to produce high quality data in order to help in the protection of tropical environments. Taking this inaccount, another application of these mass spectrometer techniques by our group is the deposition of legacy and current-use
pesticide on the top of mountains and plateau areas, we found that DDT, PCBs, dioxin/furans, several POPs pesticides as well as
endosulfan and chlorpyrifos residues are being deposited over these tropical mountains that are mostly located on natural parks.
Nowadays, since cattle grazing activities are present in these areas, in order to cope to the GAPs project and also in order to
respond to official demands on scientific results, we are presently collecting raw milk from these areas to see if this contaminationis also being transmitted to the food chain. Acknowledgements： CNPq, CAPES, FAPERJ, UERJ and UFRJ for invaluable support on
our research activities.

Oral Session
Thursday, 20th SeptemberRoom E

15:00 － 17:00

Session 40: Environment II
Chair:

Takeshi Nakano（Osaka University, Japan）

S40-1640
16:40 － 17:00

Structural Analysis of Cyclodextrin Inclusion Complexes Formed in
Nonpolar Solvents

Toshiyuki Kida, Haruyasu Asahara, Takuya Iwamoto, Mitsuru Akashi
Osaka University, Suita, Japan

Keywords:
cyclodextrin, inclusion complex, nonpolar solvent, structure
Novel aspects:
This is the first report on the structural analysis of cyclodextrin inclusion complexes formed in nonpolar media.
Abstract:
Cyclodextrins（CDs）have played a crucial role in various fields including supramolecular chemistry and analytical science due totheir unique properties to form inclusion complexes with a great variety of molecules. However, inclusion complex formation with
CDs has been limited to aqueous media and several kinds of polar organic media. On the other hand, the effective guest inclusion
into the CD caviry in nonpolar media has not been achieved yet, possibly because the main driving force for the guest inclusion ishydrophobic interactions and/or van der Waals interactions between the guests and the CD cavity, and thus the enormous
amounts of nonpolar solvents become a strong competitor for inclusion within the CD cavity.   Recently, we found that
6 -O-modifiedbeta-CD formed inclusion complexes with polychlorinated aromatic compounds in nonpolar media including
benzene and cyclohexane, possibly through the dipole-dipole interactions between the CD cavity and the guest molecule, as wellas the spatial fit of the guest into the CD cavity. In this presentation, we report the inclusion complex formation between
6 -O-modifiedbeta-CD and various aromatic guests in nonpolar media, and the structural analysis of the resulting inclusion
complexes by mass spectrometry.
Heptakis（ 6 -O-triisopropylsilyl）-beta-CD（TIPS-beta-CD）and heptakis（ 6 -O-tert-butyldimethylsilyl）-beta-CD（TBDMS-beta-CD）
were used as CD hosts. Inclusion complex formation between the CD hosts and the aromatic guests in nonpolar solvents were
evaluated by NMR spectroscopy. The association constants between the CD hosts and the aromatic guests were determinedby 1 H-NMR titration method. These CDs effectively formed 2 ： 1 inclusion complexes with pyrene in benzene-d 6 andcyclohexane-d12 with considerably high association constants. The 2 ： 1 inclusion complex formation between TIPS-beta-CD andpyrene in nonpolar solvents was also confirmed by the APPI-MS analysis. Crystallographic study of the TIPS-beta-CD/pyrene
inclusion complex obtained from the benzene solution showed the formation of a unique ternary complex among TIPS-beta-CD,
pyrene, and the solvent. Interestingly, the pyrene molecule forms a sandwich-type complex with two benzene molecules throughpi-pi interactions, and is located at the center of the dimer cavity. The crystalline structure suggested that the interaction between
the solvent and pyrene affects the mode of pyrene inclusion within the CD dimer cavity.

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 31: Native Mass Spectrometry and Structural Biology

PTh-001

11:10 － 12:20

Development, Evaluation and Comparison of Calibration Standards for
MALDI-MS in the > 100 kDa m/z Range

Simon Weidmann1, Konstantin Barylyuk1, Stefanie Maedler1,2, Renato Zenobi1
1

ETH Zurich, Zurich, Switzerland, 2Current address: York University, Toronto, Canada

Keywords:
high-mass MALDI, calibration, concatenated proteins
Novel aspects:
The use of recombinantely expressed concatenated proteins is a excellent possibility for calibration in high-mass MALDI-MS. The
masses of the calibrants are well known and span a wide m/z range.
Abstract:
Matrix-assisted laser desorption/ionization mass spectrometry（MALDI-MS）is a very versatile tool for studying protein-protein orprotein-DNA complexes and for the determination of their stoichiometry and interaction sites. The use of MALDI-MS for such
purposes has certain advantages over electrospray ionization（ESI）MS, since the spectra obtained consist of mainly low charge
states, which yield direct information about mass and stoichiometry of the sample under investigation. However, the low charge
states of the ions also lead to high m/z ratios, generally well above 100 kDa. Up to now, no calibrants are commercially available
for such a high m/z range. A suitable calibrant needs to fulfill some requirements： it must have a high and well-known mass andshould be detectable with good sensitivity and high resolution. The m/z range that is covered by the calibrant or its multimers
should be as wide as possible. Extension of the calibration range combining standards from different substance classes is possible,
but should be avoided, since such combinations lead to signal suppression caused by different ionization properties and thus thehandling of the calibration system becomes difficult.
Potential candidates that fulfill these requirements are proteins, which have either monomeric subunits with high masses or form
nonspecific multimers or complexes over a wide m/z range. Several possible calibrants were investigated including bovine
thyroglobulin, a homodimeric protein with a total molecular weight of 660 kDa, protein histidine phosphatase, and GroEL. These
two proteins form nonspecific multimers up to the decamer or pentamer, respectively. Evaluation of these candidates showedthat either the mass range covered was not wide enough or that the signals were too broad to be useful.
To obtain a new calibration standard a protein expression vector was genetically engineered and expressed in E. coli. This vector
consists of three concatenated copies of the maltodextrose binding protein（MBP）encoding gene and allows for the expressionof MBP3. The system is designed as such that the concatemer MBP3 can be cleaved into MBP and MBP2 subunits. Furthermore, aspecific interaction site is implemented to allow for the selective covalent dimerization of MBP2 or MBP3 via a disulfide bond. Since
the primary sequence is well-known, the observed signals can directly be used as calibration points.
MBP concatemers show improvements up to a factor of four in terms of peak width compared to other calibrants. The calibration
range covers the m/z range from 40 kDa（MBP）to 250 kDa（MBP6）. Optimization of the settings chosen for analysis might also
generate nonspecific multimers of the calibration standards and therefore expand the application range at least to 0.5 MDa.

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 31: Native Mass Spectrometry and Structural Biology

PTh-002

13:30 － 14:40

Relative electrospray response factor in homodimer-monomer equilibrium
system of DNA oligonucleotides

Konstantin Barylyuk1, Xueshu Xie2, Renato Zenobi1
1

ETH Zurich, Zurich, Switzerland, 2Karolinska Institutet, Stockholm, Sweden

Keywords:
electrospray, non-covalent, DNA oligonucleotides, quantification
Novel aspects:
A new model system based on monomer-dimer equilibrium in DNA oligonucleotides is introduced to directly determine relativeelectrospray response factor experimentally, which enables accurate binding affinity quantification in non-covalent complexes.
Abstract:
ESI-MS allows one to directly quantify the affinity in non-covalent complexes since all the species present in solution can readily
be detected and monitored in mass spectrum. The relative peak intensities of binding partners and complex usually correlate withthe abundances of interacting species in solution. However, differences in ionization efficiency, ion transmission, and detection
efficiency can disturb the peak ratios and compromise the result of the binding affinity determination. While equal responses canbe assumed for non-covalent complexes where a small ligand binds to a macromolecular receptor in a confined deep binding
pocket as, for instance, in case of enzyme-substrate, enzyme-inhibitor, and receptor-hormone complexes, different responses are
expected in the case of macromolecular oligomeric complexes.
We studied the relative response factors（R）in a monomer-dimer equilibrium. DNA oligonucleotides were chosen as a model
system for a number of reasons：（i）DNA exists in two distinct forms of the single strand（ssDNA）and the double helix（dsDNA）；（ii）the transition between the ssDNA and dsDNA happens in an all-or-none fashion and can be described as a simple monomerdimer equilibrium；（iii）the factors stabilizing the double helix are very well understood, so that the Gibbs free energy of
dimerization can be precisely determined experimentally or reliably predicted for a given nucleotide sequence and solution
conditions. From the known Gibbs free energy value the association constant（Ka）can be calculated.
A set of model DNA oligonucleotides with very close chemical composition but different nucleotide sequence was synthesized
and analyzed with nano-ESI mass spectrometry using a hybrid quadrupole-time-of-flight instrument（Q-TOF Ultima, Micromass,
Manchester, UK）in both positive and negative ion modes. For every sample, a dilution series was prepared in order to cover theconcentration range from high-nanomolar to middle-micromolar values. The acquired ESI-mass spectra were baseline-corrected,
smoothed and deconvoluted using the maximum entropy algorithm. The monomer-dimer peak ratios were calculated and used toestablish titration plots. A double-parametric non-linear regression was derived based on the expression for Ka and the materialbalance, and used to fit the experimental data. The K a parameter was fixed at the theoretically predicted value for a given
oligonucleotide and the R parameter was determined from the fit.
Since the chemical structure and composition of the analyzed DNAs is very close, and a dimer is built of two identical subunits
that interact through a large interface（a very typical case for biomolecular oligomeric complexes）, we expected very similar
ionization mechanism and no difference in ion transmission or detection efficiency for all the tested samples. The relative response
factors R should therefore be very similar in all cases. However, the R values determined for the positive mode varied substantially,
while those for the negative ion mode appeared to be very close to each other and were all below 1, which is consistent with ahigher response from the monomer. In the positive ion mode, analytes are charged via protonation, and in case of DNA
protonation sites are localized in the dimerization interface. The variation in R determined for the positive ion mode data cantherefore be attributed to the disturbance of the chemical equilibrium during ionization.

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 31: Native Mass Spectrometry and Structural Biology

PTh-003

11:10 － 12:20

Protein Flexibility is Key to Cisplatin Cross-linking in Calmodulin

Huilin Li, Stephen A Wells, J E Jimenez-Roldan, Rudolf A Römer, Yao Zhao, Peter J Sadler,Peter B O’Connor
University of Warwick, Coventry, UK

Keywords:
Cisplatin； Cross-linking； Calmodulin； Flexibility analysis；
Novel aspects:
Flexibility simulation as a fast and low-cost computational method can be a very useful tool for predicting cross-linking pairs inproteins and facilitating MS data analysis.
Abstract:
Chemical cross-linking in combination with Fourier transform ion cyclotron resonance mass spectrometry（FTICR MS）has
significant potential for studying protein structures and protein-protein interactions. Previously, cisplatin has been shown to be aprotein cross-linking reagent and cross-links multiple methionine（Met）residues in apo-calmodulin（apo-CaM）. However, theinter-residue distances obtained from NMR structures are inconsistent with the measured distance constraints by cross-linking：
Met residues lie too far apart to be cross-linked by cisplatin. Here, by combining FTICR MS with a novel computational flexibility
analysis, the flexible nature of the CaM structure is found to be key to cisplatin cross-linking in CaM. In both calcium-containing
and calcium-free forms of CaM, it is found that the side chains of Met residues can be brought together by flexible motions. The
possibility of cisplatin cross-linking calcium-containing CaM is then confirmed by mass spectrometry（MS）data. Therefore,
flexibility analysis as a fast and low-cost computational method can be a useful tool for predicting cross-linking pairs in protein
cross-linking analysis and facilitating further MS data analysis. Finally, flexibility analysis also indicates that the cross-linking of
platinum to pairs of Met residues will effectively close the nonpolar groove and thus will likely interfere with the ability of CaM tobind to protein targets, as was proved by comparing assays for cisplatinmodified/unmodified CaM binding to melittin. Collectively,
these results suggest that cisplatin cross-linking of apo-CaM or CaCaM can inhibit the ability of CaM to recognize its target
proteins, which may have important implications for understanding the mechanism of tumor resistance to platinum anti-cancer
drugs.

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 31: Native Mass Spectrometry and Structural Biology

PTh-004

13:30 － 14:40

High resolution hydrogen/deuterium exchange mass spectrometry maps
contact surfaces of VEGF and a novel recombinant monoclonal antibody
fragment (CIGB-166A)

Marta Vilaseca1, Marina Gay1, Andrey Dyachenko2, Claudio Diema1, Mar Vilanova1, Michael Goldflam2,
Humberto Lamdan3, Ernest Giralt2, Gabriel Padron3

Mass Spectrometry Core Facility, Institute for Research in Biomedicine, IRBBarcelona, Barcelona, Spain, 2Institute for
Research in Biomedicine, IRBBarcelona, Barcelona, Spain, 3Centro Ingenieria Genetica y Biotecnologia, CIGB, La Habana,
Cuba
1

Keywords:
Hydrogen, Deuterium, Exchange, VEGF, epitope
Novel aspects:
It is the first time that VEGF has been studied by HDX-MS and that this methodology has been applied to determine the epitopeof VEGF against a monoclonal antibody fragment
Abstract:
Vascular endothelial growth factor（VEGF）is an endothelial cell-specific angiogenic and vasculogenic mitogen. VEGF also plays arole in pathogenic vascularization which is associated with various clinical disorders, including cancer and rheumatoid arthritis.
A novel recombinant monoclonal antibody（CIGB-166A, Mab）has been designed as a VEGF antagonist, preventing its interaction
with its receptors and therefore acting as a potential drug for cancer treatment. The antibody fragment recognizes the VEGF
dimer exclusively but its binding site to VEGF is not yet known. Mapping the antigen epitope is a key step in defining antibody
specificity, predicting cross-reactivity, in assay development, rational vaccine design, and in understanding the fundamental
aspects of protein-protein interactions.
Amide hydrogen/deuterium exchange（HDX）coupled with proteolysis and high resolution MS has become a powerful method tostudy protein dynamics, protein-ligand interactions, and protein-protein interactions. In general, when performing antigen-antibody
experiments by HDX-MS, regions of the antigen with different deuterium content in the presence or absence of the antibody aredefined as the epitope.
Here we describe the HDX followed by semi-simultaneous disulfide bond reduction-proteolysis and on-line desalting LC-MS/MS
strategy used to determine the epitope of the VEGF-Mab interaction and highlight the challenges to achieve high peptide
coverage for good resolution protein structural information.
In VEGF, the monomers are covalently linked by two symmetrical disulfide bonds. Moreover, three more intrachain disulfide
linkages and intra and inter hydrogen bonds compose a highly structured dimer. Deuterium labeling of VEGF alone has alreadyprotected exchangeable regions corresponding to the contact surfaces of both monomers and those protected by the protein
folding itself. Therefore, deuterium labeling of VEGF-Mab implies that regions in VEGF protected by HD exchange are not only
those corresponding to the antigen-antibody interaction but also to those regions protected by the complex structure.
In this study, we have designed two complementary labeling strategies to get insight into the VEGF interaction sites with theantibody fragment. Strategy 1 performs the deuterium labeling of VEGF and Mab separately, before inducing their interaction.
The complex is then incubated in aqueous buffer, therefore interchanging all deuterium except those in the binding regions and inother protected regions due to the tridimensional structure of the complex. Strategy 2 induces the complex first and thanperforms deuteration. In this case, non labeled regions would indicate VEGF-Mab sites of interaction and other protected regions,
including the surface contact regions between monomers. Deuterated and non-deuterated control experiments of VEGF werealso performed in the absence of the antibody. Added to the constrain to use proteases working at low pH and low temperature
to limit back-exchange during proteolysis, proteins that contain disulfide bridges present additional difficulties to be digested.
Disulfide linkages can render extensive segments of the protein inaccessible to proteolysis, and the available proteolytic segments
would be too long to allow localization of the site（s）of H/D exchange. Therefore, the disulfide bonds need to be reduced for
effective HDX-MS analysis. The reduction cannot be performed before or during the HDX reaction, as it can change protein
conformation. Here we performed a semi simultaneous reduction and digestion after H/D exchange and under quench conditions（ 0 C, pH 2.5）. On-line desalting was then carried with an in-house double valve system, servicing a trapping and analyticalcolumn for sample resolution. All system was maintained at 0 C and column eluent was coupled to an LTQ-FT Ultra mass
spectrometer. Data processing was done with HDX Sierra Analytics software. Percentage of deuteration for each region of theprotein was calculated by comparing the results of the HDX experiments and putative regions of interaction VEGF-Mab were
determined.

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 31: Native Mass Spectrometry and Structural Biology

PTh-005

11:10 － 12:20

Following amyloid fibril formation by ion mobility spectrometry mass
spectrometry: are all pathways the same?

Charlotte A Scarff, Lydia Young, Aneika C Leney, Lucy A Woods, Sheena E Radford, Alison E Ashcroft
University of Leeds, Leeds, United Kingdom.

Keywords:
amyloid fibril, ion mobility, noncovalent
Novel aspects:
This study constitutes a thorough comparison between the structures and properties of protein monomers, oligomers and fibrils
associated with three different amyloid diseases, and potential methods of assembly inhibition.
Abstract:
Amyloid fibrils are formed when peptides or proteins self-assemble into filamentous aggregates. Such fibrils are associated withover 25 neurodegenerative and pathological human diseases. Despite extensive studies, the mechanism by which these proteinsassemble into highly ordered, cross-beta structures remains elusive, and whether the final fibrils or the intermediate oligomericspecies are responsible for toxicity is a key matter for on-going debate. The precursor proteins in each case are dissimilar in termsof their native folds and primary sequences, yet the amyloid fibrils produced share common structural and chemical features, suchas a cross-beta organisation of the polypeptide chain and the ability to bind dyes such as thioflavin-T and Congo red. The
observation of a common structural architecture for amyloid fibrils has motivated efforts to elucidate the molecular mechanismsof fibril formation in vitro with the aim of revealing possible targets for therapeutic intervention.
Electrospray ionisation ion mobility spectrometry mass spectrometry（ESI-IMS-MS）is an ideal technique for monitoring changes inthe structure and population of individual biomolecules within heterogeneous samples in real-time. Here, ESI-IMS-MS has been
employed to study the folding properties of a range of protein monomers, all of which form disease-related amyloid fibrils in vivo,
and to monitor the self-assembly of the protein monomer into oligomers over time. For each assembly pathway the mass,
stoichiometry, and collision cross-sectional area of all the species detected have been determined. Additionally, the fibril assembly
process has been monitored simultaneously using chemical tests and the architecture of the final fibrillar structures investigateduse of electron microscopy. Further insights into the structure of the final fibrils have been obtained by the use of limited
proteolysis followed by ESI-MS. For each protein, a non-amyloidogenic protein of similar sequence has been used as a control.
A comprehensive comparison between three different amyloidogenic proteins has been made in terms of the conformational
flexibility of the protein monomer, the range and dynamics of the oligomers formed en route to fibrils, and the structure of the
final fibrils. Ataxin- 3 is a 42 kDa protein that contains an N-terminal Josephin domain followed by two ubiquitin interacting motifs,
a polyQ stretch and a variable C-terminal region. It is the protein responsible for the neurodegenerative polyQ disease
Spinocerebellar ataxia type 3, or Machado-Joseph disease. Beta- 2 -microglobulin is a 99-residue, 7 -beta strand, 11.9 kDa protein
which is associated with the condition dialysis-related amyloidosis. Islet amyloid polypeptide is a 37-residue peptide hormone
produced by beta cells of the pancreas, and is one of the most amyloidogenic peptides known.
The data generated provide insights into three different fibril-producing systems. We have compared the conformational
properties of the three different wild-type protein monomers, in addition to the properties of associated protein mutants whichhave significantly different fibril-forming propensities. The array of oligomers generated in each case, their dynamic nature（assessed by sub-unit exchange）, and trends in the relationship between their collision cross-sectional areas with the increasing
number of protein sub-units, are all discussed. The structural architectures of the fibrils formed from the three proteins under
different conditions in vitro have been compared by electron microscopy and limited proteolysis-ESI-MS. Furthermore, a range ofsmall molecules has been used to test amyloid inhibition mechanisms for each protein.

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 31: Native Mass Spectrometry and Structural Biology

PTh-006

13:30 － 14:40

Structural Characterisation of Protein Complexes by Chemical Crosslinking and Ion Mobility-Mass Spectrometry

Antonio Calabrese, Danielle Williams, Yanqin Liu, Tara L Pukala
University of Adelaide, Adelade, Australia

Keywords:
Protein Complex； Ion Mobility Mass Spectrometry； Chemical Cross-linking； Negative Ion Mass Spectrometry
Novel aspects:
Advances in protein complex structure determination by a combination of IM-MS and cross-linking； Chemical cross-linking
analysis in the negative ion mode.
Abstract:
Biological processes are carried out and controlled primarily by a complex, precise network of interacting proteins, which
therefore offer potential as novel targets for therapeutic intervention in human diseases. In order to understand and modulate theprotein cellular machinery, it is useful to have a molecular understanding of its components and their binding interactions.
However, structural characterisation of such intricate protein assemblies often proves difficult due to their heterogeneity andtransient nature, and analytical limitations of current structural biology techniques.
Ion-mobility mass-spectrometry（IM-MS）has greatly enhanced our ability to study these systems and extract structural information
regarding complex stoichiometry, size and topological arrangement. In addition, chemical cross-linking can be used to discern
binding interfaces and provide restraints for molecular modelling applications in which model structures can be generated.
Here we present data demonstrating the effective use of a combination of mass spectrometric approaches to describe low
resolution structural models of protein assemblies not amenable to other technologies. We will describe the design, synthesis andapplication of novel chemical cross-linking reagents for use with negative-ion IM-MS, built around a disulfide bond that undergoes
facile cleavage in the negative mode. Such cross-linking methodology in combination with IM-MS collision cross-section
measurements can probe the topological arrangement of protein complexes of unknown structure, and we will describe
application to systems under study currently in our laboratory including calcium-calmodulin-peptide regulatory complexes, humantranscription factor assemblies such as TFIIH, and aggregation products of alpha synuclein.

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 31: Native Mass Spectrometry and Structural Biology

PTh-007

11:10 － 12:20

The effect of gas molecules for the structural dynamics of GAPDH
revealed by mass spectrometry coupled with H/D exchange

Tatsuya Yamamoto, Yasuaki Kabe, Makoto Suematsu

Japan Science and Technology Agency / Keio University, Tokyo, Japan

Keywords:
H/D exchange, Gas molecules, Protein dynamics
Novel aspects:
The effect of gas molecules for the structural dynamics of GAPDH revealed by mass spectrometry coupled with H/D exchange
Abstract:
To find new gas-sensing systems, we searches heme-binding proteins in human cell homogenate using heme-modified beads,
which decrease non-specific binding. In this experiment, we found that Glyceraldehyde 3 -phosphate dehydrogenase（GAPDH）
bound with the heme-modified beads having an iron ion. GAPDH, forms homotetrameric complex（MW： 143kDa）, is a memberof glycolytic enzymes. The activity is regulated by gas molecules such as Nitric oxide（NO）and hydrogen sulfate（H2S）, throughthe post-translational modification, feedback inhibition, and interactions with regulating proteins and ligands. Recently, we
discovered a new regulation system for GAPDH by heme and carbon monoxide（CO）. Human GAPDH undergoing suchmodifications does not only alter its catalytic activity but also triggers entering nucleus to control transcription. Since therelationship between the structural information and the aforementioned functional alterations remains largely unknown, we aimed
to determine structural movement of gas-treated enzyme in solution by hydrogen/deuterium（H/D）exchange coupled with pepsin
digestion.
GAPDH from Human was overexpressed as GST fusion protein in E. coli, and purified using affinity chromatography and filtration
after protease digestion. H/D exchange was started by mixing GAPDH solutions with D2O at 22 OC. The pH of the mixtures were
7.1 and the H：D atomic ratio was 1 ：9. GAPDH in D2O was incubated for 60 min. H/D exchange was quenched by adding aceticacid（pH 2.5）, and digested at 0 OC by pepsin for one minute. The digested GAPDH solution was frozen with liquid nitrogen.
Frozen sample was rapidly melted by mixing with 10mg/ml 3,5-dimethoxy- 4 -hydroxycinnamic acid in 50 % acetonitrile involving 0.1% trifluoroacetic acid, and set up on MALDI-TOF MS（AXIMA Performance, Shimadzu）at 104 Pa.
Peptides of the pepsin-digested GAPDH were separated in the flight tube, and 34 peaks were identified by MS/MS spectra
applying CID and TOF/TOF. We analyzed local protein dynamics of GAPDH using mass shift depending on deuterium
incorporation into these peptide, covering 96 % of the whole sequence. To discuss these structural dynamics in three dimensions,
H/D exchange map was made by painting each fragment depending on the deuterium incorporation ratio in the X-ray crystal
structure. The H/D exchange map clearly showed differences of incorporation ratio into GAPDH among untreated, NO, heme-CO,and H 2 S treated. It is known that nitrosylation at Cys152 by NO triggers apoptosis through the entering nucleus to control
transcription after interaction with Siah 1 protein. NO-treated GAPDH incorporated more deuterium atoms into interaction region（residues from Val220 to Ala238）with Siah 1. On the other hand, H/D exchange of GAPDH binding with heme-CO showed thatthe complex forms very rigid structure, which is more than GAPDH-heme complex without CO. Therefore, CO stabilizes GAPDH
structure using combination with heme. In this study, we elucidate the structural movement and stability of GAPDH controlled bygas molecules, and discovered a new function of CO.

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 31: Native Mass Spectrometry and Structural Biology

PTh-008

13:30 － 14:40

A systematic survey of protein kinase specificity by in vitro
phosphoproteomic approaches

Haruna Imamura1, Naoyuki Sugiyama2, Masaki Wakabayashi1, Yasushi Ishihama1
1

Kyoto University, Kyoto, Japan, 2Keio University, Tsuruoka, Japan

Keywords:
phosphorylation, kinase/substrate, proteomics, motif sequence
Novel aspects:
Revealing how protein kinases recognize their substrates using LC-MS/MS-based phosphoproteomics coupled with in vitro kinase
assay.
Abstract:
Cellular signal transduction regulates various biological functions. Phosphorylation, caused by protein kinases, is one of the mostubiquitous post-translational modifications, and the kinase/substrate pairs are the main components of cellular signaling network.
Recent development of liquid chromatography-tandem mass spectrometry（LC-MS/MS）with effective enrichment methods forphosphopeptides has enabled to identify thousands of phosphorylation sites on substrates. Although all kinases catalyze
essentially the same phosphoryl transfer reaction, they are known to display remarkable diversity in their substrate specificity.
However, MS-based phosphoproteomics cannot afford to obtain upstream kinase information while it excels at identifying theirsubstrates. This makes difficult to intertwine the kinases with their substrates for revealing the whole picture of signaling network.
In this study, we examined how protein kinases recognize their substrates using LC-MS/MS-based phosphoproteomics coupledwith in vitro kinase assay.
The proteins were extracted from cultured cells, and were subsequently dephosphorylated by alkaline phosphatase to eliminate
the intrinsic phosphate group. After dephosphorylated proteins were converted into their substrate peptide forms, the in vitro
kinase assay was performed using recombinant kinases. After the enzymatic digestions, the control and reacted samples werechemically labeled with stable isotope containing tags individually and were mixed. Phosphorylated peptides were enriched by
TiO2 chromatography followed by LC-MS/MS analysis to identify and quantify peptides phosphorylated by the spiked kinases.
In this experiment, recombinant kinases were reacted in vitro with two sets of the substrate pool, which contain the wide rangeof substrate peptides with the variety of sequence and length. As a first step, we evaluated the effect of the substrate forms on
kinase recognition using peptide substrates with full or partial motif sequences. Motif sequence is known to exist aroundphosphorylated residue and is recognized by their upstream kinases. This examination validates the specificity and affinity ofkinases to sequences in substrates. As motif sequences are mainly classified into three groups, which are rich in acid residues（eg.
Asp and Glu）or basic residues（eg. Lys and Arg）motif, and motifs with Pro. Among those motifs, we have generated the basic oracid motif-disrupted peptides using glutamyl endoproteinase Glu-C（protease V 8 ）, lysyl endoproteinase Lys-C and trypsin. V 8
specifically digests peptides at C-termini of Asp and Glu. For base-rich motifs, Lys-C and trypsin, which specifically cleaves at
C-termini of Lys and Lys/Arg, respectively, were selected. Next, for the evaluation of the substrate length effect to kinaserecognition, we have made two kinds of peptide substrate pools using the combination of Lys-C and trypsin. The proteins digested
by Lys-C are supposed to be longer（cleaved at C-termini of lysine）than the peptides that are doubly digested by both of Lys-C
and trypsin（cleaved at C-termini of Lys and Arg）. The increase or decrease of phosphopeptides in respective substrate pool was
evaluated by comparing with the result in intact proteins.
Eight kinases including both serine/threonine and tyrosine kinases such as PKA, CaMK, CDK 2 /CycA2, Erk2, CK 1 d, CK 2 a2, EPHB 1
and JAK 2 were performed with fragmented peptide sets and proteins. Consequently, we found that the contribution of thesubstrate sequence and length to the substrate specificity were highly varied, depending on each kinase. According to theobtained properties of each kinase, we extracted a set of rules governing the substrate specificity. The information will be usefulto predict upstream kinases from the properties of phosphorylated peptides identified by LC-MS/MS, leading to unveil signaling
network.

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 31: Native Mass Spectrometry and Structural Biology

PTh-009

11:10 － 12:20

Cellobiose dehydrogenase represents a real challenge for hydrogen/deuterium exchange mass spectrometry

Petr Man1,2, Alan Kadek1,2, Roland Ludwig3, Petr Novak1,2, Petr Halada1

1Institute of Microbiology v.v.i., Prague, Czech Republic, 2Faculty of Science, Charles University in Prague, Czech
Republic, 3University of Natural Resources and Applied Life Sciences, Vienna, Austria

Keywords:
H/D exchange, FT-ICR mass spectrometry, sample preparation, cellobiose dehydrogenase
Novel aspects:
A special sample treatment including disulfide bond reduction, deglycosylation and optimized digestion strategies is required prior
H/D exchange mass spectrometry on cellobiose dehydrogenase protein.
Abstract:
Cellobiose dehydrogenase（CDH）, the only currently known extracellular flavocytochrome, is produced by a number of wooddegrading and phytopathogenic fungi. CDH is typically a monomeric glycoprotein consisting of two domains joined by a flexible
linker region. The larger catalytic（dehydrogenase）C-terminal domain is FAD-associated while the smaller（cytochrome）
N-terminal domain contains a haem. The unique combination of one haem and one flavin within a single protein molecule gives
CDH the ability to exchange electrons with both two- and a diversity of one-electron acceptors and thus makes CDH a perspective
enzyme for applications in biotechnology and in the field of biosensors and biocatalysis.
To date the crystal structure of full-length flavohaemoprotein has not been solved, because crystallisation of the intact CDH ishampered by the flexible interdomain linker. Therefore, the crystal structures of the flavoprotein and the cytochrome domain were
elucidated separately and a model structure of the intact CDH was suggested by molecular modelling and docking studies.
However, the exact orientation of the domains in native conformation, possibly being different from the theoretical model, has notbeen described yet.
Our research strategy is based on the combination of hydrogen/deuterium exchange and mass spectrometry. This technique isroutinely used to study protein conformation changes and protein interaction surfaces. However, majority of the proteinsappearing in publications are rather easy cases because of their smaller size and absence of heterogeneous modifications. CDH ison the other hand large protein（100 kDa）representing a real challenge due to presence of several disulfide bonds andN-glycosylation. In order to unify glycosylation but retain protein native structure we employed deglycosylation using EndoH.
Disulfide bond reduction was achieved by addition of TCEP into the quench solution followed by short incubation on ice.
However, this treatment was not offering complete sequence coverage. As we found out, the N-terminal part of the protein（cytochrome domain）requires high concentration of denaturing agents to become digested. But even this treatment is notenough（coverage approx. 70%）. Therefore we are now working on other digestion strategies involving slightly elevated digestion
temperatures and use of different acid proteases. H/D experiments will start as soon as we find conditions providing completesequence coverage.
Acknowledgement:
The work was supported by grant P206/12/0503（GACR）and by Institutional Research Project RVO61388971（IMIC）.

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 31: Native Mass Spectrometry and Structural Biology

PTh-010

13:30 － 14:40

Improved analysis of biopharmaceutical samples using an MS-only
Orbitrap mass spectrometer

Maciej P Bromirski, Jan-Peter Hauschild, Eduard Denisov, Eugen Damoc, Alexander Makarov
Thermo Fisher Scientific, Bremen, Germany

Keywords:
Orbitrap, Biopharma, Intact Proteins
Novel aspects:
An Orbitrap MS-only instrument was optimized for detection of proteins and a secondary charge detector was added to improve
AGC.
Abstract:
Introduction
This work is dedicated to improve capabilities of an MS-only Orbitrap mass spectrometer（second generation Exactive）in analysis
of very complex mixtures and biopharmaceutical samples. As intact proteins create fast decaying beat patterns in FourierTransform（FT）image current detection systems, the instrument needs to be modified in order to get ability to detect the mostabundant signal from the very first beat. When dealing with very complex samples, a dedicated C-Trap charge detection（CTCD）
system is shown to improve the accuracy of the prescan-based automated gain control（AGC）. Together with the advanced
signal processing, the hardware improvements show a significant improvement for several applications.

Methods
To detect the very first beat within the transient, the insulators within the Orbitrap assembly were modified to allow very fast
voltage settling after the ion injection pulse. The design of the entire assembly was made completely symmetrical with respect tostray capacitances. The pre-amplifier circuitry was redesigned to improve recovery time from saturation caused by pulses during
injection. In conjunction with advanced signal processing shorter transients can be used to record only the very first beat at highabundance while maintaining high resolving power. To improve AGC, periodic detection of ejected charges by a charge detector
was employed. This allows an internal monitoring and correction of the AGC results when necessary.
Preliminary data
Although voltage switching during ion injection into the Orbitrap analyzer disturbs image current detection, the implemented
hardware changes reduced the duration of this disturbance from four milliseconds to 250 microseconds. With this improvement,
the entire first beat of the image current of intact proteins can be used for data acquisition.
Intact Humira antibody（Abbott Laboratories）was used to show the increase in signal due to detection of the entire first beat of
the transient. Shorter transients showed higher signal-to-noise ratio as ion signal is concentrated in first few milliseconds of thetransient and remainder of transient adds nothing except noise. This also has an influence on mass calibration of such heavy
proteins.
In clinical samples, used for the rapeutical drug monitoring（TDM）, higher molecular species coeluting with the analytes ofinterest may lead to reduced sensitivity and mass accuracy of the instrument due to wrong estimation of the number of charges in
the C-trap by the AGC. It could be shown that with the CTCD system in place, AGC remains effective even under criticalconditions so that mass accuracy and correct signal intensity maintain under all conditions.

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 31: Native Mass Spectrometry and Structural Biology

PTh-011

11:10 － 12:20

Topology of cytochrome P450 2B4 and cytochrome b5 interaction andmembrane orientation by photoactivable nanoprobes/chemical crosslinking and mass spectrometry

Tomas Jecmen1,2, Monika Koberova2, Bozena Kubickova2, Petr Novak1,2, Petr Hodek2, Jiri Hudecek2,
Miroslav Sulc1,21Institute of Microbiology, Prague, Czech Republic, 2Charles University, Prague, Czech Republic

Keywords:
photoactivable/chemical cross-linking, membrane protein, HRMS
Novel aspects:
The determination of transient interaction between two membrane proteins using combination of photoactivable/chemical crosslinking and HRMS.
Abstract:
The photoactivable nanoprobe or chemical cross-linking in combination with the high resolution mass spectrometry（HRMS）has
been employed to characterize contacts and membrane regions involved in the transient interaction between two membraneproteins, cytochrome P450 2 B 4 （P450 2 B 4 ）and cytochrome b5. The methionine analog, L- 2 -amino-5,5-azi-hexanoic acid（pMet）,
incorporated to the membrane domain of recombinant cytochrome b5 was used as the photoactivable nanoprobe. The chemical
cross-linking by soluble carbodiimide（EDC）, a“zero length cross-linking reagent“, was employed to form the covalent amidebond between closely located or interacting amino and carboxy-group of amino-acid residues. Although, the photoactivation or
chemical cross-linking were accomplished in equimolar catalytic complex of P450 2 B 4 ：cytochrome b5 reconstituted in DLPC
vesicles, SDS-PAGE revealed  the covalent P450 2 B 4 ：cytochrome b 5 hetero-dimers in molar ratio 1 ：1, 2 ： 1 and 1 ：2. The
excised protein bands from SDS-PAGE were analyzed（after in-gel digestion with trypsin or chymotrypsin）using LC-HRMS（FTICR）
mass spectrometry to identify crosslinked amino-acid residues. The protein native structures（at the same conditions of proteinreconstitution）were confirmed by observation of the stimulatory effect on enzymatic P450 2 B 4 O-depentylation activity of
7 -pentoxy resorufine in both systems.
Based on our findings with the EDC cross-linking, the determined covalently linked amino-acid residues were located on P450
2 B 4 proximal surface. On the other hand, using pMet incorporated into membrane domain of cytochrome b5 revealed aminoacid residues situated on predicted P450 2 B 4 hydrophobic regions. The computed in silico models of P450 2 B 4 ：cytochrome b5
complex using amino-acids detected in cross-links suggest two different types of cytochrome b5 orientations： the first allowing
potential cytochrome b 5 electron donation to P450, the second one only cytochrome b 5 induced modulation of P450 structuralchanges. Obtained data are discussed from the aspect of P450 function in organism, the participation in xenobiotics detoxification,
drug metabolism and unfortunately carcinogen activation.
The project was supported by Grant Agency of the Czech Republic (P207/12/0627), by Charles University in Prague (UNCE #42)
and by Institutional Research Project RVO61388971.

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 31: Native Mass Spectrometry and Structural Biology

PTh-012

13:30 － 14:40

Structural Examination of hexadecameric and octameric G-quadruplexes,
exchange-studies and H/D-exchange on Supramolecular Architectures

Dominik Sattler1, José M Rivera2, Christoph A Schalley1
1Freie Universität Berlin , Berlin , Germany, 2University of Puerto Rico, Río Piedras, San Juan, Puerto Rico

Keywords:
G-quadruplexes, H/D-exchange, Self-assembly； Ionophores, ESI-FTICR-MS
Novel aspects:
H/D-exchange on supramolecular complexes. Examination of the exchange-behavior between G-quadruplexes.
Abstract:
The synthesis and self-assembling properties of 8 -aryl- 2 ’-deoxyguanosine derivatives are described. Our studies suggest that aproperly placed acetyl group can increase the stability and specificity of the resulting Guanosine-quadruplex supramolecules byenhancing noncovalent interactions such as hydrogen bonds and π-stacking. The self-assembly of lipophilic dG-analogues could
be modulated by replacement of the H 8 in the guanine base with a functionalized phenyl group.
The Guanosine-rich sequence of telomeres, ends of chromosomes, have a propensity to form G-quadruplex structures in vitro. In
the areas of supramolecular chemistry and nanotechnology, the groups of Davis and Gottarelli have made various lipophilic
G-analogues for the construction of self-assembled ionophores, self-assembled liquid crystals, and other molecular devices. The
stated importance of Guanosine-quadruplexes makes it appropriate to determine structure function relationships with
G-analogues. In particular, G-analogues that are modified at the guanine base could be used to modulate the properties ofoligonucleotides or other self-assembled structures containing them.
In our studies we show different buildingblocks of 8 -aryl-20-deoxyguanosine
derivatives, which form homoleptic, self-assembled, supramolecular complexes with discrete sizes characterized by ESI-FTICR-MS.
With microreactor experiments in front of our ESI-source we were able to estimate the kinetics of exchange between complexesof different buildingblocks in solution.
It also shows that mixtures of hexadecameric and octameric complexes lead to mixed hybrid complexes, which have adodecameric arrangement. In following MS/MS-studies and theoretical calculations we are also able to draw conclusions about
the exchange-mechanism of the buildingsblocks exchange between two complexes.
In ongoing H/D-exchange-studies were perform in gas-phase H/D-exchanges on G-quadruplexes with NH4 + and K+ and subsequent
MS/MS-studies to recive a better understanding of the different intrinsec nature of the hydrogenbonds network in the complex.

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 31: Native Mass Spectrometry and Structural Biology

PTh-013

11:10 － 12:20

Identification of on-pathway intermediates of beta-2 microglobulin fibril
formation

Lucy A Woods, Claire J Sarell, Sheena E Radford, Alison A Ashcroft
University of Leeds, Leeds, UK

Keywords:
ion-mobility, protein-protein, protein-ligand
Novel aspects:
Use of ESI-IMS-MS to identify species on-pathway to the fibril formation of beta- 2 microglobulin
Abstract:
The ability of amyloidogenic proteins to assemble into large, insoluble fibrillar species is associated with over twenty-five different
disease states, such as Alzheimer’sdisease, Parkinson’s disease and type II diabetes. In each case, a different protein or peptide
self-assembles to form amyloid fibrils, although the final fibril architecture is distinct. The formation of amyloid fibrils in vitro can beinitiated using a variety of conditions, for example, by denaturation, upon the addition of metal ions or through the introduction of
mutations and deletions within the protein sequence. Under each of these conditions, various monomeric protein confirmations
and oligomeric precursors exist together in solution in dynamic equilibrium. Identifying which of these species are key precursors
to amyloid formation is vital to understand more about the mechanism of formation, an aspect which is essential for thedevelopment of amyloid inhibitors to be used as therapeutics, in addition to characterising potentially toxic oligomeric species.
However, identifying which states initiate fibril formation can be challenging, as few techniques are able to both separate andcharacterise such transient species.
Here, the power of ion mobility spectrometry, combined with mass spectrometry, has been harnessed to achieve the separationand characterisation of both monomeric and oligomeric precursors of beta- 2 microglobulin fibril formation. Through comparing
species key to fibril formation under a number of different conditions, such as through variation of pH and ionic strength,
differences in the pathway of assembly have been identified. Further insights into the importance of these oligomeric species havebeen gained through analysing the effect of a small-molecule inhibitor of amyloid formation, revealing these oligomeric species are
crucial to initiate amyloid formation. In addition, a comparison of the final fibril architecture formed under different conditions has
been achieved through the use of limited proteolysis, revealing subtle differences in final fibril structure that are indistinguishable
using other techniques.

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 31: Native Mass Spectrometry and Structural Biology

PTh-014

13:30 － 14:40

Mapping the interaction between Hsp90 and TOM by chemical crosslinking coupled to mass spectrometry

Fabio C Gozzo, Alana R Figueiredo, Carlos I Ramos, Leticia M Zanphorlin, Lisandra M Gava
University of Campinas, Campinas, Brazil

Keywords:
cross-linking, Hsp90, TOM
Novel aspects:
Chemical cross-linking was used to map the interaction region of Hsp90/TOM complex
Abstract:
The molecular chaperone Hsp90 is a homodimer, where each monomer contains a N-terminal domain（~30kDa）that contains a
ATP binding site, followed by an intermediate domain（~30 kDa）and a C-terminal domain（~20 kDa）that is responsible for the
dimer formation. The C- and N-terminal domains are also responsible for the interaction with several binding partners. The list of
known interacting partners are extensive, however little is known about how these proteins interact with Hsp90.
A recent approach to study protein-protein interactions is the use of chemical cross-linking coupled to mass spectrometry to map
the interaction regions of complex subunits. This technique has several experimental advantages, namely high sensitivity, shortanalysis time and large applicability.
In this work, we described the study of Hsp90/TOM complex by chemical cross-linking and the complex model generated fromcross-linking data. The model is compatible with several experimental data already obtained for this complex and reveals how
these proteins interact with each other.

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 31: Native Mass Spectrometry and Structural Biology

PTh-015

11:10 － 12:20

Chemical Cross-linking Coupled to Mass Spectrometry Applied to theCharacterization of the Interaction Domains in FimX-PilZ Complex

Mariana Fioramonte1, Cristiane R Guzzo2, Shaker C Farah2, Fabio C Gozzo1
1

University of Campinas, 2University of São Paulo

Keywords:
Chemical cross-linking, PilZ, FimX, Structural Proteomics
Novel aspects:
Chemical cross-linking was used to characterize the PilZ-FimX complex.
Abstract:
The PilZ protein was originally identified as necessary for type IV pilus（T 4 P）biogenesis. Since then, a large and diverse family ofbacterial PilZ homology domains have been identified, some of which have been implicated in signaling pathways that control
important processes, including motility, virulence and biofilm formation. Furthermore, many PilZ homology domains, though not
PilZ itself, have been shown to bind the important bacterial second messenger bis（ 3 ’-> 5 ’）cyclic diGMP（c-diGMP）. Someworks show that PilZ binds to the EAL domain of FimX, which regulates T 4 P biogenesis and localization in other bacterial species.
These interactions were confirmed in NMR, two-hybrid and far-Western blot assays and are the first interactions observed
between any PilZ domain and a target protein.
Chemical cross-linking coupled to mass spectrometry is an attractive tool for structural proteomics when no high resolution
method is applicable to the system of interest. In recent years, this approach has been widely used in the study of protein folding,identification of binding partners, monitoring of conformational changes upon ligand binding, characterization of surfaces inprotein complexes and probing for solvent residues accessibility. In this work we used chemical cross-linking along with highresolution method（X-Ray crystallography）to map the interaction between FimX and PilZ proteins. The complex crystal was
obtained and chemical cross-linking was used to probe the complex structure in solution.

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 31: Native Mass Spectrometry and Structural Biology

PTh-016

13:30 － 14:40

Inter-molecular interactions of SMC proteins studied by MS under nondenaturing condition and HDX-MS

Susumu Uchiyama1, Masanori Noda1, Rie Takino1, Yuya Miyahara1, Kazuki Kawahara2, Yuki Hosokawa1,
Daisuke Motooka1, Shota Nakamura1, Tadayasu Ohkubo1, Kiichi Fukui1
1

Osaka University, 2Nara Women’s University

Keywords:
MS under non-denaturing condition, HDX-MS, chromosome protein, condensation
Novel aspects:
Dimer formation and interaction sites of SMC proteins in solution are firstly identified.
Abstract:
Formation of metaphase chromosomes is essential for proper segregation of genomic information to daughter cells. Pioneering
work of Hirano showed that structure maintenance of chromosomes（SMC）proteins play a central role in chromosome
condensation. In case of higher eukaryotes, three non-SMC proteins bind to SMC dimer to form condensin complex composed offive subunits. Our proteome analysis of human metaphase chromosomes（Uchiyama, et al., JBC, 2005, Takata et al, 2007 etc.）and
RNAi analyses of identified chromosome proteins（Matsunaga et al, Cell Rep., 2012, etc.）showed condensin is one of major
proteins in chromosome structure.
Here, we analyzed inter-molecular interactions of hinge domain of human SMC（hSMC-h）by mass spectrometry under nondenaturing condition and hydrogen exchange mass spectrometry（HDX-MS）.
MS under non-denaturing condition showed that hSMC 2 -h forms homo-dimer while most of hSMC- 2 forms hetero-dimer with
hSMC- 4 h. These dimer formations were confirmed in solution by analytical centrifugation sedimentation velocity. The homo-dimer
formation was disrupted in hSMC 2 -h-GD variant of which three glycine residues were replaced to aspartic acid residues.
We then carried out HDX-MS analyses of hSMC 2 -h homodimer and hSMC 2 -h-GD variant. The results specified three regions
which would be responsible for the dimer formation. The regions well reflected the interaction sites for the homo-dimer formationin the x-ray crystal structure of hSMC 2 -h homodimer. The comparison of HDX-MS results between hSMC 2 -h homodimer andhSMC 2 -h/hSMC 4 -h hetero-dimer identified regions which would be involved in the formation of the hetero-dimer.

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 34: MS Informatics for Identification and Characterization

PTh-017

11:10 － 12:20

StavroX - a software for analyzing crosslinked products in protein
interaction studies.

Michael Götze, Jens Pettelkau, Sabine Schaks, Konstanze Bosse, Christian Ihling, Fabian Krauth,Romy Fritzsche, Uwe Kühn, Andrea Sinz
Martin-Luther-University,Halle,Germany

Keywords:
crosslinking, protein, complexes, analysis, software
Novel aspects:
StavroX is suitable for a wide variety of crosslinking-MS applications. It combines automatic data analysis with an easy-to-use
graphical user interface.
Abstract:
Chemical crosslinking in combination with mass spectrometry has matured into an alternative approach to derive low-resolution
structural information of proteins and protein complexes. Yet, one of the major drawbacks of this strategy remains the lack ofsoftware that is able to handle the large MS datasets that are created after chemical crosslinking and enzymatic digestion of thecrosslinking reaction mixtures. Here, we describe a software, termed StavroX, which has been specifically designed for analyzing
highly complex crosslinking datasets. The StavroX software was evaluated for three diverse biological systems：（ 1 ）the complex
between calmodulin and a peptide derived from Munc13,（ 2 ）an N-terminal ß-laminin fragment, and（ 3 ）the complex between
guanylyl cyclase activating protein- 2 and a peptide derived from retinal guanylyl cyclase. We show that the StavroX software isadvantageous for analyzing crosslinked products due to its easy-to-use graphical user interface and the highly automated analysis
of mass spectrometry（MS）and tandem mass spectrometry（MS/MS）data resulting in short times for analysis. StavroX is expected
to give a further push to the chemical crosslinking approach as a routine technique for protein interaction studies.

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 34: MS Informatics for Identification and Characterization

PTh-018

13:30 － 14:40

Electrospray mass spectrometry for detailed mechanistic studies of analdolreaction catalyzed with L-proline

Mohammed Wasim Alachraf, Wolfgang Schrader
Max Planck Institut for Coal research

Keywords:
Organocatalysis, L-proline, Mechanistic study, Electrospray-ionization.
Novel aspects:
Mechanistic studies of catalyzed aldol reaction
Abstract:
In the last few years, organocatalysis has emerged as a new catalytic methods based on metal-free organic molecules. In manycases, these small compounds give rise to extremely high enantio selectivities. Usually the reactions can be performed under anaerobic atmosphere with wet solvents. The catalysts can be easily synthesized in both enantiomerically pure forms and they areoften more stable than enzymes or other bioorganic catalysts. Also, these small organic molecules can be anchored to a solid
support and reused more conveniently than organometallic/bioorganic analogues. Herein, we present a mechanistic study of theformation of the aldol condensation product as side product of organocatalytic aldol reaction with L-proline by ESI-MS, and ESIMS/MS.
The mechanistic studies of the organocatalytic aldol reaction was accomplished by ESI-MS using a Thermo TSQ Quantum Ultra
AM triple quadrupole.
Reaction of hexanal was carried out with L-proline as a catalyst（20 % mol）in acetone, which was acting both as solvent andreactant. The reaction was performed at room temperature for 24h. Samples were taken after different time intervals for mass
spectrometric study. The reaction was performed at different conditions and deferent techniques were applied（e.g. online
monitoring reaction）to study the mechanism of this reaction.
The aldol reaction produce two deifferent products, an addition product and a condensation product, the first one is the desired
product of this reaction. The aim of this work is to understand the formation mechanism of the side product and hopefully
determine how to avoid production of this side product. There are three potential mechanisms to form this condensation product.
The first mechanism is an aldol mechanism, in which the catalyst forms an enamine intermediate with the ketone（here, acetone）.
This intermediate reacts in the next step with the aldehyde to yield the second intermediate, which is then hydrolyzed in the last
step to form the final condensation product and the regenerated catalyst. The second potential mechanism is a Mannich
mechanism, in which the catalyst reacts with the aldehyde to form an iminium cation as a first intermediate. An acetone molecule
attacks this intermediate in a nucleophilic attack to produce the second intermediate. In the last step the catalyst splits off to form
the final condensation product. The third mechanism consists of the first steps of both the aldol and Mannich mechanisms, where
the enamine intermediate of the aldol mechanism reacts with the iminium intermediate of the Mannich mechanism to form thesecond intermediate. This intermediate has two molecules of catalyst, from which one catalyst splits off and the other hydrolyzed
to form the final condensation product. The first step in determining the proper mechanism of such a reaction is to find selective
intermediates that can be used as reaction markers for each mechanism. Reaction markers of two pathways were discovered.
Although the empirical formula and the mass of the intermediates of both mechanisms is identical, it is possible to determinate
the intermediates structure by using ESI-MS/MS according to McLafferty rearrangement fragmentation. Using different techniques,
like deuterium exchange in the retro-reaction we were able to exclude one of the two remaining pathways to ensure that only
one pathway controls this condensation reaction.
The final results will be presented.

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 34: MS Informatics for Identification and Characterization

PTh-019

11:10 － 12:20

LC/MS and surface ionization techniques in classification of red wine

Petr Bednar1, Barbora Papouskova1, Lucie Hartmanova1, Karel Hron2, Jan Stavek3, Josef Balik3,
Renata Myjavcova1, Petr Bartak1, Eva Tomankova3, Karel Lemr1

Regional Centre of Advanced Technologies and Materials, Department of Analytical Chemistry, Faculty of Science,
Palacky University, Olomouc, Czech Republic, 2Department of Mathematical Analysis and Applications of Mathematics,
Faculty of Science, Palacky University, 3Department of Post-Harvest Technology of Horticultural Products, Faculty of
Horticulture in Lednice, Mendel University in Brno
1

Keywords:
LC/MS, Anthocyanin, Variety, MALDI, nanoDESINovel aspects:
LC/MS profiling of anthocyanins combined with AAEs and PCA gives robust data for variety classification. MALDI and nanoDESI
allow fast wine evaluation directly from intensity of signals of particular anthocyanins.
Abstract:
Profiling of flavonoids and products of their transformation is effective tool for evalutation of authenticity and quality of foodproducts. This task, however, can hardly by done without a reliable and robust method providing long-time consistent results.
Combination of（ultra）high performance liquid chromatography with high resolution tandem mass spectrometry undoubtedly
represents such a method. Recently we have shown on a complete set of certified red wine varieties that LC/MS profiling ofanthocyanins in combination with evaluation of activity of enzymes involved in anthocyanin biosynthesis（AAEs）［ 1 ］andadvanced statistical data treatment（clr transformation, PCA）allows efficient classification of red wine varieties in northernmost
European viticultural area. To our knowledge the study is the first evidence of data structure stability related to anthocyanin
profile over long-time（evaluation of four vintages； analysis of each in respective year）and comprehensive wine sample set ingiven region［ 2 ］. Main features and relations observed during the study can be summarized as follows. Higher content ofpeonidin derivatives（a higher activity of related methyltransferase）refers to a potential occurrence of teinturiers（i.e. varieties
Alibernet, Neronet and Rubinet）when included in the set of analyzed wine samples. It was found out that variety Blaufrankish has
low content of acetylderivatives of anthocyanins（lower activity of acetyltransferase）and this fact can be utilized for resolution ofthis variety. Two-dimenzional display of data（PCA in the from of biplot）emphasizes differences among individual teinturiers,
Blaufrankish and partially separate the group of Blaufrankish, Pinot Noir, Blauer Portugieser and Domina varieties. Moreover,suitable division of biplots in a direction of a principal component based on enzymatic activities further allowed a more detailed
classification of Merlot and Cabernet Moravia varieties and led to an unambiguous differentiation of variety Saint Laurent from
Blaufrankish which has indispensable meaning from the viewpoint of their high occurrence on the Czech market. The above
mentioned approach is based on precise quantification of particular anthocyanins from LC/MS data. Surprisingly, a similar degreeof classification based on evaluation of AAEs and PCA was allowed one year later simply by direct evaluation of signals ofanthocyanins in MALDI spectra of particular varieties. Beside, desorption nanoelectrospray（nanoDESI）allowed revelation of
addition of a teinturier（variety Rubinet）into a common red wine for the purpose of its articial color enhancement［ 3 ］. Those
results indicate a high potential of surface ionization techniques for wine authenticity evaluation and the approach will be also
discussed in this communication.
Acknowledgement
Authors thank to Grant Agency of Czech Republic（project P206/10/0625）. This work was supported also by the projects MSM
6198959214 and Operational Program Research and Development for Innovations European Social Fund （project
CZ.1.05/2.1.00/03.0058）of the Ministry of Education, Youth and Sports of the Czech Republic.
References［ 1 ］W. Heller, G. Forkmann, Biosynthesis. In： J.B. Harborne（Ed）, The Flavonoids, advances in research since 1980. New York：
Chapman and Hall, pp. 399425（1988）.
［ 2 ］B. Papouskova, P. Bednar, K. Hron, J. Stavek, J. Balik, R. Myjavcova, P. Bartak, E. Tomankova, K. Lemr, J. Chromatogr. A 1218
（2011）7581-7591.
［ 3 ］L. Hartmanova, V. Ranc, B. Papouskova, P. Bednar, V. Havlicek, K. Lemr, J. Chromatogr. A, 1217（2010）4223-4228.

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 34: MS Informatics for Identification and Characterization

PTh-020

13:30 － 14:40

Higher-Order Structural Characterization of an IgG2 Monoclonal Antibody
by Disulfide Mapping and Hydrogen Deuterium Exchange Mass
Spectrometry

Taiji Kawase1, Kenji Hirose1, Joomi Ahn2, Stephane Houel2, Asish Chakraborty2, Ying Qing Yu2,
Justin B Sperry3

1
Nihon Waters, Tokyo, Japan, 2Waters Corporation, Milford, MA, 3Analytical R&D, Biotherapeutics Pharm. Sci., PfizerInc., Chesterfield, MO

Keywords:
Antibody, IgG2, Disulfide bond, HDX
Novel aspects:
An analytical workflow combining proteolytic mapping and HDX MS analyses definitively characterized several batches of an IgG 2
mAb.
Abstract:
Introduction:
Recombinant human monoclonal antibodies（mAbs）are widely used in biotherapeutics research and development as potential
therapies for various diseases. After antibody production several analytical techniques focused on primary and higher-order
structural analyses are conducted to adequately characterize these complex therapeutics. Recent reports detailing thecharacterization of IgG 2 mAbs revealed the presence of disulfide-linked structural isoforms, termed IgG 2 -A, IgG 2 -B, and IgG 2 -A/
B. In this study, we present an analytical workflow to automatically map out the disulfide -linked isomers using data independent
LC/MS acquisition and disulfide bond mapping informatics tools. Once the peptide map information is obtained, hydrogen
deuterium exchange mass spectrometry（HDX MS）using on-line pepsin digestion and peptic peptide mapping was used toinvestigate the consistency of multiple IgG 2 batches.
Method:A recombinant human IgG 2 mAb was produced in several batches at Pfizer（Chesterfield, MO）. Each IgG 2 batch was digested
with trypsin without reduction. The digest was separated using UPLC directed into a Q-Tof mass spectrometer. The LC-MS data
were processed in software that assigns disulfide bonds based on peptide theoretical masses and high collision energy fragment
ions. For HDX MS experiments, each IgG 2 batch was diluted into deuterated solutions, quenched at certain times and injected
onto a pepsin column for online digestion. The peptic peptides were separated by UPLC into a Q-Tof mass spectrometer tomeasure the deuterium incorporation. The amount of deuterium in several peptic peptides was compared using differential plots
produced by automated HDX data processing software.
Preliminary Data:
Informatics tools developed at Waters confirmed the identification of the disulfide-linked structural isoforms in the IgG 2 mAb.
Several interchain and intrachain linked peptides were automatically identified from the LC/MSE data. This workflow improves the
speed and consistency of identifying predicted and non-predicted disulfide linked peptides in mAbs.
The HDX MS experiments, which combined online pepsin digestion and automated peptide assignment, afforded high sequence
coverage of both light and heavy chains（88% and 93% respectively）. The proteolytic digestion with pepsin produced consistent
peptide cleavages over each labeling time-course and IgG 2 lot. The automated software assigned deuterium uptake levels toglycopeptides containing G 0 F, G 1 F, and G 2 F N-linked glycans, the most common post-translational modifications in mAbs. We
observed interesting deuterium exchange kinetics for a peptide representing the C-terminus of the light chain（VTHQGLSSPVTKSFNRGEC, residue numbers 196 to 214）. The major IgG 2 isoforms present in this mAb are the IgG 2 -B and
IgG 2 -A/B. Each of these isoforms contain an interchain disulfide bond between the light chain（at position 214）to the heavy chain
hinge region. The deuterium exchange profile of this particular light chain peptide exhibited EX 1 -like behavior, as observed ashaving multiple deuterium distributions at long deuterium exchange times. We attribute this behavior to the presence of multiple
disulfide isoforms present in the mAb. Furthermore, the same exact deuterium uptake profile was also observed for this peptide in
the other batch of IgG2. HDX MS combined with disulfide bond mapping was found to be a powerful approach to assess theprimary and higher-order structure IgG 2 mAbs across multiple production batches.

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 34: MS Informatics for Identification and Characterization

PTh-021

11:10 － 12:20

Compound search technique for overlapping peaks of LC/MS with aunique algorithm for simultaneous multicomponent identification

Yuka Noritake1, Haruo Shimada1, Yoshimasa Nakatani1, Rakan Matsui1, Kazumasa Kinoshita2,
Satoru Kamikubo2, Yasuo Shida3

1
Shiseido Research Center, Yokohama, Japan, 2Bio Chromato, Inc., Fujisawa, Japan, 3Clean Energy Research Center,
University of Yamanashi, Koufu, Japan

Keywords:
search algorithm, multicomponent identification, rapid analysis, DART-MS, LC/MS
Novel aspects:
The key to efficient compound search is how to analyze overlapping spectra. We showed the simultaneous multicomponent
identification of complex mass spectra from cream and emulsion using the unique algorithm.
Abstract:
Introduction: Compound analysis is important for companies producing products such as cosmetics, medicine, and food throughresearch and development, product development and quality control management. Under the current industrial situations, therapid and simple analysis techniques have been required to increase their efficiency and cost performance.
In general, liquid chromatography/mass spectrometry（LC/MS）and gas chromatography/mass spectrometry are widely usedmethods for the analysis of complex mixture. Although these methods are beneficial for compound analysis, they have twobottlenecks from the view point of analytical efficiency. One is a time consuming process such as sample preparation anddevelopment of LC conditions for separation. Another is a difficulty in data analysis such as peak separation because of limited
capacity of conventional algorithms in search software for compound identification. For example, they can identify completely
separated peaks by focusing on the extraction of a single mass spectrum derived from one component in a mixture. On the other
hand, it is a time consuming process to analyze data that have peaks consisted of two components with the same retention timeand an apparently single peak that is actually overlaps of multiple compounds. For the shortcut to data analysis, currently wehave developed the unique algorithm to search mass spectra of mixtures simultaneously, which enables us to identify multiple
components easily and exclude the bottlenecks as described above. In this study, we showed rapid identification of components
in complex spectra obtained from MS analyses of the cream and emulsion with the algorithm. The result indicated that it could beversatile with a direct analysis in real time（DART）and an infusion analysis of LC/MS for the rapid compound analysis. Our poster
focuses on a new compound search technique with the unique algorithm for the compound analysis of complex materials（pharmaceutical cream and emulsion）and its application data with the visualization of peak information.
Materials and Methods: Cream and emulsion samples containing active ingredients（lidocain, diphenhydramine, tocopherol
acetate）and cream base（methyl benzoate, ethyl benzoate, etc.）were dissolved in acetonitrile. The identification of thecompounds was carried out by a quadruple time-of-flight mass spectrometer（Bruker MicrOTOF-Q 2 ）equipped with HPLC system（Shimadzu prominence XR）with an electrospray ionization（ESI）interface or a DART interface. Separations were performed on areversed-phase C18 column at 40 degrees C. The mobile phase was a simple gradient of acetonitrile-water. For DART analysis, thesample was applied on the glass rod, which was followed by exposure to metastable helium gas. The data obtained from themeasurements of LC-ESI-MS and DART-MS were successively assigned to the respective standard mass spectra by using thealgorithm.
Results: The active ingredients in the cream and emulsion were eluted successively within ten minutes by LC/MS. Components
appeared in the mass spectra were assigned using the algorithm for simultaneous multicomponent identification. The peaks oflidocain and diphenhydramine were identified as a single component, respectively. The peak of tocopherol acetate was assignedwith overlapping components of cream base. In spite of the simultaneous elution of many components with the infusion analysisof ESI-MS, each component was identified and corresponded to each standard spectrum. The components in the spectrum
obtained from DART-MS were also identified.
Conclusion: In conclusion, the algorithm used in this study enabled simultaneous search of mass spectrum of mixture without
peak extraction from complex peaks. The components in cream were not only identified without any pre-examination of HPLC for
the peak separation, but also identified without any separation process in the case of DART and an infusion analysis of LC/MS.
Our study indicated that this algorithm could be useful for the rapid identification of multicomponent.

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 34: MS Informatics for Identification and Characterization

PTh-022

13:30 － 14:40

To determine the activity of Essential Oil from Blumea eriantha from
Indian origin by HSGC and GCMS.

Jitendra G Kelkar1, Deepti Kulkarni1, Ajit Datar1, Dheeraj Handique1, Aarti Karkhanis1,
Prajakta Pednekar2

1
Shimadzu Analytical (India) Pvt Ltd, Mumbai, India, 2(2)Guru Nanak Institute of Research and Development, G. N.
Khalsa College, Matunga, Maharashtra, India

Keywords:
Essential Oil phyto-constituents by HSGCMS
Novel aspects:
Blumea eriantha has shown excellent anti microbial activities. Its components have been identified by HSGC and GCMS. The
absorption of Essential oil by plasma shows selectivity.
Abstract:
Introduction
Aromatherapy is a healing art whose roots are laying in antiquity. It is a form of alternative medicine that uses volatile plantmaterials, known as essential oils, and other aromatic compounds for the purpose of altering a person’s mind, mood, cognitive
function or health. The essential oils have been used therapeutically for centuries； there is a little published research on many ofthem. A significant body of research on pharmaceutical and therapeutic potentials of essential oils is therefore necessary.
In the present work we have selected the following plant of Indian origin to study various healing potentials of its essential oil：Blumea eriantha Family: Compositae; Asteraceae.
This plant is selected as detailed systematic work on therapeutic properties of essential oils has not been reported in theliterature.
In the current research work the antibacterial potential of the essential oil, specifically against skin pathogens are evaluated. The
antioxidant potential and the safety of the essential oil are also evaluated by cyototoxicity and dermal toxicity study. In this article,
we are discussing the analysis and identification of ingredients present in Essential oil from the above plant using Head Space GC
and GCMS.
The plant material was collected from Seawoods, around Mumbai which grow in plenty between the months of October toMarch.
MethodThe Essential oil was extracted from the plant by hydro distillation using Clevenger Apparatus. The extraction conditions like
temperature, weight of plant material, rate of distillation were optimized so as to get maximum yield of essential oil.
The chemical properties were analyzed using following techniques：
The method was developed on Gas Chromatography（GC）, Shimadzu GC2010 with FID equipped with Tekmar Teledyne HT 3
Head Space Accessory.
The GC method then was transferred to Shimadzu, GCMS（QP 2010 Ultra）for identification of various phyto-constituents ofessential oil using EIMS library（NIST）and Terpenoids Library using relative retention indices.
Absorption of Essential oil by Plasma was studied by using the raw rabbit plasma spiked with known amount of essential oil andthe absorption of the components of essential oil was studied by using above Head Space GC and GCMS System.
The Microbial Activity of Essential oil was studied on skin pathogens and cancer cells.
Preliminary Data
Shimadzu GCMS model QP2010 Ultra with Tekmar HT 3 Head Space Analyzer played a great role in identification of componentsof essential oil from plasma. The phyto-chemicals were identified. The absorption of Essential Oil Analysis in plasma has revealed
the selective absorption of some of the components. Further work on the selectivity and active ingredient in the oil responsible
for the activity is being done.
The microbial activity has been carried out and the oil has shown very good activity on the skin pathogens namely： Propioni
bacterium acnes, Staphylococcus epidermidis, Staphylococcus aureus, Streptococcus pyogenes and it showed activity against allof these at low concentration levels.
It has also shown activity against the cancer cells, namely： ACHN（Renal Cancer）,Panc 1 （Pancreatic cancer）, Calu 1 （lungcancer）, H460（Non small cell lung cancer）, HCT116（Colon cancer）.

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 34: MS Informatics for Identification and Characterization

PTh-023

11:10 － 12:20

Screening of antioxidants present in unripe Manilkara zapota fruit ofIndian origin by using LCMS/MS.

Shailendra A Rane1, Deepti Bhandarkar1, Rashi Kochhar1, Shruti Raju1, Bhairavi Saraf1, Ajit Datar1,
Jeetendra Kelkar1, Bhagyashree Jadhav2

1
Shimadzu Analytical (India) Pvt Ltd, Mumbai, India, 2(2)Bioanalytical Department, Ramnarain Ruia College, Matunga,
Maharashtra, India.

Keywords:
Antioxidant screening by using LCMS/MS
Novel aspects:
LCMS/MS can be used for screening of antioxidants present in unripe Manilkara zapota fruits, for their potential application as
new source of natural antioxidants for food, nutraceutical and pharmaceutical industries.
Abstract:
Introduction
Antioxidants are the compounds that act as radical scavengers, prevent the radical chain reactions of oxidation, delay or inhibit
the oxidation process and increase shelf life by retarding the process of lipid peroxidation.
Antioxidants found in fruits and vegetables play an important role via their protective effects against the onset of aging-related
chronic diseases. The objectives of this study is to screen the antioxidants present in unripe Manilkara zapota fruit, which iscommonly available in India, and to indicate that it can become a new source of natural antioxidants for food, nutraceutical andpharmaceutical industries. The LCMS/MS is used to identify the presence of antioxidants from the plant.
MethodAntioxidants were extracted from unripe Manilkara zapota fruit followed by purification using Solid Phase Extraction（SPE）. Eluate
from SPE was subjected to Low Volume Evaporator（LVE）with final reconstitution in methanol.
The extract was then analysed using Ultra High Performance Liquid Chromatography（UHPLC）Nexera system coupled with LCMS8030 triple quadrupole system of Shimadzu Corporation. The use of LCMS-8030 ultra high speed mass spectrometer, which iscompatible with UHPLC system, aided in keeping the ultra fast and high sensitivity without any deterioration in the quality of data.
The use of Nexera with High Speed LCMS/MS has facilitated not only in achieving fast method development but also in gettinghigh sensitivity in MS/MS mode.
Preliminary Data
Antioxidants were extracted from unripe Manilkara zapota fruit and UHPLC method was developed for the analysis of the same.
The extract was also subjected to mass spectrometric analysis using triple quadrupole system.
Polyphenol antioxidants like catechin, epicatechin were observed in the extract. Their presence was confirmed by comparing m/z
values obtained with those cited in the literature. Product Ion Scan spectra for selected precursor ions gave further confirmationof their presence.

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 34: MS Informatics for Identification and Characterization

PTh-024

13:30 － 14:40

Rapid development of functional extensions for mass spectrometry using
freeware Mass++

Howell E Parry1, Satoshi Tanaka1, Tsuyoshi Tabata2, Ken Aoshima2, Yoshiya Oda2, Shinichi Utsunomiya1,
Shigeki Kajihara1, Koichi Tanaka1
1

Shimadzu Corporation, 2Eisai Co., Ltd.

Keywords:
freeware, plug-in, proteomics, metabolomics, multi-vendor
Novel aspects:
Mass++’s unique plug-in architecture makes it easy for third parties to extend the extensive core feature set with originalfunctionality which may be distributed freely.
Abstract:
Introduction
Mass++ was originally funded by CREST（Core Research for Evolutional Science and Technology）and developed by Eisai.
Mass++ 2.0.0 development was taken over by Shimadzu Corporation with continued support from Eisai, and funding from FIRST（Funding Program for World-Leading Innovative R&D on Science and Technology）.
Mass++ is freeware for mass spectrometry analysis that can be downloaded from http://www.first-ms3d.jp/. Mass++’s primaryobjectives are：
1. To provide essential functionality for proteomics and metabolomics analysis.
2. To support a wide range of vendors’ data file formats.
3. To be easily extendible using plug-in technology.
Mass++’s rich feature set, implemented as plug-ins to the core software, includes chromatogram, spectrum, heatmap and 3 D
viewers, data pre-processing, advanced peak detection and quantitative analysis.
Support for a wide range of data file formats has been implemented - Shimadzu（LCMSsolution, GCMSsolution）, Applied
Biosystems（Analyst, AnalystQS）, Thermo Fisher（Xcalibur）, Waters（MassLynx）, mzXML/mzML and MSB（original format）.
Mass++ is distributed as freeware. Mass++’s unique plug-in architecture makes it easy to use Mass++ as a base platform onto
which original functionality may be implemented as plug-ins.
MethodMass++ has a“plug-in type“ concept； by handling plug-in types, a plug-in can respond to Mass++ events（for example, when aspectrum is displayed, a DRAW_SPEC event occurs and any plug-in could respond by annotating additional information onto thegraph）or it can define itself as a certain type of plug-in, and it will appear as a new option in a dropdown list within Mass++. The
types of plug-in that can be created include：- Data file input- File export- Peak detection（spectrum, chromatogram, 2 D）
- Charge detection- Database search- Peak filter- Peak analysis- RT（retention time）alignment
To support third party developers who want to write original plug-ins, Mass++ ships with：- Comprehensive documentation- Microsoft Visual Studio development wizard.
This presentation will show, step-by-step, how easily new functionality can be developed with the Mass++ platform, using as an
example a simple plug-in to eliminate unwanted detected peaks based on a user-specified height threshold.
The steps for creating a new plug-in are as follows：
1. Create a plug-in project using the development wizard
2. Define the user interface and handler functions etc. in definition XML files
3. Implement handler functions
4. Copy the built plug-in DLL and XML files into the Mass++ plug-ins folder
ResultNewly-created plug-ins are auto-detected the next time Mass++ is run, and their functionality automatically appears in Mass++,for immediate use. It is hoped that third party developers will be encouraged to take advantage of Mass++’s rich corefunctionality via its SDK to develop their own original plug-ins. New plug-ins may be distributed freely.
References
1 ）S.Tanaka et al., MSSJ 57th 3 P-44（2008）.
2 ）S.Tanaka et al., MSSJ 58th 2 P-004（2009）.
3 ）S.Tanaka et al., ASMS 59th ThP383（2011）4 ）H.Parry et al., MSSJ 59th 2 P-01（2011）

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 34: MS Informatics for Identification and Characterization

PTh-025

11:10 － 12:20

UHPLC-MS/MS, an Alternative Solution to Conventional Biosensor
Approach for Quorum Sensing Signalling Molecules Detection in Complex
Environmental Samples

Chuan Hao (Grant) Tan2,4, Kai Shyang Koh4, Scott A Rice2,4,5, Yan Zhou2,3, Staffan Kjelleberg4,5,
Wun Jern Ng*2,3, Peiting Zeng1, Zhaoqi Zhan1

1Shimadzu (Asia Pacific) Pte Ltd, Singapore, Singapore, 2Advanced Environmental Biotechnology Centre, Singapore,
Singapore, 3Nanyang Environment and Water Research Institute, Singapore, Singapore, 4Singapore Centre on
Environmental Life Sciences Engineering, Nanyang Technological University, Singapore, Singapore, 5The Centre forMarine Bio-Innovation, The University of New South Wales, Sydney, Australia

Keywords:
Quorum Sensing, Biosensor, Quantification, UHPLC-MS/MS
Novel aspects:
Identification and quantification of thirteen AHLs in a complex activated sludge system were demonstrated for the first time using
UHPLC-MS/MS
Abstract:*Corresponding author
Introduction
Quorum sensing（QS）signalling is critical for coordinating the social behaviours of bacteria, i.e., regulating biofilm development［ 1 ］. Acyl-homoserine lactones（AHLs）consist of a homoserine lactone ring and an acyl side chain, at variable lengths, oxidation
states and saturation levels, are the most common signals employed by the bacteria. As AHLs are highly susceptible to elevatedtemperatures, alkaline conditions, and are often degraded rapidly by other microbes living within the same niche, it is always areal challenge to characterize and quantify AHLs in the natural environments［ 2 ］. Conventionally, AHLs are detected bybiosensor-dependent assays, which are time-consuming and with limited sensitivity detecting all range of AHLs［ 3 ］. Here, a
UHPLC-MS/MS method is developed to overcome the limitations of the biosensor-based AHL detection.
Experimental
Naturally occurring AHLs, from the activated sludge of a lab-scale bioreactor system, were extracted by dichloromethane. The
samples extracted were analysed on UHPLC-MS/MS tandem quadrupole system（Shimadzu LCMS-8030）using a Shim-pack XR-ODS
column（ 2 x 100 mm, 2.2µm）. A total of thirteen synthetic AHLs, ranging from C 4 to C14, with various oxidation states, were used
as reference AHLs for the analysis. Automatic MRM optimization was applied to each standard to determine the MRM transitions
for subsequent sample analysis. A pair of MRM transitions was selected for each standard. The MRM transition that exhibited
higher intensity was used for quantification analysis, while the other for confirmation of the AHL identity. In addition, MS full scancoupled with synchronised survey scan was employed to identify possible existence of other AHL structures with predicted m/z
values.
Results and discussion

A rapid and sensitive quantitative MRM method for the AHLs was established using UHPLC-MS/MS. This UHPLC-MS/MS based
method allows for detection and identification of AHL identities with high level of confidence, which would not be achievable
otherwise by the conventional biosensor-dependent assays that generally require the AHL samples to be first separated on a thinlayer chromatography（TLC）plate, followed by the detection with a specific bacteria biosensor overlay. Using the synthetic AHLs
as standards, in the blank sample matrix, good repeatability of the UHPLC-MS/MS method was demonstrated. Despite thepresence of complex matrix components, which are highly inhibitory to the ionization of the AHL molecules with ESI interface, thelimit of detection（LOD）for all the thirteen AHL standards were found to be approximately 0.1 ppb to 1.0 ppb. These LOD values
are comparable to various biosensor-based AHL detection methods ranging from 0.05 ppb to 1000 ppb. However, unlike thebiosensor assays, which are often bias towards specific types of AHLs with different acyl side chain lengths and oxidation states,
the UHPLC-MS/MS method is unequivocally sensitive to all the AHLs studied at various oxidation stages. This was demonstrated
by the detection of naturally occurring AHLs in activated sludge samples, with acyl side chains ranging from C 4 to C10-HSL andwith or without carbonyl-substitution at low concentrations. On the other hand, the analysis using Agrobacterium tumefaciens
A136 biosensor was only able to detect the presence of carbonyl-substituted C 8 -HSL（C 8 -OXO-HSL）in the same samples
extracted. Moreover, an additional MS full scan coupled with synchronized survey scan approach allowed further detection ofother AHL structures beyond the thirteen synthetic AHL standards that were present in the samples, which would be otherwise
not detectable by the biosensor methods.
Conclusions
It was clearly demonstrated that UHPLC-MS/MS as a rapid, sensitive and high-throughput alternative to the conventional
biosensor-dependent approach in validation and quantification of quorum sensing signalling molecules in the complex
environmental samples.

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 34: MS Informatics for Identification and Characterization

PTh-027

11:10 － 12:20

Strategy for key odor component discovery using GC×GC-TOFMS

Fumihiko Tsuchiya, Fumie Kabashima, Wataru Uchikawa, Michiko Kanai
LECO Japan, Tokyo, Japan

Keywords:
GCxGC, TOFMS, Fisher Ratio, Flavor, Foods
Novel aspects:
The workflow by non-targeted analysis using GCxGC TOFMS is an efficient method to extract potential candidate compounds
from the huge quantities of peak data.
Abstract:
Introduction
The investigation of aroma compounds has been growing in importance in several analytical fields such as food science and theenvironment, as well as clinical medicine. In such samples, there are many kinds of flavor compounds which contribute to theoverall sample flavor profile in a synergistic way. Therefore, comprehensive profiling of the aroma components is essential. GCxGC
TOFMS allows non-target analysis and comprehensive fragrance screening due to the enhancements in selectivity and sensitivity
provided by two dimensional GC（GCxGC）and by Time of Flight mass spectrometry（TOFMS）. In this presentation we show an
example of the comparison of the fragrance components of Awamori, specialty distilled liquor in Okinawa, that has been stored inthree different types of vessel, stainless steel, glass and pottery bottles, for twenty years. There are significant differences in flavor
between samples due to the long aging process and the method of storage. We applied a non-target analytical method using
GCxGC TOFMS to characterize each sample and determined typical compounds defining sample characteristics. We present a
workflow for this study and discuss the efficiency of the method for flavor/fragrance analysis.
Method and Materials
One micro liter of each Awamori sample was dispensed into a tall vial（10 mL）and diluted with 1 mL of distilled water. After
capping of the vials, aroma components were collected using SPME（DVB/CAR/PDMS）at 60°C, for twenty minutes. The GCxGC
data were collected on a Pegasus® 4 D GCxGC-TOFMS system（LECO Corporation）and processed using ChromaTOF® Version
4.41 software. We repeated the measurements four times for each sample to allow a variance analysis. All peak lists tables for the
samples were subjected to a peak alignment step in the software to generate a spread sheet which could be exported into themultivariate analysis software JMP08.
Results
More than three thousands peaks were detected for each sample. In order to compare samples we summarized all peak lists
into a spreadsheet using the peak alignment function in ChromaTOF 4.41 software. Additionally we calculated Fisher Ratios foreach compound in the list. The Fisher Ratio which is a variance ratio between class-to-class and within-class variance is a powerfultool to eliminate peaks that do not show significant difference among the data sets. We sorted the Fisher Ratio of each compoundin the peak list in descending order. Since the higher Fisher Ratio is generated by good repeatability and significant differences,
the top 150 compounds were assumed to be important candidates as that contribute to the characteristics of the sample. The listof candidates includes many aromatic compounds（e.g. esters, terpenes, sulfur compounds, etc.）. The compound name and peakarea data in the list can be used to express the relative abundance ratio of each compound with a 100% stacked bar graph. This
workflow provides not only information about compound identity but also quantitative data. The candidate list can also be
subjected to multivariate analysis, and showed definite differences between samples in a principal component analysis. The result
indicates that this workflow by non-targeted analysis using GCxGC TOFMS is an efficient method to extract potential candidate
compounds from the huge quantities of peak data.

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 34: MS Informatics for Identification and Characterization

PTh-028

13:30 － 14:40

Coordination Polymer Formation Observed by Cold-Spray Ionization Mass
Spectrometry

Kazuaki Ohara, Kentaro Yamaguchi

Tokushima Bunri University, Sanuki, Japan

Keywords:
CSI-MS, coordination polymer, crystal, halogen interaction
Novel aspects:
Coordination species, which extends to form coordination polymer, was detected by cold-spray ionization mass spectrometry.
Abstract:
Coordination bonding is one of the most attractive interactions for the construction of nanometer-sized molecules such as
catenane, rotaxane which is dumbbell shaped molecule, and guest encapsulating compound. The interaction provides thereaction profiles as well as selective molecular recognition capability. Analytical techniques such as NMR spectroscopy, X-ray
analysis and other spectroscopic approach have been developed with a progress of macromolecular synthesis and applied toreveal coordination structure or selective molecular recognition. However, conventional mass spectrometry（MS）has not been
adopted for these labile complexes even by using soft ionization, such as electrospray ionization（ESI）. Cold-spray ionization（CSI）
at low temperature has made it possible to observe labile coordination species without any dissociation. CSI-MS has been applied
to unstable compounds, Grignard reagents, host-guest molecules, multiply stranded DNAs, and other unstable biologicalcompounds.
Metal-organic porous materials mainly categorized as metal-organic frameworks（MOFs）and coordination polymers（CPs）are
generally obtained by hydrothermal synthesis and the diffusion method, respectively. After the material is synthesized, structure of
the product, generally obtained as the solid, is determined by powder or single-crystal X-ray structure analysis.
However, the coordination species which is soluble and grown up to be insoluble coordination polymer has not received any
attention for a long time. In recent research, the crystal growth of MOFs was investigated by time-resolved in situ X-ray scattering.
Although this analytical method yields a part of the metal-organic structure determined by the combination of the ligand and themetal before crystallization, it is not sufficient to discuss the detailed structures of the coordination species in solution. In ourprevious study, it was succeeded to observe organic network structure with hydrogen bonding of steroid compounds over 10-mer
in solution by CSI-MS and confirms its network structure accommodated into crystal. The molecule having weak interaction eachother in solution generally exhibits good agreement with the structure observed in crystal.
In this work, we observed the coordination behavior in solution by CSI-MS comparing to the crystalline coordination structure. To
discuss coordination, Cu（I）iodide was selected as a metal which potentially had two competitive weak interactions：coordination bonding and metal-halogen interaction. Although Cu（I）iodide was a widely used catalyst for the 1,3-dipole
cycloaddition reaction to obtain multiply substituted 1,2,3-triazole, the tetrahedral CuI easily gave 1 -D chain cluster with metalhalogen interaction. Complexation of organic ligand having multiple nitrogens with the copper potentially gives several
coordination compounds. In addition, the two competitive weak interactions furnish highly complicated pathways for theconstruction of coordination polymers. We investigated a soluble coordination species in CSI-MS and discuss coordination
structure in solution compared with coordination polymer.

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 34: MS Informatics for Identification and Characterization

PTh-029

11:10 － 12:20

Mass Spectral Libraries for Analysis of Complex Plant Metabolomics
Datasets - Algorithms

Wm Gary Mallard1, Steve Stein1, Yuri Mirokhin1, Nirina Rabe Andriamaharavo1, John Halket2
1

National Institute of Standards and Tecnology, Gaithersburg, MD USA, 2Kings College, London, UK

Keywords:
AMDIS, plant metabolomic libraries, deconvolution,
Novel aspects:
A library of plant metabolites is developed of recurrent unidentified spectra. Specific algorithms to improve the data quality forcomplex data files are discussed.
Abstract:
A principal problem in plant metabolomics is the large number of unidentified components in the samples. However, thesecomplex mixtures may be characterized by frequently recurring components in terms of the spectral and retention parameters of
the components. We have developed a series of methods for the analysis that provides high quality spectra with wellcharacterized retention parameters for recurrent compounds that can be used in search libraries. Here we discuss the algorithms
that are used to filter and sort these data. The example data used here is a set of essential oil samples of Vetiver taken frommultiple commercial sources and analyzed at multiple dilutions.
The GC/MS data files are deconvoluted using the NIST deconvolution software AMDIS and a target library composed of spectrafrom the NIST MS library（NIST11）as well as spectra from the literature for components in Vetiver. The analysis was limited to aregion of the chromatogram between 16 and 31 min on DB-5. Typically 120 to 200 components were extracted by AMDIS with
about one half identified using the target library. All unidentified components were then analyzed to determine which recurred.
AMDIS was run in a mode that rejected data if there were too few ion peaks that had similar time histories（model peaks）, if the
S/N of the spectra was so low that the minimum ion peak that could be extracted from the noise was not at least 0.5% of thebase peak. The resulting set of unidentified peaks provided between 200 and 400 unknowns that were recurring. These unknowns
were then searched against the entire NIST database and where identification was made（initially about 5 % of the unidentified
components）, the new compounds were placed in the target library.
The spectra from the recurring unknowns were further processed using a set of algorithms that examined the distribution of peaksin the spectrum. These algorithms were designed to find spectra that a human expert would find“unlikely“. The preliminary
version of the algorithm focused on the appearance of clusters of peaks. Thus spectra with numerous isolated peaks with noapparent isotope peaks were rejected. In addition, spectra with very few peaks were rejected as were spectra that appeared tohave unreasonable neutral losses. Preliminary application of these criteria rejected almost 80 % of the unidentified spectra. The
resulting 20% were incorporated into a target database and then rerun against the data files to confirm the quality of the data. The
resulting data sets were examined exactly as was done with the initial data set and the same set of filters were applied to define
any remaining high quality spectra that could be added to the library.
The challenge in analyzing these complex mixtures is that there are two common problems that give poor spectra - strong overlap
between components so that it is not clear that there are multiple components（resulting in extra peaks in a component）andsmall components that are partially coeluting with larger components containing common ions that make the deconvolution of
the these ions difficult（resulting in missing peaks in the component）. Specific algorithmic tools were developed to identify cases
where one or the other of these problems was occurring and spectra where these problems could be identified were rejected.
The success of these algorithms was tested on the Vetiver data prior to their being applied to a far larger set（over 1000 data files）
of essential oils.
The library of these recurrent, high quality spectra is available for download.

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 34: MS Informatics for Identification and Characterization

PTh-030

13:30 － 14:40

Structure estimation of triacylglycerol by comparing calculated peak
pattern with high energy CID spectrum using MALDI-TOF-TOF tandem MS

Takaya Satoh, Kubo Ayumi, Hizume Takuhiro, Sato Takayoshi, Ueda Yoshihisa
JEOL Ltd.

Keywords:
Structure estimation, MALDI-TOF-TOF, lipid, triacylglycerol
Novel aspects:
Structure estimation of triacylglycerol by comparing calculated peak pattern with high energy CID spectrum using MALDI-TOF-TOF
tandem MS was performed
Abstract:
Introduction：
Lipids are the molecules consisted of backbone structure, such as glycerol and, which combined with one or more fatty acids
combined with backbone. Characters of lipids vary according to structure of fatty acids in those. Structural analysis of lipids havebeen performed using tandem mass spectrometer. Especially tandem mass spectrometers which are capable of high energy CID
have been well performed for obtaining detail structure of fatty acid moieties, such as position of double bond and branching, byobserving signal yielded by charge remote fragmentation. However, product ion spectra of lipids which have two or three fatty
acids observed by those instruments is quite complex to interpret structural information, because signals derived from each fatty
acids are overlapped in a product ion spectrum. Therefore, simple interpretation methodology of product ion spectra is necessary.
In this study, automatic interpretation based on database search was performed. There are some databases of product ion
spectra of lipids, but those which has enough product ion spectra of lipids by high energy CID is not found. And, development of
new database required the much time, effort, and many money to acquire product ion spectra of lipids standards. So, we
attempted to develop the estimation method of product ion spectrum of lipid based on the empirical rule. This empirical rule wasestablished by comparing the product ion spectra between the several kinds of lipids that have a similar structure each other.
We report the estimation method of high energy CID product ion spectra of lipids and the validation result of the method byemploying triacylglycerol as a test sample.
Method：A JMS-S3000“SpiralTOF“ MALDI-TOF-TOFtandem MS（JEOL Ltd., Tokyo, Japan）was employed for obtaining product ion spectraof several kinds of TAG whose chemical structure have already known. The product ion spectrum of one of TAGs in low or middle
mass region were estimated by consulting past literature［ 1 ］. And the product ion spectra in high mass region were estimatedbased on the observed product ion spectra of TAG that has similar structure. The product ion spectra of several kinds of TAG（54： 3 ）were estimated by using above method, and those were stored into the database. MassBank search system were used
for product ion search between observed product ion spectra and estimated spectra.
Result：
The product ion spectra of several kinds of isomer of TAGs（54： 3 ）were estimated and stored on to the private database of
MassBank. The observed product ion spectrum of TAG（16：0,18：1,18： 2 ）were applied for MassBank search by using the private
database. As a result of database search, correct candidate was clearly distinguished from some structural isomers of TAG（54：3 ）.
On the other hand, identification of positional isomers was not clearly performed（MassBank search gave almost same score foreach isomers）because product ion spectra in high mass region were exact same that was induced by CRF of fatty acid moieties.
But, characteristic peaks which enable identification of positional isomers were observed. If the search algorithm can be improved
for TAGs by using such characteristic peaks, product ion spectrum search system based on MassBank has possibly to be useful
tool for lipids identification.
［ 1 ］. Pittenauer,E.； Allmaier, G.； J. Am. Soc. Mass Spectrom. 2009, 20, 1037-1047.

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 34: MS Informatics for Identification and Characterization

PTh-031

11:10 － 12:20

Identification of antioxidants in Fructus aurantii and its quality evaluation3
using a new on-line combination of analytical techniques

ZONGTAO LIN2, JING DONG1, HONG WANG2, YING XU2, YUKI HASHI3, SHIZHONG CHEN2

Shimadzu(China), Beijing, China, 2School of Pharmaceutical Sciences, Peking University, Beijing, China, 3Shimadzu(China) Co., Ltd., Shanghai Office,ShangHai, China

1

Keywords:
Fructus Aurantii, On-line derivatisation, Flavonoids, Antioxidant identification, HPLC-DAD-ESI-MS-PCD-LPFCL
Novel aspects:
An on-line system developed here can be applied for rapid, accurate identification of multiple active constituents in Fructus
Aurantii
Abstract:
A new on-line method for simultaneous identification and monitoring of antioxidants in Fructus aurantii was established bycoupling high performance liquid chromatography-diode array detector-electrospray ionisation-ion trap-time of flight-mass
spectrometry with post-column derivatisation and luminol-potassium ferricyanide chemiluminescence（HPLC-DAD-ESI-IT-TOF-MSPCD-LPFCL）. While the HPLC fingerprint, structural identification and radical scavenging profile were rapidly obtained by an online assay using ultraviolet（UV）absorption, MS and LPFCL, details of the precise substitution patterns of various structures wereachieved through UV absorption using PCD addition of shift reagents. Twenty-five flavonoids were identified by either their PCD
and MS data or comparison with reference substances. Data collected both from chromatograms and activity profiles of 12
samples revealed significant differences among samples from different habitats. The results showed that this method was rapid
and precise, and therefore would be an effective and sensitive method for biocompounds analysis and quality evaluation forcomplex food and medicinal samples.

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 34: MS Informatics for Identification and Characterization

PTh-032

13:30 － 14:40

Use specific proteins of Taiwan tea as biomarkers for the certification oforiginHan-Ju Chien, Chien-Chen Lai

Institute of molecular biology/National Chung Hsing University, Taichung, Taiwan

Keywords:
Oolong tea / 2 D-PAGE / MS / biomarker
Novel aspects:
To our knowledge, this is the first report of using the proteomic strategy to distinguish Taiwan teas from the foreign teas.
Abstract:
Oolong is a traditional Chinese tea (Camellia sinensis), one of the major teas in Taiwan, processing with partially fermentation, themajor components are tea polyphenols and caffeine. Many studies have proofed that drinking of tea appropriately can prevent
cardiovascular disease, obesity and cancer. Recently, the cheap cost and the poor-quality of the foreign tea have made anenormous impact on Taiwan teas. The blend of low-quality foreign teas with Taiwan teas resulted in the low competitiveness of
Taiwan teas exportation. Furthermore, consumers are worried about the unclear place of production and indistinct safety ofimport foreign teas. In this study, we focus on finding the key biomarkers to differentiate between import foreign teas and Taiwan
teas. The sources of Taiwan oolong teas come from Nantou, Chiayi, Taitung, New Taipei City, Taichung, Ilan, Hualien, Yushan,
Lishan, Alishan, Deer Valley, Lushan, Dong Ding Mountain, and ones of foreign oolong teas come from Thailand, Indonesia, andSouth Vietnam. Until now, several studies have showed that the proteome expression profiles of plant leaves altered with thevariant environment, including harvest time, composition of the soil, insect damage, disease, cultivation, and the pollution ofpesticide and heavy metals, it have been carried out on different kinds of stress in plants and biomarker discovery. Herein,
2 D-PAGE（two-dimensional polyacrylamide gel electrophoresis）was applied to the comparative proteomics analysis of the teaswith different production place. These specific proteins could be used to distinguish Taiwan teas from the foreign teas.

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 34: MS Informatics for Identification and Characterization

PTh-033

11:10 － 12:20

JobRequest - an easy-to-use software platform for proteomic analysis and ProteoAnalysis, its application for protein identification.

Tsuyoshi Tabata1, Akiyasu C Yoshizawa2, Takayuki Kimura1, Tatsuji Nakamura1, Ken Aoshima1,
Yoshiya Oda1, Shigeki Kajihara2, Koichi Tanaka2

1
Eisai Product Creation Systems / Eisai Co., Ltd., 2Koichi Tanaka Laboratory of Advanced Science and Technology,
Shimadzu Corporation

Keywords:
Platform Proteomics Identification JobRequest ProteoAnalysis
Novel aspects:
We developed an easy-to-use and scalable software platform for proteomic analysis, and its application for protein identification.
They will be publicly released on the web.
Abstract:
Introduction
For proteomic analysis, it is often necessary to modify the analysis procedure for the large amount of data acquired. However, ittakes time and effort to rewrite the“controlpart“ of the entire process, such as the control part of the execution sequence formultiple programs, or the administration for multiple computational servers, etc. Moreover, this rewrite is not related to theproteomic analysis itself. For the UNIX environment, some tools for these purposes are already available, although utilizing thempresents a significant hurdle for experimental scientists to overcome, because they require an advanced knowledge of computer
systems. Hence, an easy-to-use software platform for the bioinformatics analyses of proteomic data is widely anticipated.

Implementation of JobRequest
We developed the“JobRequest“ analysis platform in order to overcome the situation mentioned above： this program has ascalable architecture that works on one or more PCs, and controls the hardware and the entire process on both Windows andUNIX compatible（Linux）environments via a graphical user interface. JobRequest itself can be run on both Windows and Linux,
accompanying the Apache web server. This platform automatically executes system-related tasks and scalability-related tasks
such as designing parameter setting screens, scheduling tasks and assigning low load PCs to each analysis process： hence users
are relieved from the burden of implementing these functions, and can focus on writing the actual analysis programs. For the
analysis programs in JobRequest, web-standard languages, such as PHP, Ruby, and Perl, are supported. We will introduce thebasic functions of JobRequest in the poster presentation session.

Implementation of ProteoAnalysis
As a practical application of JobRequest, we also constructed the“ProteoAnalysis“ software suite on the JobRequest platform
for the identification of proteins from raw mass spectrometry data. By inputting multiple raw data, this suite automatically
executes several processes as follows, in parallel：（ 1 ）Making peak lists using Mass++［ 1 ］functionality.
（ 2 ）Identification of proteins from MS/MS spectra using X! Tandem as the search engine andsearching using MSPTM-DB, which we have developed［ 2 ］,is also available.
（ 3 ）Filtering results by search scores.
（ 4 ）Adding detailed annotations such as Gene Symbols, Gene Ontology terms, literature
information etc.
Each result is automatically merged and integrated into a single file and displayed on the screen or saved in a comma separated
value（CSV）file which can be opened in Excel. Both JobRequest and ProteoAnalysis will be freely available to download in theimmediate future. ProteoAnalysis will also be published as a web service.

References［ 1 ］Tanaka, S., et al., Development of a client tool for a mass spectra database. 59th ASMS Conference on Mass Spectrometry
and Allied Topics, 2011, Denver, USA.
［ 2 ］Yoshizawa, A.C., et al., MSPTM-DB： a known PTM database for high-speed and accurate search available on the“ProteoAnalysis“ web site. submitted to this IMSC.

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 34: MS Informatics for Identification and Characterization

PTh-034

13:30 － 14:40

Fragmentation analysis of glucuronide metabolites using hybrid ion-trap/
orbitrap mass spectrometer with electrospray ionization

Kazuyoshi Nozaki1, Mitsuo Takayama2
1

Astellas Pharma Inc, Tsukuba, Japan, 2Yokohama City University, Yokohama, Japan

Keywords:
Fragmentation, Glucuronide, CID, ESI, ISD
Novel aspects:
An MS approach for determining the site of glucuronidation in glucuronide metabolites was found by using CID, pos/neg ion
mode, and ESI-ISD methods.
Abstract:[Introduction]
Identifying metabolic pathways of a drug candidate is important not only for its pharmacokinetic characterization, but also for theestimation of its safety and efficacy. The chemical structures of metabolites are mainly estimated based on mass fragmentation
data in drug discovery. It is known that glucuronidation is one of major non-CYP metabolic pathways. However the metabolic siteof glucuronidation is difficult to determine using mass fragmentation data, because the glucuronide is easily decomposed during
electrospray ionization（ESI）process and rapidly dissociated during fragmentation process. In this study, the in-source decay（ISD）
in ESI and collision-induced dissociation（CID）of glucuronide metabolites were investigated using eight model compounds.
[Method]
Acyl-glucuronides of probenecid, ketoprofen, naproxen, and telmisartan, and 0 -glucuronide isomers of raloxifene and ezetimibe
were used as model compounds. Experiments were performed using a LTQ Orbitrap Velos hybrid mass spectrometer withelectrospray ionization interface（Thermo Fisher Scientific Inc., San Jose, CA）. The solution of glucuronide standards at aconcentration of 20 μmol/L, delivered by a syringe pump at a flow rate of 5 μL/min, was mixed through a T-piece with an HPLC
effluent. The mobile-phase flow rate was set at 0.2 mL/min. The heated capillary was set at 330°C, and the spray voltage was kept
at 4.5 kV（positive ion mode）or 3 kV（negative ion mode）. The sheath gas flow（N2）and auxiliary gas flow（N2）were set to 50
and 10 arbitrary pressure units, respectively. Capillary and lens voltages were systematically optimized for the protonated or
deprotonated molecule using autotune. The CID was used to acquire the product ion spectra using helium as a collision gas in theion trap.
[Results and discussion]
The ionization efficiency of each deprotonated molecule［M-H］- was little different among eight model compounds in negative ion
mode. However that of protonated molecule［M+H］+ was so much different in the positive ion mode. In the case of thecompounds ketoprofen and naproxen, the protonated molecule was not detectable at any conditions. This may be due to lowefficiency of protonation since they have no nitrogen atoms in their chemical structures. The extent of the deconjugation in ESI-ISD
was very low in the negative ion mode except for a compound probenecid, On the other hand, the glucuronide of the compounds
ezetimibe, probenecid and telmisatan was dissociated easily in positive ion mode. The extent of the deconjugation of telmisartan
glucuronide was higher under acidic conditions than neutral conditions. The results obtained about the telmisartan in the positive
ion MS data and chemical conditions suggested that the dissociation or deconjugation takes place through the attack of proton
to the analyte molecules. The fragmentations in the CID spectra of six model compounds showed that the glucuronide moiety
was rapidly dissociated even when lowest collision energy was applied, while the CID spectrum of two ezetimibe glucuronides
did not show a rapid dissociation. When the compounds have a beta-lactam ring like the ezetimibe, a rapid dissociation of thelactam moiety occurred. Here we obtained the information about the MS and chemical conditions for the dissociation ordeconjugation of glucuronide metabolites, and using the information obtained here the glucuronidation sites could be obtainedon the basis of the fragmentation analysis.

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 34: MS Informatics for Identification and Characterization

PTh-035

11:10 － 12:20

Proteomic analysis of salt-responsive ubiquitin-related proteins in riceroot.

Yen-Fu Chen1, Chang-Sheng Wang2, Chien-Chen Lai1

Institute of Molecular Biology College of Life Science, National Chung Hsing University, Taichung, Taiwan, 2Departmentof Agronomy, National Chung Hsing University, Taichung, Taiwan

1

Keywords:
Rice, salt stress, ubiquitination, LC-MS/MS
Novel aspects:
These salt-responsive ubiquitinated proteins may be helpful in further elucidating the molecular mechanisms involved in protecting
plants from salt stress.
Abstract:
Ubiquitination of proteins plays an important role in regulating a myriad of physiological functions. In plants, ubiquitination governs
such processes as xylogenesis, senescence, cell cycle control, and stress response. However, only a limited number of proteins inplants have been identified as being ubiquitinated in response to salt stress. Rice（Oryza sativa）seedlings exposed to salt stress,
and the proteins of roots were extracted then analyzed using Western blotting against ubiquitin. Differentially expressedubiquitinated proteins were identified by LC-MS/MS and quantified by the Exponentially Modified Protein Abundance Index. In
addition, we performed a gel-based shotgun proteomic analysis to detect the total ubiquitinated proteome that may be involved
in response to salt stress. We found ubiquitinated proteins that were expressed in response to salinity such as pyruvate
phosphate dikinase 1, heat shock protein 81-1, probable aldehyde oxidase 3, plasma membrane ATPase, and cellulose synthase A
catalytic subunit 4 ［UDP-forming］. Most of those ubiquitinated proteins were found to be involved in metabolism and transport
functions. The result demonstrates that the differentially expressed ubiquitinated proteins with different rice species in salt stressby using proteomics technology and mass spectrometry.

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 34: MS Informatics for Identification and Characterization

PTh-036

13:30 － 14:40

Application of high speed LC-MSMS technology towards unambiguous
characterization of degraded products of Lenalidomide, an anticancer
molecule

Janani Thyagarajan, Raman Palavannathan, Saravanan Subramanyam, Govindarajan Chandramohan,
Mohan Kasi, Manohar VENKAT
Indian Institute of Chromatography & Mass Spectrometry

Keywords:
High Speed LC-MSMS Technology
Novel aspects:
The detailed charaterization of base degraded impurities of Lenalidomide has been accomplished for the first time, the structureof the impurities is elucidated unambiguously.
Abstract:
Lenalidomide RS（ 3 -（ 4 -amino- 1 -oxo1, 3 -dihydro- 2 H-isoindol- 2 -yl）piperidine-2,6-dione）is a potent immunomodulatory drugwith antineoplastic, antiangiogenic and anti-inflammatory properties. It is also used in combination with dexamethasone for thetreatment of multiple myeloma. It is being evaluated in combination with other agents for the treatment of other type of tumors,
including solid one. One of the requirements of drug substance as well as the drug product is their stability under harshenvironments such as temperature, humidity, light and oxidizing agents. It is also important that the drug substance / drug producthas to be evaluated at wider pH range to understand the degradation of the product. Following ICH guidelines, degradation
studies are carried out by acid hydrolysis, base hydrolysis, UV irradiation studies and peroxide degradation. Knowing the potencyof the lenalidomide, many attempts have been made to understand the stability of the compound simulating different
environmental conditions towards separation and identification of degradation products using HPLC and LC-MS methods. Though
the molecular weight of some of the degraded products has been identified, no attempts have been made to fully characterize
the degraded products to understand the mechanism of degradation of lenalidomide. The high speed LC-MSMS technology isapplied, for the first time, to unambiguously characterize the forced degraded products by appropriately proposing the MS
fragmentation pathways for the degraded products.
Lenalidomide was in-house synthesized following a four step synthetic process. Briefly, esterification of 2 -methyl- 3 -nitrobenzoic
acid in the presence of thionyl chloride in methanol provided the methyl ester of 2 -methyl- 3 -nitrobenzoic acid. Benzylic
bromination of this methyl ester using N- Bromosuccinimide provided the bromo compound, isolated bromo compound was
coupled with 3 - aminopiperidine-2, 6 -dione hydrochloride using triethylamine in the mixture of acetonitrile anddimethylformamide gave 3 -（ 4 -nitro- 1 -oxoisoindoline- 2 -yl）piperidine-2,6-dione. Hydrogenation of 3 -（ 4 -nitro- 1 -oxoisoindoline2 -yl）piperidine- 2,6-dione using palladium on carbon in dimethylformamide provided crude lenalidomide, crude was purified
using methanol to obtain a pure lenalidomide. The purity of the lenalidomide was found to be 99.96%（% area）by HPLC.
The degradation was simulated under（a）acid hydrolysis,（b）base hydrolysis,（c）peroxide oxidation and（d）photo degradation
under sunlight. It was observed that the degradation was the highest and instantaneous when base hydrolysis simulation was
carried out, whereas acid hydrolysis did not show any degradation products in HPLC analysis. The purity of lenalidomide got
reduced from 99.96% to about 97.0% under peroxide oxidation and exposure to sunlight. The number of degraded products formed
under these conditions were 7 to 8 components of intensity varying from 0.02 % to 1.6 % . However, in this presentation thecharacterization of degraded products due to base hydrolysis is discussed in detail.
Though the instantaneous formation of degradation products of lenalidomide by base hydrolysis is known, the application of highspeed scanning capability of Shimadzu LCMS-8030 has enabled us to characterize these products. We observe two major
impurities as degradation products having the relative retention time（RRT）of 0.47 and 0.67 minutes respectively. It is interesting toobserve that the molecular weight of both the degradation products is found to be 277 Daltons. The negative MSMS analysis ofboth the components has shown that the structures are different with unique fragmentation pattern. The product at RRT 0.47 isfound to be 4 -（ 4 -amino- 1 -oxoisoindolin- 2 -yl）- 4 -carbamoylbutanoic acid and the product at RRT 0.67 is found to be 2 （ 4 -amino- 1 -oxoisoindolin- 2 -yl）- 4 -carbamoylbutanoic acid. The structures are assigned unambiguously by exploiting the highspeed scanning rate of LCMS8030 under MSMS environment.

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 34: MS Informatics for Identification and Characterization

PTh-037

11:10 － 12:20

Metabolite ID analysis using UNIFI, a novel database driven software
platform approach for screening and understanding metabolism

Milar Alan1, Mark Wrona1, Stephen Mcdonald1, Robert Plumb2, Joanne Mather1
1

Waters Corporation, 2Imperial College, London, UK

Keywords:
Metabolite ID, LC/MS, Informatics,
Novel aspects:
Metabolite identification for processing and sharing of high resolution mass spectrometry datasets using a scientific library
controlled LCMS software package
Abstract:
Current software platforms have been limited generally to single experiment analysis types, although these approaches have andcontinue to serve us well, the majority were designed for linear experimental designs and answers. As individual assay
performance in Met ID and DMPK departments have continued to improve with LCMS platform advances in recent years,
researchers are focusing on ways to increase value from their complex datasets. The approach defined is a Met ID softwaresystem that uses the latest generation PC hardware（64bit, multi-core capabilities）, latest generation hardware（High resolution
UPLC/MS Quant of platforms）and software（scientific library data management）to more accurately screen, confidently
interrogate datasets and report the data to the user in a highly customizable format.
Samples were analyzed on G 2 and G 2 -S Quant of MS platforms equipped with I-class AcquityUPLC systems. Metabolite analysisof several compounds was performed using a prototype version of UNIFI 1.6 software. Software contains a scientific library which
is used to enter and retrieve information about the compounds being analyzed and is able to interface. Samples wereautomatically processed to provide a comprehensive list of metabolites which can then be filtered or interrogated to suit both theanalytical and reporting needs of the user. Integrated and flexible mass accuracy filtering, decimal mass filtering, isotopic
recognition and fragment analysis along with metabolite relationship mapping will be discussed.
Dataset processing using UNIFI was compared and contrasted to current generation software product, Metabolynx XS version 2.0.
Data was analyzed using latest Apex peak picking algorithms with advanced multicore processing capabilities. Processing takes
advantage of builtin scientific library functionality which manages both compound and metabolite identification specific criteria
and allows users to define and generate metabolite relationships within a single LCMS software package. Current methodology
relies heavily on linear single compound, single dataset, single answer approaches. With a scientific library underpinning, users areable to both build and retrieve metabolite relationship information across datasets and samples that were historically difficult tomanage as a single report. Stronger integration of in silico tools, metabolite prediction, isotopic pattern analysis and more flexible
mass defect filtering will be discussed. Will demonstrate how drug metabolism departments will have the ability to manage
instrument and server assets remotely across the network. The network based infrastructure also facilities scientifically sharing
and disseminating data and knowledge across labs and across networks. DMPK datasets are processed for both generating
qualitative structural and relationship pathways as well as for quantitative analysis and putting the datasets into meaningfulcontext within a single, integrated processing and reporting environment.

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 34: MS Informatics for Identification and Characterization

PTh-038

13:30 － 14:40

Metabolite Identification Workflow Software

Graham A McGibbon1, Vitaly Lashin2, Alexey Aminov2, Dmitry Mitushev2, Hans de Bie1
1

Advanced Chemistry Development (ACD/Labs), Toronto, Canada, 2ACD Ltd., Moscow, Russia

Keywords:
software, metabolite identification, biotransformation database
Novel aspects:
Association of structures from metabolism data including fragmentation and involvement of software to manage fullbiotransformation schema linked to interpreted spectra.
Abstract:
Background

Metabolite structure determinations made by review of LC/MS extracted chromatograms and spectra are vital but timeconsuming. Gains in sensitivity, acquisition rates and resolution of mass spectrometry instruments offer more rather than simpler
data, so the identification process must increasingly involve software for data, structure and information management to satisfy
mounting pressure for workplace efficiency. However, software absolutely needs to handle chemical structures and results
documentation as well as facilitating the processes of mass spectrometry data interpretation to truly be effective and reliable.
While elemental composition assessments are improving, finding metabolic ’soft spots’ of new chemical entities and summarizing
biotransformation pathways for drug candidates remain key activities in pharmaceutical discovery and development. This work
describes a new software approach facilitating overall structure assignment workflows including biotransformation knowledge
management for metabolite identification.

Methods

New ACD/Labs software was investigated for processing mass spectrometry data acquired from any major instrument system.
Workflows for LC/MS data from several vendor instruments, including different quadrupole time-of-flight analyzers, were examined.
Parent structures were provided and metabolites were identified by automated feature finding and subsequent determination ofmass and from accurate mass systems also elemental composition. The software made comparisons of parent and metabolites tofurther localize sites of modification when possible. The software was able to conveniently handle structurally ambiguous
metabolites using Markush representations. The structures were stored together with extracted mass spectra, including assigned
fragment ions, and could be reported or exported to a biotransformation database. Various manual searches of the saved
structures and data were then conducted to demonstrate that feasibility.

Preliminary Results

LC/MS data files from metabolite identification studies were processed post-acquisition. Along with the data files from various
samples, the appropriate parent structure was imported and translated to a new software format capable of managing the various
channels of LC/MS, LC/MS/MS, LC/MSn data and associating chemical structures with those data. With parent structures provided,
metabolites were identified by automated feature finding then their masses determined and, if mass accuracy permitted,
elemental compositions. The software made comparisons of parent and metabolites to further localize sites of modification whenpossible. The software was able to conveniently handle structurally ambiguous metabolites using Markush representations. Files
storing structures together with extracted mass spectra, including assigned fragment ions, could be saved or reported. Also, amajor aspect for workflow is that any data could also be exported along with the structure assignments to another softwaremodule designed for biotransformation knowledge management. The latter accommodates entire metabolic pathways, withsearchable spectra and structures. Various manual searches of the structures, substructures and data saved in a biotransformation
database were then conducted to demonstrate that feasibility.

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 34: MS Informatics for Identification and Characterization

PTh-039

11:10 － 12:20

Integration of spectral library in standard data processing pipeline forshotgun proteomics data

Junko Kimata1, Kai Fritzemeier2, Torsten Ueckert2, Bernard Delanghe2
1

Thermo Fisher Scientific K.K., Yokohama, Japan, 2Thermo Fisher Scientific (Bremen) GmbH, Bremen, Germany

Keywords:
Pre-search Spectral library, Shotgun proteomics
Novel aspects:
The integration of a spectral library, used as a pre-search, in standard data processing pipeline for shotgun proteomics data.
Abstract:
Introduction
Over the past years the usage of spectral library search tools was established in shotgun proteomics. Instead of identifying thesame peptide over and over again by a sequence database search engine like SEQUEST, spectral library search uses a library ofspectra identified with high accuracy. The restriction to previously measured spectra avoids a lot of computational overhead.
Search tools like SpectraST, MSPepSearch or BiblioSpec are freely available and a lot of work was spent on building accurate andconfident libraries, which is the crucial part of the identification procedure. Here we present the integration of a spectral library
search engine（SpectraST）into an automated pipeline for shotgun proteomics data analysis（Proteome Discoverer）.

Methods
The usability of spectral library search as fast and sensitive first level search to speed up the peptide identification in a standard
shotgun proteomics data analysis workflow will be presented.
All samples were measured on a Thermo Scientific LTQ Orbitrap Elite instrument coupled to a Thermo Fisher Easy nano-LC. The
data was analyzed using a pre-release version of Thermo Scientific Proteome Discoverer. All spectral libraries used were from
NIST. For data analysis different workflows were used. Search results of spectral library searches were compared to classicaldatabase search engines like SEQUEST and Mascot. The benefit of the usage of spectral library search as pre search to a standard
database search is investigated in a sequential Proteome discoverer workflow.

Abstract
A HeLa and a E.Coli sample were processed using the different workflows. Comparing spectral library search to classical databasesearch shows a decrease in search time. The number of spectra identified in the spectral library search is smaller compared to the
database search due to the limitations of the used libraries to previously identified high confident peptides, but almost all
identifications from spectral library search have a corresponding highly confident identification in the database search. Using aspectral library search as an identification method for a subset of spectra prior to a database search is still considerably fasterthan a method based on identifying peptide sequences by database search alone.

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 34: MS Informatics for Identification and Characterization

PTh-040

13:30 － 14:40

Application of MALDI-TOF Mass Spectrometry Characterization in theDevelopment of Nano-porous Metallophthalocyanin Sensor ElectrodesSiau Gek Ang, Chuan Ming Yap, Guo Qin Xu
National University of Singapore

Keywords:
Metallo 4 ’, 4 “, 4 ’“, 4 ““ tetraamine Pthalocyanines, nitric oxide（NO）detection, Nanoporous Sensors
Novel aspects:
Application of MALDI-TOF mass spectrometry as a conclusive characterization tool for Metal Tetranitro- and Metal TetraaminoPthalocyanins
Abstract:
Metallo 4 ’, 4 “, 4 ’“, 4 ““ tetraamine Pthalocyanines（MTAPcs）have attracted attention for the preparation of chemicallymodified electrodes for electrocatalytic reactions and sensor applications and they are typically immobilized onto the electrodesurface as a stable polymeric film by electropolymerization that proceeds through the oxidation of the -NH 2 substituent. In thiswork we report the successful synthesis of MTAPc monomers（using a 2 -step synthetic protocol）via reduction of thecorresponding Tetra-nitro Phthalocyanine（MTNPc）intermediates using oil bath or microwave heating methods for M=Cu, Mn, Zn,
Fe and Pt. The synthesis of Metal tetranitropthalocyanins（MTNPcs）involves the cyclotetramerization of pre-functionalized
phthalonitrile, phthalic acid and phthalic anhydride precursors together with an appropriate metallic salt, with subsequent
reduction to give the desired amino monomers（MTAPcs）.
Apart from the use of UV-Vis and 1 H-NMR spectroscopies and elemental analysis, the products of synthesis were also extensively
characterized using MALDI-TOF mass spectrometry. In the analysis of experimental MALDI-TOF mass spectrometric data of
MTNPcs and MTAPcs, attention was paid to compare our results with simulation results of expected products（“IsotopeViewer
version 1.0“ was used to provide the simulation of the［M］+· isotopic clusters）. MALDI-TOF mass spectrometry has been foundto be an exceptionally useful characterization tool in providing conclusive evidence of success of the synthesis.
In general, MALDI-TOF mass spectra of the MTNPc and MTAPc complexes provided intense signals due to the molecular radicalion （［M］+·）. The mass spectra of MnTNPc, PtTNPc and ZnTNPc complexes show minor fragmentations due tophotodeoxygenation of the peripheral nitro substituents. The mass spectra of FeTNPc and FeTAPc show the［M］+· of the metal
complex as well as those of the metal-free H 2 TNPc and H 2 TAPc compounds, strongly suggesting the demetallation of FeTNPc
and FeTAPc under the intense laser pulses. For PtTNPc, the［M］+· species（calculated： 887.05489, found： 886.9318）is 23 % in
intensity relative to the base peak of 903.9324（100%）. The base peak is deduced to be due to the［M+OH］+ species, where M is
the molecular weight of PtTNPc.
The second part of our work involves studies on the electrochemical detection of dissolved nitric oxide（NO）in phosphatebuffered saline（pH 7.4）by electropolymerized-Metallo 4 ’, 4 ’’, 4 ’’’, 4 ’’’’ tetra-amine Phthalocyanine（poly-MTAPc）modified
electrodes. For fabrication of electrodes for electrocatalytic reactions and sensor applications, MTAPcs are typically immobilized
onto the electrode surface as a stable polymeric film by electropolymerization that proceeds through the oxidation of the -NH 2
substituent. The electrocatalytic poly-MTAPc coating is in the form of arrays of nanotubes and fibers - this gives rise to improved
sensitivity and detection limit through the provision of more electroactive area.
Two modified electrode systems（the flat glassy carbon electrode（GCE）and a Pt-coated nanoporous anodic aluminium oxide（AAO）membrane）were selected as the bare substrates for poly-MTAPc modification. Our Differential Pulse Voltammetry andAmperometry（DPV and DPA）results showed that of the two, the high-density poly-MTAPc nanotube array within the modified
AAO electrode provided a high faradaic（signal）to charging（background）current ratio leading to 10-15 times improvement insensitivity and a 10 times drop in detection limit.

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 34: MS Informatics for Identification and Characterization

PTh-041

11:10 － 12:20

Highly accurate chemical formula prediction tool utilizing high-resolution
mass spectra, MS/MS fragmentation, heuristic rules, and isotope patternmatching

Tomas Pluskal1, Taisuke Uehara2, Mitsuhiro Yanagida1
1

Okinawa Institute of Science and Technology, Onna, Japan, 2Eisai Co. Ltd., Tsukuba, Japan

Keywords:
chemical formula, heuristics, isotope pattern, MS/MS, prediction
Novel aspects:
The problem of chemical formula elucidation has been addressed previously, but this is the first comprehensive and user-friendly
implementation in an open source mass spectrometry profilingtool（MZmine 2 ）.
Abstract:
Mass spectrometry is commonly applied to qualitatively and quantitatively profile small molecules, such as peptides, metabolites,or lipids. Modern mass spectrometers provide accurate measurements of mass-to-charge ratios of ions, with errors as low as 1
ppm. Even such high mass accuracy, however, is not sufficient to determine the unique chemical formula of each ion, andadditional algorithms are necessary. Mass spectrometry vendors provide their own software modules for chemical formulaprediction, the capabilities of which vary by vendor. We developed a universal software tool for predicting chemical formulas fromhigh-resolution mass spectrometry data. The tool is based on the use of a combination of heuristic techniques, including MS/MS
fragmentation analysis and isotope pattern matching. The performance of the tool was evaluated using a real metabolomic
dataset obtained with the Orbitrap MS detector. The true formula was correctly determined as the highest-ranking candidate for
79% of the tested compounds. For compounds smaller than 250 Da, the true formula was always predicted correctly and, in 67% of
the cases, as the only candidate conforming to the heuristic rules. On average, the heuristic algorithms reduced the search spacefor potential formula candidates 40-fold. The novel isotope pattern-scoring algorithm outperformed a previously published method
in 64% of the tested Orbitrap spectra. Great emphasis was put on user-friendly operation of the tool, which is now freely available
as part of the open-source MZmine 2 framework and its source code can be accessed within the MZmine 2 source code
repository.
Reference： Pluskal T., Uehara T., Yanagida M., Highly Accurate Chemical Formula Prediction Tool Utilizing High-Resolution Mass
Spectra, MS/MS Fragmentation, Heuristic Rules, and Isotope Pattern Matching. Anal Chem（2012）, 84（10）：4396-403

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 35: Environment I

PTh-042

13:30 － 14:40

SBSE probe desorption GC-IT-MS analysis of degradation products of
esfenvalerate obtained by chemical oxidation process

Renata Colombo, Marcos Roberto V Lanza, Janete H Yariwake
University of Sao Paulo/IQSC, Sao Carlos, Brazil

Keywords:
SBSE-GC-IT-MS； esfenvalerate； chemical oxidative degradation
Novel aspects:
Study of degradation of esfenvalerate by using a chemical oxidative process. Utilization of SBSE-GC-IT-MS for extraction of
degradation products of esfenvalerate and their identification.
Abstract:
A growing number of investigations have been reported recently showing the widespread occurrence of agrotoxics in theenvironment, notably in the aquatic compartment. The treatment of these pollutants by oxidative process using hydroxyl radicals（·OH）have been highlighted because of its high efficiency in the degradation of numerous organic compounds and low operatingcost. To evaluate the efficiency of this process and monitoring of intermediates and final products, hyphenated chromatographic
techniques are indispensable. However, due to the low concentration of pollutants and their degradation products in aqueous
medium associated to the incompatibility of the aqueous matrix with conventional GC-MS techniques, some steps of preparationand pre-concentration of the samples are necessary. The SBSE（stir bar sorption extraction）technique combined with hyphenatedchromatographic techniques such as GC-MS resulted in rapid analysis, low solvent consumption, higher analytical precision andsensitivity, and has been successfully employed in the extraction of agrotoxics in water at the concentration range of sub-ng L-1.
However, SBSE-GC-MS analysis requires thermic desorption of the analytes, and nowadays only one commercial SBSE thermal
desorption system is available. On the other hand, an approach called“SBSE probe desorption“ is being investigated by us as analternative to integrate the advantages of SBSE and the power of GC-IT-MS（gas chromatography-ion trap-mass spectrometry）as
the detection technique. In this procedure, the SBSE bar, containing the analytes sorbed（extracted）is placed into an
appropriated probe and thermally desorbed, without using the commercial SBSE thermal desorption system. In this work thedegradation of esfenvalerate, a pyretrhoid insecticide, was studied by using a chemical oxidation process and the degradation
products were monitored by using SBSE probe desorption GC-IT-MS. Degradation was performed by using 50% hydromethanolic
solution containing 45 mg L -1 esfenvalerate, pH 11.25, 25 mg L -1 of hydrogen peroxide, within a 4 hour reaction period. Afterdegradation process, the products were extracted by SBSE by using stir bars（10mm × 0.5mm, 24 µL PDMS coating, Twister,
Gerstel）at room temperature（25oC）, for 120 min, with 15% methanol, 12% NaCl and stirring at 1000 rpm. After extraction, the stir
bar was placed into the probe of a GC CP 3800 Varian, coupled to an ion trap MS Saturn 2000（Varian）. Thermal desorption of theanalytes were done at 250oC, and the probe heating was held for 18 min. GC analysis were performed on a DB- 5 ms fused silica
capillary column（30 m x 0.25 mm i.d., 0.5 μm film thickness, Agilent）. The oven temperature was programmed from 70oC（held for
0.5 min）to 300oC/min（held for 6 min）, at 20oC/min. Helium was used as carrier gas at flow rate of 1.2 mL min-1. The MS analyses
were done in the scan mode（m/z 40 to 450）using electron impact ionization（70 eV）. The temperature of transfer line, ion trap
and manifold were set at 300 o C, 220 o C and 40 o C, respectively. By using the SBSE probe desorption-GC-IT-MS method, it waspossible to fully identify two products of chemical oxidation of esfenvalerate, 3 -phenobenzoic acid and 3 -phenoxbenzaldeyde.
These compounds were previously described as being metabolites of esfenvalerate, with small estrogenic（endocrine-disrupting）
activity and possibly with small environmental impact（McCarthy et al., J. Environ. Monit. 2006, 8,197）. The structural elucidationof the other oxidation products of esfenvalerate is still in progress.
Acknowledgements: FAPESP, CNPq

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 35: Environment I

PTh-043

11:10 － 12:20

Adosorption property of PCB 209 congeners by gamma-cyclodextrin
polymer

Yasunori Fukuda1, Kazuhiro Miyawaki1, Eiichi Kato1, Toshiyuki Kida2, Takeshi Nakano2, Mitsuru Akashi2
1

NEOS Co.,Ltd., Konan, Japan, 2Osaka University, Suita/Japan

Keywords:
Atropisomer,Congener specific analysis,GC-MS/MS,PCBs and other toxic substances,Persistent Organic Pollutants（POPs）
Novel aspects:
Nonpolar organic solvents containing PCBs and other toxic substances which passed gamma-CD polymers solid phase wereanalyzed congener-specifically using the GC-MS/MS.
Abstract:
Polychlorinated biphenyls（PCBs）production in Japan was started by Kaneka Chemical in 1954. The infamous incident of Yusho,
rice-oil poisoning, took place in western Japan in 1968. In 1970’s, regulations over PCBs production and use became enforced and
PCBs disposal measures have been in operation since 1980’s. PCBs waste destruction is still going on today. PCBs were widelyused as insulating fluids in capacitors and transformers. Although their manufacture and commercial use have been prohibited inmany countries since the 1970s because of their strong toxicity, environmental persistence, and bioaccumulation, large amounts ofinsulating oils contaminated with PCBs are still being used or are kept without being appropriately treated in many countries,
including Japan. In the Stockholm Convention on Persistent Organic Pollutants（POPs）, more than 150 countries have agreed todestroy PCBs until 2025. Thus, the efficient and safe treatment of PCB-contaminated insulating oils is a crucial problem from a
global viewpoint.
Cyclodextrins（CDs）are a class of cyclic oligosaccharides consisting of several gamma-（1,4）-linked D-glucopyranose units. They
have a hydrophobic cavity into which a guest molecule of an appropriate size and shape can be incorporated. The ability of CDs
to form inclusion complexes with organic molecules has found applications in many areas, including the food and pharmaceuticalindustries and analytical chemistry. However, in most cases, inclusion complex formation with CDs has been achieved in aqueous
media. On the other hand, much less attention has been paid to inclusion complex formation with CDs in nonpolar organic media,
because it has been believed that inclusion complex formation in nonpolar organic media would be very difficult due to theunfavorable competition with enormous amount of nonpolar organic solvents against the guest molecules for inclusion into the
CD cavity.
We were reported here the removal of PCBs from insulating oil or nonpolar organic solvents by gamma-CD polymers as a newadsorbent. Nonpolar organic solvents containing PCBs and other toxic substances were analyzed adsorption charactor which
passed gamma-CD polymers solid phase using the GC-MS/MS. PCB 209 congeners were analyzed by product ion obtained bydestroying precursor ion using MS/MS method. Other toxic substances were analyzed in the same way. The gamma-CD polymers
were prepared by the reaction of gamma-CD with various kinds of crosslinkers. Among the gamma-CD polymers thus obtained,
the polymer crosslinked with terephthaloyl units showed the highest adsorption capability towards PCBs. Using this type ofpolymer（more than 45 wt% of insulating oil or nonpolar organic solvents）as an adsorbent, PCB 209 congeners and other toxic
substance, whose initial concentrations were 1 &#65374；100 ppm, were completely removed from isooctane solution.

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 35: Environment I

PTh-044

13:30 － 14:40

Charge exchange ionization in reversed-phase liquid chromatographyatmospheric pressure photoionization mass spectrometry of hormones

David Matejicek

Mendel University in Brno, Brno, Czech Republic

Keywords:
APPI, charge exchange,
Novel aspects:
The use of charge exchange reactions in APPI-MS/MS detection of hormones under reverse-phase HPLC conditions.
Abstract:
Atmospheric pressure photoionization（APPI）has been introduced as an innovative ionization source for LC-API-MS systems tobroaden the range of analytes that could be determined using LC-MS even if having weak polarity. From the literature data it isobvious that APPI provides a better ionization for the non-polar analytes than ESI and APCI. Furthermore, photoionization is lesssusceptible to ion suppression compared to ESI or APCI. Several methods employing APPI-MS detection have been suggested forthe analysis of environmentally relevant estrogens, predominantly with toluene as dopant.
Under reversed-phase liquid chromatography conditions, toluene has become a dominant solvent used in the dopant-assisted
photoionization（DA-APPI）. Toluene allows charge exchange in solvents with low proton affinities, however, in solvents commonlyused in reverse phase chromatography（acetonitrile, methanol）； ionization via proton transfer prefers to via charge exchange
reaction. The use of APPI-MS with a dopant enabling ionization via charge exchange will be evaluated for the determination ofseven environmentally relevant estrogens and progestogens（preferably hormones cited in U.S. EPA contaminant candidate list 3 ）in drinking and river water. Finally, the comparison between APPI-MS using ionization via proton transfer（toluene as dopant）or
charge exchange（e.g. anisole, chlorbenzene, brombenzene, 2,4-difluoroanisole, 3 -（trifluoromethyl）anisole）will be performed interms of sensitivity and ion suppression assessment.
The financial support from the Grant Agency of the Czech Republic（Grant Reg. No. GAR P503/10/0975）is gratefully acknowledged.

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 35: Environment I

PTh-045

11:10 － 12:20

Identification of Biodegradation Products of High-Molecular-Weight
Perfluorinated Compounds Using Two-Dimensional Liquid
Chromatography/High-Resolution Mass Spectrometry

Atsushi Yamamoto1,2, Hirotaka Hisatomi1, Daisuke Ono3, Shuusuke Takemine4, Mikiya Kitagawa5,
Hideya Kawasaki1, Ryuichi Arakawa1

Kansai University, Suita, Japan, 2Osaka City Inst. Pub. Health Environ. Sci., Osaka, Japan, 3Osaka Municipal Tech. Res.
Inst., Osaka, Japan, 4Hyogo Pref. Inst. Environ. Sci., Hyogo, Japan, 5Nippon Dionex K.K., Osaka, Japan

1

Keywords:
2 D-LC, Orbitrap, PFCs, biodegradation
Novel aspects:
Two-dimensional liquid chromatography and high-resolution mass spectrometry could demonstrate the occurrence of
biodegradation of huge PFCs to small PFCs in aerobic biodegradation.
Abstract:
Perfluorinated compounds（PFCs）have been considered an environmental problem. Early studies on PFCs focused on thepresence of perfluoroalkyl carboxylic acids（PFCAs）and sulfonic acids（PFSAs）in environmental matrices and their possible
biological effects. However, more complicated PFCs were usually used rather than simple molecules such as PFCAs and PFSAs.
There is limited information about such complicated PFCs. Although major fluorochemical companies now have phased out of
production and use of PFOA, PFOS and PFOS-related products, PFOA and PFOS still have been found in various contexts. Bothfluorochemical products and environmental samples always contain complicated matrices. It is difficult to determine what kinds of
PFCs really exist.
In the past few years, two or multidimensional liquid chromatography has been attractive for analyses of complex mixtures.
Multidimensional chromatography coupled with mass spectrometry notably offers comprehensive analysis and has been applied
to the characterization of natural products and industrial materials. In this study, we used an off-line 2 D liquid chromatographcoupled with a high-resolution mass spectrometer（ 2 D-LC/HR-MS）to identify commercial fluoroproducts and their biodegradation
products.
Fluoroproducts examined consisted of four products. Three of them were products sold before the regulation. All of them werepurchased in Japan. Liquid samples were diluted in water by 3000-fold. Activated sludge was added to the diluted samples at aconcentration of 30ppm. The samples were transferred in closed containers, which were made to be able to supply oxygen foraerobic biodegradation. Ten-mililiter portions of samples were taken from each stirred sample once a week for a month. The
portions passed through solid phase extraction cartridges, OASIS WAX（Waters）, to extract PFCs. PFCs were eluted from the
cartridges with 0.1 % NH4OH methanol. The eluates were analyzed by liquid chromatograph/mass spectrometer, Ultimate 3000/
Exactive（ThermoFisher）.Ultimate 3000 and Exactive were used for acquisition of mass spectra with a high resolving power of
100,000. In addition, Acquity and Xevo TQ（Waters）were used for quantitation of PFCAs and PFSAs and acquisition of product ion
spectra. Two-dimensional LC was carried out with two different kinds of columns. A TSK-Gel ODS-100S from TOSOH and an EpicFO column from ES-industries were used for chromatography.
Acquired mass spectra with single LC included numerous ions. The interpretation of mass spectra was, therefore, very difficult.
Two-dimensional LC could effectively separate these compounds depending on the interaction strength with two different typesof columns. The mass spectra that generated from separated compounds were easier to interpret than the spectra from
unseparated compounds. Fragment ion spectra were also acquired.
The chemical structures were determined from the interpretation of the data of samples before biodegradation. A dominant ion
with an m/z value of 1574.2809 was found in one product examined. Because three fragment ions that had equal spaces of 357.007
were found in the observation with collision gas, the compound was the molecule that had three C4F9SO2NCH3C2H4O groups. If
triisocyanate such as Tolonate reacts with an alcohol, C4F9SO2NCH3C2H4OH, the chemical formula of the molecule yielded will be
C45H60N9O15S3F27. The exact mass of the ion is 1574.2867. The two values were well-matched.
Biodegradation products were also examined by analyses of the samples after biodegradation. Two out of four products
examined showed unequivocal increase of concentration of PFCAs. Although the above-referenced sample did not show any
increase of PFCAs and PFSAs, a degradation product that had a carboxyl group at the terminal was identified. The result
complemented the presumption of original structure. However, further degradation structures such as sulfonamide were notfound.
Two-dimensional LC/HR-MS demonstrated the occurrence of biodegradation of huge PFCs to small PFCs in aerobic
biodegradation. We need further investigation of fluoroproducts that have been used so far.

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 35: Environment I

PTh-046

13:30 － 14:40

Current status of organophosphorus compounds contaminants in Maizuru
Bay, JapanEmi Yatsuzuka1, Hiroya Harino1, Chiaki Yamao1, Masaaki Ueno2, Madoka Ohji3
1

Kobe College, 2Kyoto University, 3Tokyo University of Agriculture and Technology

Keywords:organophosphorus compounds, water, sediment, mussel
Novel aspects:
Organophosphate esters and pesticides were detected in water, sediment and mussel samples from Maizuru Bay, Japan.
Abstract:
Organophosphorus（OP）compounds which have been utilized as flame retardants, plasticizers, stabilizers, antifoaming andwetting agents, and additives in lubricants and hydraulic fluids etc. are the useful chemical compounds and are used in the various
areas. It is well known that aquatic environment has contaminated by volatilization, leaching or abrasion from the broad
application range of these compounds. In this study, the concentrations of eight species of organophosphate esters（OPE）and OP
pesticides（diazinon, fenitrothion, chlorpyriphos and iprobenphos）are measured in water, sediment and green mussels from
Maizuru Bay and the distribution of these compounds in water environment is discussed.
Water sediment and mussel samples in Maizuru Bay were taken from July 7 th to 15th, 2009. Subsurface water samples were takenin 7 sites and the surface sediment samples（ 0 5 cm）were taken using a Ekman-Birge type bottom sampler in 13 sites. The
mussel（Mytius galloprovincialis）samples were taken at 9 sites. The shell lengths of the green mussels were in the range of 380 750 mm. Three mussel samples in each site were homogenated before analysis. Water samples were stored in a fridge at 3 oC andtheir samples were analyzed within a few day. Sediment and mussel samples were stored in a freezer at -20 oC for until chemical
analysis.
Water samples were extracted with dichloromethane by shaking for10 min. The aqueous layer was dried with anhydrous
Na2SO4and was concentrated up to 0.5 ml after the addition of Atradine-d25 as an internal standard. The analytes were measured
by GC-MS. OPs in sediment and mussel samples were extracted with acetone. After centrifugation, the supernatants were cleaned
by addition of distilled water, celite and zinc acetate. After removal of suspended matters by filtration, the analytes in liquid layer
were extracted dichloromethane. After the addition of Atradine-d25, the organic layer was concentrated up to 0.5mL. The analytes
were measured by GC/MS.
The distribution of OP compounds in Maizuru Bay was surveyed. The concentrations of OP in water samples from Maizuru Bay
were in the range of 2.7 62 ng/L, which these level was under a thousandth of acute toxic level for aquatic organisms. The
concentrations of OPEs were high the order of TBXP > TDCPP >TCEP > TBP > TCP > TPP > TEP. This trend is similar to those in
1976-1996. Diazinon, fenitrothion and chlorpyriphos of OP pesticides were detected in water samples.
The concentrations of OP compounds in sediment from Maizuru Bay were in the range of <0.5 - 56 ug kg -1 dry weight（dw）. The
patterns of OPEs in sediment are classified two groups. The patterns of OPEs concentrations in a group are characterized that
OPEs concentrations are high the order of TBXP > TDCPP or TBP, and the other OPEs are similar concentrations. The patterns of
OPEs concentrations in the other group characterized that TDCPP concentrations is the highest among OPEs. Diazinon was
detected in all sediment samples from Maizuru Bay and the concentrations of diazinon were in the range of 1.8 and 71 ug kg-1 dw.
The detection frequencies of fenitrothion and chlorpyriphos were low in sediment.
The concentrations of OP in mussel from Maizuru Bay were in the range of <0.5 and 34 ug kg -1 wet weight. The concentrations of
OPEs were high the order of TBP > TDCPP >TCP >TBXP = TPP = TCEP.
The partition rate between water and sediment（Kws）of diazinon and fenitrothion were in the range of 200 and 1300, and in therange of 200 and 300, respectively and the partition rate between water and biological samples（Kwb）of diazinon and fenitrothion
were in the range of 700 and 3300, and in the range of 450 and 700, respectively, suggesting that these pesticides prefer biota tosediment.

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 35: Environment I

PTh-047

11:10 － 12:20

Tandem SPE clean up/extraction: strategy to minimize matrix effects on
LC-MS determination of endocrine disrupters and pharmaceuticals insewage samples

Robson J Afonso1, Fernanda B Queiroz2, Julio C Silva3, Rafaela P Gomes1,2, Mauricio X Coutrim1,2,
Sergio F Aquino1
13

Federal University of Ouro Preto/Minas Gerais/Brazil, 2Molecular Characterization/Mass Spectrometry Laboratory,
Federal University of Minas Gerais/Minas Gerais/Brazil

Keywords:
Emerging Contaminants； Matrix Effects； Sewage
Novel aspects:
The work uses tandem SPE（SAX+HLB）. This approach was efficient at removal of LAS and to minimize their matrix effects on ESI
responses of analytes in sewage samples extracts.
Abstract:
In recent years, a variety of organic compounds, also called emerging contaminants, have being detected by LC-MS, at low
concentrations（ng L- 1 ）, in samples of surface water, wastewater, groundwater, and even drinking water［ 1 - 3 ］. According toTaylor［ 4 ］, the matrix effect is the“Achilles’ heel“ of the HPLC-MS techniques. Therefore, it is extremely critical to evaluate and/or minimize the influence of complex matrix composition on the HPLC-MS responses of analytes. Few articles have evaluatedthoroughly the matrix effect in analysis of emerging contaminants in sewage samples. In this work a new method for thedetermination of three endocrine disrupters （estradiol, ethinyl estradiol, and bisphenol A） and five pharmaceuticals（sulfamethoxazole, trimethoprim, diclofenac, bezafibrate and miconazole）in raw and treated sewage samples was developed
and validated. The method consisted of the application of tandem SPE procedure that uses both a strong ion exchange sorbent（SAX）and a modified divinylbenzene-pyrrolidone sorbent to reduce the levels of linear alkylbenzenesulphonate（LAS）and toconcentrate the analytes of interest from sewage samples, prior to analysis by liquid chromatography combined with highresolution mass spectrometry（LC-HRMS）. The influence of matrix composition on the ionization efficiency, the SPE recoveries,
and the sensitivity of the method was determined. The SAX sorbent was capable of significantly removing LAS content in sewage
samples extracts. It was also capable of retaining the analytes that were eluted with ethyl acetate with recoveries that varied from17 to 35 %（CV < 9 %）. This approach was very efficient at minimizing the matrix effects. The sum of the recoveries from bothsorbents varied from 30 to 70%（CV < 9 %）. The mass spectrometry detection was performed using a LC-ESI-IT-TOF/MS instrument
working at high-resolution（10.000 FWHM）and high mass accuracy（< 5 ppm）. The instrumental limits of quantitation varied from
0.4 µg L- 1 to 3.3 µg L-1. The method limits of quantitation ranged from 3.3 ng L- 1 to 41 ng L-1. The method was successfully applied
to the determination of analytes in raw sewage samples at the Arrudas Sewage Treatment Plant, Belo Horizonte, Brazil and also toevaluate the efficiency of different experimental sewage treatment systems.
［ 1 ］M.J.L. deAlda, S. Diaz-Cruz, M. Petrovic, D. Barcelo, J. of Chromatography A 1000（2003）503-526.
［ 2 ］D. Barcelo, M. Petrovic, Trac-Trends in Analytical Chemistry 26（2007） 2 -11.
［ 3 ］M.A. Moreira, R.J.C.F. Afonso, S.F. Aquino, Environmental Technology 32（2011）1409- 1417
［ 4 ］P.J. Taylor, Clinical Biochemistry 38（2005）328-334.

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 35: Environment I

PTh-048

13:30 － 14:40

Simultaneous analysis of anionic, amphoteric and non-ionic surfactants
using ultra-high speed LC-MS/MS

Keiko Matsumoto, Jun Watanabe, Junko Iida
Shimadzu Corporation., Kyoto, Japan

Keywords:
surfactant, LAS, Betain,Heptaethyleneglycoldodecylether,TQ
Novel aspects:
Simultaneous analysis method of anionic, amphoteric and non-ionic surfactant using ultra-high speed polarity switching techniqueof LC-MS/MS was developed.
Abstract:
Introduction
Surfactant chemistry has made a considerable impact on a number of household products including detergents, shampoos andtoothpaste. Products are generally classified by the type of each hydrophilic substructure into anionic, cationic, amphoteric andnon-ionic surfactants. Either anionic or non-ionic surfactants are typically used as synthetic detergents, however, to better
elucidate the potential risk in environmental samples, mainly in agricultural soils and sediments, methods need to take into
account a range of surfactant chemistries. Current surfactant monitoring methodologies tend to focus on a specific surfactant.
Here, we have developed the simultaneous analysis method for typical anionic, amphoteric and non-ionic surfactant using LC-MS/
MS.
MethodCommercially available surfactants were used for this experiment. Standards of surfactants were diluted with water to an
appropriate concentration and then determined by LC-MS/MS. As an LC-MS/MS system, UHPLC was coupled to triple quadrupole
mass spectrometer（Nexera MP with LCMS-8030, Shimadzu Corporation, Kyoto, Japan）. Separation was achieved using a YMCTriart C 8 column（100mmL., 2.0mmI.D., 1.9um particles）and column oven temperature was maintained at 40 C. Samples were
eluted at flow rate 300uL/min with a binary gradient system； the mobile phase consisted of（A）10mM ammonium acetate bufferand（B）mixture of 10mM ammonium acetate / acetonitrile / isopropanol（ 1 / 4 / 5 ）. LC-MS/MS with electrospray ionization was
operated in multiple-reaction-monitoring（MRM）mode with ultra-fast polarity switching.
Preliminary data
The following standard surfactants were selected and analyzed； anionic surfactant： linear alkylbenzene sulfonate（LAS）C10-C14
mixture, amphoteric surfactant： EMPIGEN BB Detergent （Betaine） C10, C12, C14 mixture and non-ionic surfactant：heptaethyleneglycoldodecylether（HEDE）. Full scan measurement by flow injection analysis（FIA）was conducted to determine
the optimum ionization polarity of target compounds followed by MRM transition optimization by FIA. As a consequence, all LASs
were detected as the de-protonated ions and m/z 183 was selected as the product ion of MRM transitions for all LASs（C10-C14）.
All Betaine were detected as protonated ions and m/z 104 was selected as the product ions of MRM transitions for all Betaine（C10, C12 and C14）. HEDE yielded the protonated ion as the precursor and m/z 133 was selected as product ion for MRM
transition. As compounds selected in this experiment formed either positive or negative ion, high-speed polarity switching is an
important parameter to consider in developing an optimized method. The dilution series of these compounds were analyzed andall compounds were detected at sub ppb level with excellent linearity. In addition, the quantitative analysis of real world sample
using the kitchen detergents and liquid soap was achieved using this method

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 35: Environment I

PTh-049

11:10 － 12:20

Limitations of a commercially available plasma air purifier

Stefan Schmid1, Andreas Gerecke2, Herbert Haechler3, Hubert Hilbi4, Simon Weidmann1,
Renato Zenobi1

1
ETH Zurich, Zurich, Switzerland, 2Swiss Federal Laboratories for Material Science and Technology, Dubendorf,
Switzerland, 3University of Zurich, Zurich, Switzerland, 4Ludwig-Maximilians-Universitaet, Munich, Germany

Keywords:
Non-thermal plasma, Cleaning efficiency, Air-cleaning
Novel aspects:
Efficiency study of a commercially available plasma air purifier over a wide range of different substances
Abstract:
The molecular processes in a commercially available AC, driven plasma air purifier（PAP）were studied in detail. Such air purifiers
are supposed to break down all air contaminants to small, nontoxic molecules（e.g. H2O and CO2）. However, the degradation
mechanisms in such PAPs are not known yet. In this study, we examined the exhaust of a commercially available PAP to determine
its efficiency and the molecular processes taking place. Three different classes of substance were studied： environmental toxins
representative for of low MW molecules, a high-mass protein, and various bacteria to represent very high masses. One goal of thisproject was to examine the limitations of such commercially available air purifying systems.
A setup was designed in such a way that the PAP could be studied under realistic conditions, simulating common heating andventilation systems. Phenanthrene and methyltriclosane were chosen as small molecules. Bovine serum albumin（BSA）was chosen
as a model high mass protein. Legionella Pneumophila and Bacillus anthracis were used to cover the class of airborne infectious
agents. The sampling method was adapted to the respective compounds. Adsorption tubes were used for the low MW
molecules； bubbling 10% of the PAP exhaust through water was used to quantify the amount of degraded BSA； L. pneumophila
and B. anthracis were sampled using agar strips, which were afterwards wrapped in sterile containers and incubated.
The study of environmental toxins using GC-MS showed a degradation of 31.0% and 16.9% for phenanthrene and methyltriclosane,
respectively（relative error, 13 %）. However, no characteristic degradation products could be found. Therefore, a mass balancewith methyltriclosane was conducted which yielded surprising results. On the 4 copper electrode surfaces of the PAP exactly 17%
were methyltriclosane found. Since the degradation was determined by performing an experiment with and without activated
PAP, the decreased amount of methyltriclosane was considered as degraded. However, our experiments do not support the
hypothesis of degradation for small molecules anymore.
Measuring the PAP degradation performance of BSA showed a reduction of 81.1±30 % , when comparing experiments with andwithout the plasma activated. The copper electrodes showed a white film after the experiment with the plasma activated. Afterdissolving the white precipitate in water, LC-MS and MALDI-MS experiments identified it as BSA. 1 % of white and water insoluble
crystals which were identified as polymerized BSA using MALDI-MS equipped with a high mass detector. The reduction in amountof various bacteria showed, that the PAP is capable of reducing aerosolized bacteria in air. However, it seems that the reduction ismainly occurring due to adherence to the copper electrodes.

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 35: Environment I

PTh-050

13:30 － 14:40

Multi-component quantitative analysis of pharmaceuticals and personalcare products in the environment by LC-MS/MS with fast polarity
switching

Natsuyo Asano, Kiyomi Arakawa, Shinjiro Fujita, Manabu Ueda, Kazuo Mukaibatake, Ichiro Hirano
Shimadzu Co., Kyoto, Japan

Keywords:
LCMS-8080, higher sensitivity, multi-component quantitative analysis , fast polarity switching, and pharmaceuticals and personalcare products（PPCPs）
Novel aspects:
Fast polarity switching results have been equivalent to dedicated single polarity experiments for the analysis of PPCPs inenvironmental samples.
Abstract:
Pharmaceuticals and personal care products（PPCPs）constitute a group of emerging contaminants which have received
considerable attention in recent years. Monitoring of PPCPs in the environment is vital as many of these compounds are
ubiquitous, persistent and biologically active with recognised endocrine-disruption functions. Given the hazardous nature of thesecompounds, there is a need to provide fast and sensitive multi-residue methods that are able to analysis multiple classes ofcompound within one analytical procedure. Here we report a new multi-residue UHPLC-ESI-QqQ method that utilises fast polarity
switching with an optimised chromatographic gradient that removes matrix effects and results in excellent ng/L detection levels.
Furthermore, we evaluate the performance of polarity switching in comparison to dedicated single polarity experiments.
Natural river and lake water was collected from the Shiga region（Japan）and spiked, without any sample pretreatment, at a
range of concentration levels（ 1 - 10000 ng/L）with 15 PPCPs（e.g. Carbamazepine, Dehydronifedipine, Gemfibrozil, Triclocarban）.
Separation was achieved using a Shim-pack XR-ODS III column（50 x 2.0 mm, 1.6 µm）maintained at 40 °C on a UHPLC system,
Nexera（Shimadzu, Japan）. The method was maintained at a flow rate of 0.4 mL/min with mobile phase A： water/0.1 % formic
acid and B： acetonitrile. The gradient（detailed in preliminary data）was optimized to minimise matrix effects. A higher sensitivity
triple quadruple mass spectrometer, LCMS-8080（Shimadzu, Japan）operating in SRM mode with fast polarity switching（20 msec）
was used for the detection of positively and negatively charged analytes.
As a result of the complex matrix PPCPs are present； the occurrence of ion suppression/enhancement is commonly described inliterature and results in reduced MS/MS detection limits and inferior precision. For this reason, a gradient was optimised that
focused target analytes at the head of the chromatographic column while allowing the interfering environmental matrix to beeluted； this resulted in excellent recoveries of around 100 % for almost all studied compounds. This was achieved using a gradient
which held the aqueous mobile phase at 100 % for 6 min, and subsequently increased the organic mobile phase to 80 % over 10
minutes and then to 100 % organic mobile phase.
PPCPs encompass a wide range compound classes and chemical properties and consequently it is necessary to employ both
positive and negative electrospray ionization for complete analysis in a single run. All compounds were measured by SRM with
fast polarity switching（20 msec）for multi-component analysis. Excellent limits of quantification were achieved in the range 1 - 50
ng/L for nearly all studied compounds, with outstanding linearity（R2> 0.999）.
To evaluate the capability of polarity switching the data quality obtained was compared to dedicated negative or positive
analysis. Comparisons were made by selecting compounds that prefer positive（Carbamazepine and Dehydronifedipine）andnegative ionisation（Gemfibrozil and Triclocarban）. Findings showed that the data quality obtained during polarity switchingexperiments was directly comparable to that achieved during dedicated positive or negative analysis. In addition, long term
stability was investigated by making 100 injections over >10 hours, with polarity switching presenting excellent stability.

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 35: Environment I

PTh-051

11:10 － 12:20

Screening and library search of Environmental pollutants in Japaneseenvironmental water using LC-MS/MS

Ushio Takeda1, Kozue Asakura1, Yuji Kakazu1, Andre Schreiber2
1

AB SCIEX, Tokyo, Japan, 2AB SCIEX, Concord, Canada

Keywords:
PPCPS Environment LC/MS/MS Water
Novel aspects:
Fast Aqcuisition time, High Sensitivity, Direct measurement, Over 100 compounds
Abstract:
Introductions
In recent years, many kinds of chemical substances are polluting environmental water. When people take a medicine, use insect
repellent, or apply cosmetics, these chemicals flows into the water in the environment, finally. These compounds are called PPCP
（Pharmaceuticals and Personal Care Products）as a whole. Furthermore, it is known that the pesticides originally used for
agricultural products are polluting environmental water. These compounds are also contained in PPCP in a broad sense.
The diversity and the numerousness of these compounds make method development challenging. But In order to properly assess
the effects of these compounds on our environment, it is necessary to accurately monitor their presence. Liquid Chromatography
coupled to tandem Mass Spectrometry（LC-MS/MS）is able to analyze polar, semi-volatile, and thermallylabile compounds
covering a wide molecular weight range, such as pesticides, antibiotics, drugs of abuse, x-ray contrast agents, drinking water
disinfection by-products etc. In addition, state-of-the-art LC-MS/MS instruments operated in selective Multiple Reaction Monitoring（MRM）mode, offer unmatched selectivity and sensitivity to quantify PPCP reproducibly at trace levels without time consuming
and extensive sample preparation.
AB SCIEX QTRAP®4500 LC/MS/MS System can measure many compounds simultaneously because of its fast MRM scanning, and its
can measure compounds high-sensitive because its hasQ-jet® 2 ion guide.
More recently, it is discussed that detection only by MRM cause false positive and false negative. To avoid false detection, one
useful process is library search for a fragment ion spectrum. This process judges positive or negative by comparing the spectrumof standard with the spectrum of the compound detected in the sample. At 1 ch MRM, while judging from one fragment ion, by
the library search using a spectrum, a judgment is performed from all fragment ions which the compound has. Therefore, library
search can do more precise judging.
ABSCIEX QTRAP ® 4500 LC/MS/MS System can acquire fragment ion spectrum fast and sensitive because its has Linear Ion
Trap  technology with Linear Accelerator TM Trap. And AB SCIEX has fragment ion spectrum library that contains large number ofcompounds.
LC-MS/MS is suitable for measurement of PPCP, and judgment by library search is desirable. Therefore, AB SCIEX QTRAP® 4500
LC/MS/MS System suitable for PPCP screening.
Methods and results
20 Japanese environmental water samples were injected directly into AB SCIEX QTRAP® 4500LC/MS/MS System to detection PPCP
at low parts-per-trillion（ppt）levels（ng/L）. 4500 quick scanning speeds supported simultaneous analysis of 304 compounds（273for positive, 31 for negative）were acquired by using MRM. Enhanced Product Ion Scan（EPI）was used for acquiring the fragment
ion spectrum at ppt levels, and“Mega-library“ spectrum library was used to judge compounds detected by MRM were true or
false. EPI is high-sensitive and high-speed fragment ion scan mode, supported by Linear Ion Trap  technology with Linear
AcceleratorTM Trap.“Mega-library“ is fragment ion spectrum library that contains over 1240compound’s spectrums.
From Japanese environmental water, some compounds were detected by MRM. The detected compounds did not related
sampling point. According to results of the library search, some compounds were truly detected, but some compounds were false
positive.
As a conclusion, the PPCP exist Japanese wide area environmental water. So it is necessary to measure much more samples toknow a trend, such as regionality.

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 35: Environment I

PTh-052

13:30 － 14:40

OPTIMIZATION OF SOLID PHASE EXTRACTION FOR PERFLUORINATEDCOMPOUNDS ANALYSIS IN WATER SAMPLE

Chinagarn Kunacheva, Tanaka Shuhei, Fujii Shigeo, Suzuki Yuji, Chularueangaksorn Pattarawan
Kyoto University, Kyoto, Japan

Keywords:
Micropollutant, Perfluorinated compounds（PFCs）, PFOA, PFOS, Solid Phase Extraction
Novel aspects:
The solid phase extractionwas optimized for analysis of twelve PFCs in water sample, especially tap water and river water（withlow matrix）.
Abstract:
In recent years, perfluorinated compounds（PFCs）have appeared as a new class of global contaminants. Researchers havereported PFCs contamination in surface water, tap water and bottled water around the world. They are particularly difficult todeal with once released into the environment, because they do not break down easily, they can travel long distances carried byair or in water and they can accumulate in human and animal tissue. To understand the sources and fate of these compounds, it
is essential to optimize the analytical methods for a wide range of perfluorinated compounds in water sample. Solid phaseextraction（SPE）coupled with high-performance liquid chromatography-tandem mass spectrometry（HPLC-MS/MS）is generally
used to analyze PFCs in environmental matrices. Researchers have reported the difficulties in analysis of short chain（C 4 C 6 ）andlong chain（C10 C12）PFCs. The ineffective SPE procedure and matrix interferences were the two major difficulties for analyzing
environmental water sample. To overcome these problems, the optimization of SPE process is needed. The objective of this study
was to evaluate the optimum SPE condition for two ion exchange（OasisWAX and PresepPFC-II）cartridges for analyzing 12 PFCs
in water sample. These cartridges were also compared with PresepC-Agri（C 18 ）and OasisHLB, which are generally used for
perfluorooctane sulfonate（PFOS）and perfluorooctanoic acid（PFOA）analysis. Several options for optimizing SPE were evaluatedsuch as adjusting sample loading flow rate, washing sample’s bottle by methanol, optimizing cartridges drying step, adding matrix
washing step, conducting sequential elution, adding volume of solvent and optimizing nitrogen purging. Ion-exchange cartridges（OasisWAX and PresepPFC-II）were showed better result comparing to both PresepC-Agri（C18）and OasisHLB. Flow rate 10 mL/
min showed better recoveries of most chemicals comparing to flow rate 5 and 20 mL/min. Comparison of drying cartridges andwithout drying cartridges was conducted for both PresepPFC-II and OasisWAX. The average increase of all twelve chemicals was
9 % . An experiment was conducted for washing the sample bottles two times after loading to the cartridges. Five milliliter ofmethanol（ 2 times）was applied for each washing. Recoveries of C10 C12 increased by 2 %, 12% and 33%, respectively for both
cartridges. The elution was done three times by using 2 mL 0.1%NH 4 OH in methanol each time. More than 97% of all compounds
were eluted in the first elution. An experiment on the effect of PFCs lost during nitrogen purging step was also examined. There
was no significant in the loss of PFCs during nitrogen purging for 1 hr, 2 hrs and 3 hrs. The solid phase extraction was optimized
for analysis of twelve PFCs in water sample, especially tap water and river water（with low matrix）. Coupled with the use of
HPLC-MS/MS, a method detection limit in the range of several tens of parts-per-quadrillion（pg/L）in water can be achieved.

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 35: Environment I

PTh-053

11:10 － 12:20

Analytical method and homolog distribution of OH-PCBs in ambient Air

Yuki Haga1, Motoharu Suzuki2, Masahiro Tsurukawa1, Yoshiya Yamoto3, Chisato Matsumura1,
Takeshi Nakano4

Hyogo Pref. Inst. of Env. Sci., Kobe, Japan, 2Kobe University, Kobe, Japan, 3Kansai University, Osaka, Japan, 4Osaka
University, Osaka, Japan

1

Keywords:
OH-PCBs, air, GC/MS
Novel aspects:
OH-PCBs in ambient air was detected using QFF and PUF which were set to Hi-Volume air sampler.
Abstract:
This analytical method is suitable for the determination ofHydroxylated Polychlorinated Biphenyls（OH-PCBs）in ambient air by gas
chromatography- high-resolution mass spectrometer with selected-ion monitoring（GC/HRMS-SIM）. OH-PCBs are considered as
one of the endocrine disturbing chemicals because OH-PCBs have negative impacts on the some thyroid and female hormones.
OH-PCBs are formed by metabolism of PCBs by the cytochrome P450 enzyme-mediated oxidation and OH radical reaction of PCBs.
The concentrations of OH-PCBs in the environmental waters, air, sediments and living things have been investigated and OH-PCBshave been detected from every medium. In Canada, OH-PCBs were also detected from the rain and snow. In this study, we
modified the analytical method and investigated the concentration of OH-PCBs in ambient air. The air was introduced into quartz
fiber filter（QFF）and poly urethane foam（PUF）which were set to Hi-Volume air sampler at a flow rate of 700 L/min for 24 hr（total
volume was 1000 m3）. After collection was completed, clean-up spike（OH-Di-Hp-CB-13C 12 3.0ng）was added to the PUF. BothQFF and PUF were simultaneously extracted with acetone by ASE. The acetone extract was added 6 mL of 5 % sodium chloride
solution, and extracted with 2 mL of hexane, twice. The hexane phase was clean upped with pre-washed Sep-Pak Plus Florisil.
Sep-Pak Plus Florisil was eluted with 0.5% diethyl ether/hexane 8 mL（for PCB）, and then with 50% acetone/methanol 10 mL（forOH-PCBs）. The eluate was concentrated until just before dryness. After derivatization and alkaline digestion, added 6 mL of 5 %sodium chloride solution, and extracted with 2 mL of hexane, twice. Concentrated to about 1 mL and dehydrated withanhydrous sodium sulfate. The concentrate was applied to a pre-washed Sep-Pak Plus Florisil, and eluted with 8 mL of 5 %diethyl ether/hexane. The eluate was concentrated to100 μL and added syringe spike（MBP-70- 13 C 12 0.50 ng）. The method
detection limits（MDL）and the method quantification limit（MQL）were 0.067 - 0.13 and 0.17 - 0.33 pg/m3, respectively. The averageof recoveries（n = 3 ）from 1000 m 3 of air sample added with 3.0ng OH-PCBs were almost 60-110 % , and the relative standard
division was 1.2 - 24% . Lowly chlorinated OH-PCBs tend to be collected in PUF and highly ones tend to be collected in QFF. In the
air samples, OH-PCBs were detected 1 pg/m3 order and the lowly chlorinated OH-PCBs were dominant.

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 35: Environment I

PTh-054

13:30 － 14:40

Application of combined UPLC-TOF-MS and combustion ion
chromatography for electrolytic degradation mechanism of PFOS in water

Shogo Taniguchi1, Ayumi Hashiguchi2, Rabindra R Giri1, Hiroaki Ozaki1
1

Osaka Sangyo University, Osaka, Japan, 2Kyoto University, Kyoto, Japan

Keywords:
Activated-carbon, bond cleavage, byproducts, TOF-mass
Novel aspects:
Combined UPLC-TOF-MS and combustion ion chromatography technique used for first time in elucidating electrolytic degradation
mechanism of PFOS.Carbon-carbon bond cleavage adjacent to sulfonate functional group is the first reaction step.
Abstract:
Perfluorooctanoic acid（PFOA）and perfluorooctane sulfonate（PFOS）are the most extensively investigated representative
compounds of perfluoroalkyl carboxylate（PFAC）and perfluoroalkyl sulfonate（PFAS）groups respectively. PFOA can be degraded
by UV photodegradation in water while PFOS is extremely resistant to the degradation. But the latter in water can be degradedvery easily by electrolytic method than by UV photodegradation. However, electrolytic degradation mechanism and pathways for
PFOS are not still known.
Mechanism of electrolytic degradation of PFOS in water is investigated and discussed for the first time using ultra-pressure liquid
chromatography time of flight mass spectrometry（UPLC-TOF-MS）combined with combustion ion chromatography. Batch tests forelectrolytic degradation（current = 1.0 A, DC voltage： 33.0 V, surface area of circular platinum electrodes = 236 mm2, current
density = 4.23 A/m 2 ）of PFOS（concentration = 1.0 mg/L, volume = 250 ml）were conducted using a cylindrical air-tight and heatresistant glass vessel for 60 min durations. Sodium bicarbonate（50.0 mmol/L）was added to the reaction solution for enhancing
electrical conductivity. Liquid samples were collected and analyzed（UPLC-TOF-MS）for PFOS and its degradation intermediates.
Head space gas samples were adsorbed to activated carbon columns by continuous nitrogen gas flow through the space. Total
fluoride ion concentration and short carbon-chain intermediates in the sample（i.e. activated carbon after head space gas
adsorption）were analyzed using combustion ion chromatography. PFOS degradation mechanism in water is discussed based on
the identified intermediates.
Though it has already been demonstrated that fluorinated short carbon-chain byproducts of PFOA degradation remain mostly ingas phase（i.e. head-space gas）, no such products of PFOS electrolytic degradation in activated carbon columns were observedin this investigation. Absence of fluorinated short carbon-chain compounds and additional fluoride ion contents due to oxidationof the compounds during combustion ion chromatographic analysis strongly suggested that either the compounds were actuallyabsent during PFOS degradation or the compounds in gas phase were not adsorbed to activated carbon. This point still remains
to be elucidated. Analysis of water samples by UPLC-TOF-MS showed fragments（with seven carbon atoms）of PFOS havinghydroxyl functional group suggesting carbon-carbon bond cleavage adjacent to sulfonate functional group followed by its
hydrolysis during electrolytic degradation of PFOS. The present investigation needs to be continued further to understand whether
fluorinated short-carbon chain byproducts of PFOS electrolytic degradation do not really exist in head-space gas and elucidate oncomplete degradation mechanism.

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 35: Environment I

PTh-055

11:10 － 12:20

2,3,7,8-tetrachlorodibenzo-p-dioxin congener in breast milk among 3 hotspots in Vietnam

Manh D Ho1, Teruhiko Kido1, Rie Okamoto1, Tai T Pham2, Muneko Nishijo2, Hideaki Nakagawa2,
Shoko Maruzeni2, Hung N Nguyen3, Nhu D Dang3
1

Kanazawa University, Kanazawa, Japan, 2Kanazawa Medical University, Japan, 3Ha Noi Medical University, Vietnam

Keywords:
TCDD, dioxin congener, hot spot, Vietnam
Novel aspects:
Investigating dioxin and furan congener profile in 3 dioxin - contaminated areas in VIetnam, which are known as hot spots, wefound TCDD contribution were different in these hot spots.
Abstract:
Using GC-MS to quantify 7 congener of polychlorinateddibenzo-p-dioxins（PCDDs）and 10 congener of polychlorinated dibenzo-pfurans（PCDFs）in breast milk of Vietnamese primiparous mothers who are living in dioxin-hot spots in Vietnam. These areas areformer United State Airbases in Southern Vietnam, where herbicide was stored during the Vietnam War. These airbases are
knowns as hot spots because of extremely higher level of dioxin in comparison with sprayed and non-sprayed areas. There major
hot spots are Bien Hoa, Da Nang and Phu Cat. 52 breast milk samples in Bien Hoa（BH）, 43 in Da Nang（DN）, and 23 in Phu Cat（PC）, and 19 in Kim Bang（KB）- a non-exposed area were collected between 2008 and 2010. Mean total toxicity equivalence（TEQ-WHO 2005）of PCDDs/PCDFs in BH, DN, PC were 10.9, 16.0, 14.8 pg-TEQ/g lipid, respectively while it was 4.51 pg-TEQ/g lipid
in KB. Mean of TCDD concentration in BH, DN, PC and KB were 3.60, 2.47,1.79 and 0.64 pg/g lipid, respectively. DN and PC havesame congener dioxin and furan profile but different from BH. In DN and PC the relative abundance of
1,2,3,7,8-pentachlorodibenzo-p-dioxin（PeCDD）is about 2 - 3 times higher than 2,3,7,8-tetrachlorodibenzo-p-dioxin（TCDD）；
however relative abundance of TCDD is higher than that of PeCDD in BH. When TCDD concentration were categorized,
percentage of samples have TCDD in the highest category（>10 pg/g lipid）or lowest category（< 1 pg/g lipid）in BH are higher
than PC and DN, which mean that there is a large variation in TCDD level from BH, especially a small number of mothers in BH are
highly exposed. Mean percentage of TCDD contribution to TEQ in BH, DN, PC and KB were 24.6 % , 15.1 % ,12.2 % and 13.3 % ,respectively. Various herbicides were used during the Vietnam War including Agent Orange, Agent Purple, Agent White, Agent
Blue, Agent Pink.These herbicides were contaminated with different concentration of TCDD, which may explain the different in
TCDD levels from those hot spots.

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 35: Environment I

PTh-056

13:30 － 14:40

Profiling waters from natural sources and areas of oil sands activity using
Fourier transform ion cyclotron resonance mass spectrometry

Mark P Barrow1, John V Headley2, Kerry M Peru2, Brian Fahlman2, Richard Frank3, L Mark Hewitt3

University of Warwick, Coventry, United Kingdom, 2Environment Canada, Saskatoon, Canada, 3Environment Canada,
Burlington, Canada

1

Keywords:
Fourier transform ion cyclotron resonance mass spectrometry, atmospheric pressure photoionization, environment, oil sands
Novel aspects:
Usage of APPI and FTICR mass spectrometry for the characterization and comparison of organic components, including
compounds of low polarity, found in waters from different sources in the Athabasca region
Abstract:
Due to pressures on a finite supply of petroleum as consumption continues to grow, it is necessary to turn to less conventionalsources, such as oil sands. The Athabasca oil sands are located in Alberta, Canada, and consist of clay, sand, water, and bitumen.
An alkaline hot water extraction process can be used to separate the bitumen, which can then be upgraded to synthetic oil.
Approximately three barrels of water are consumed during the production of one barrel of oil, resulting in intensive water usage
by the oil sands industry. This oil sands process water（OSPW）must be stored in vast tailings ponds, as there is a zero discharge
policy, and there is a need to monitor potential effects upon local water quality. It is important to be able to differentiate between
those organic components found in the aquatic environment due to natural processes, such as expected seepage of oil sands
material, and those which arise due to human activity.
Samples were acquired from a range of natural water and oil sands process water sites in the Athabasca Basin and analysis was
performed using a 12 T solariX Fourier transform ion cyclotron resonance（FTICR）mass spectrometer（Bruker Daltonik GmbH,
Bremen, Germany）. Previous research into the characterization of OSPW has typically relied upon the usage of electrosprayionization（ESI）, usually due to the targetting of naphthenic acids within the complex mixtures. Whilst ESI is best suited to thestudy of polar and ionic species, atmospheric pressure photoionization（APPI）can be applied to the study of less polar species
and can produce radical ions in addition to protonated/deprotonated ions. ESI and APPI represent complementary methods,
where APPI is an amenable ionization method for the study of a broad range of compounds, such as less polar, sulfur-containing
compounds and hydrocarbons which do not incorporate heteroatoms. Mass spectra of complex mixtures that have been acquired
using APPI are typically more complex, due to the greater number of components observed and the fact that radical ions areobserved in addition. High field FTICR mass spectrometry offers ultra-high resolving power and mass accuracy, which afford highconfidence in assignments of species within complex mixtures, which is particularly important for mass spectra generated using
APPI.
Following assignments of elemental compositions, it is possible to visualize the data using different methods of categorization,such as heteroatom content, carbon number,“hydrogen deficiency“（Z）, or double bond equivalents（DBE）. Principal
component analysis（PCA）has been used to highlight similarities and differences between water sources. Differences were foundbetween river waters, ground waters, and OSPW, indicating potential for determining anthropogenic influences on the aquatic
environment.

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 35: Environment I

PTh-057

11:10 － 12:20

Analysis of biomarkers of the pesticide imazalil using LC/MS/MS.

Moosa Faniband, Bo A Jönsson, Christian H Lindh

Occupational and Environmental Medicine, Lund University, Sweden

Keywords:
Biomarkers, Pesticides, LC/MS/MS, Quatification, Urine
Novel aspects:
This study presents a new quantitative method for quantification of the pesticide imazalil and its major metabolite DCPI in humanurine using LC/MS/MS.
Abstract:
Introduction - Imazalil is a widely used post-harvest fungicide applied on a variety of crops, especially on fruits. Imazalil is also
used in veterinary medicine as an antimycotic drug.
Imazalil is classified as“Likely to be carcinogenic inhumans,“ according to EPA. Exposure to imidazole derivatives is also
suspected to produce craniofacial malformations in vertebrate development. Studies on rats have shown that imazalil ismetabolized into 25 different metabolites with 1 -（2,4-dichlorophenyl）- 2 -（ 1 H-imidazole- 1 -yl）- 1 -ethanol（DCPI）as the major
metabolite. There are very few studies in humans, however a case study of a patient treated with imazalil indicated a half-time of
2 h in serum. Various methods have been published for determination of imazalil residues in agricultural and food products.
However, no methods have been described for determination of imazalil or its metabolites in human biological samples. Humanoccupational exposure can be substantial especially in developing countries were the use is high. It is especially common thatwomen in fertile age are exposed during post-harvest treatment. Thus, methods for measurement of biomarkers of exposure isvaluable. The aim of this study was to develop a simple liquid chromatography-tandem mass
spectrometry（LC/MS/MS）method for the analysis of imazalil and DCPI in human urine.
Method Several SPE columns were evaluated and optimized for sample pretreatment. The eluates were analyzed using
electrospray ionization and selected reaction monitoring（SRM）in positive ion mode after separation on a C18 LC column. The
mobile phase used was water and methanol with0.1% formic acid. D5-labeled imazalil was used as an internal standard.
Results Two SPE columns could be used for samples pretreatment, C 2 and Oasis HLB. The sample was retained at neutral pH andeluted using 1 % formic acid in methanol. For quantification of imazalil the transition atm/z 297.1-159.0 and for DCPI the transition
m/z 257.0-69.2 was used. The method have an LOD of 1 ng/ml for both imazalil and DCPI. Data on levels found in a population will
be presented.

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 35: Environment I

PTh-058

13:30 － 14:40

Mass spectrometric investigation of mechanisms for methane formationfrom epicuticular wax under aerobiosis and UV

helge Egsgaard1, Dan Bruhn2,3, Teis MIkkelsen2, Morten M Rolsted3, Per Ambus2

1
Department of Chemical and Biochemical Engineering,, 2Department of Chem. and Biochem. Engineering, Technical
University of Denmark, Denmark, 3Department of Biology,University of Copenhagen, Denmark

Keywords:
GCMS, photolysis, methane , greenhouse gases
Novel aspects:
The combination of GCMS analyses snd photolytical studies has resulted in detailed understannding of the underlying mechanisms
for aerobic formation of methane from plants.
Abstract:
Introduction
Methane（CH4）is the second most important long lived greenhouse gas. The total global CH4 source strength is well described,
whereas substantial uncertainties exist about the strength of individual source components. When it was discovered thatterrestrial plants emit CH4 under aerobic conditions it presented quite a conundrum as CH4 is a highly reduced molecule. The
ubiquitous plant structural component pectin is a highly activated methyl donor, and in purified form it emits CH4 in response to
UV-radiation. Therefore, pectin is regarded as one of the most likely precursors in plants to aerobically emitted CH4 . In vivo,
however, pectin is situated under the cuticle and in between primary cell walls and is very well protected against UV-radiation
due to absorption and reflection by the outer epicuticular wax layers. This raises the question to what extent UV-induced CH 4
production may occur at the plant surface wax layer. In this presentation we report the mass spectrometric investigations of thephotolytic pathways.
Materials and methods
Seeds of Brassica oleracea capitata f. alba, donated by Nordic Gene Bank, were germinated in potted soil and grown for sevenweeks in a climate controlled chamber. The B. oleracea surface wax was gently removed and exposed to 17 W m-2 of UV-B（309314 nm）for 331 h using Philips PL-S 9 W/01 2 P 1 CT tubes while incubated in gas tight, UV transparent glass vials.
Analysis
Head space aliquots of 500 µl were sampled and identification of the gaseous products（C1-C3）was performed with a Varian 3400
gas chromatograph interfaced to a Saturn II ion trap mass spectrometer（GCMS）. The compounds were separated using a
PORAPLOT U fused silica column
The leaf surface wax was analyzed by GCMS using a Hewlett Packard HP 6890 gas chromatograph interfaced to a HP5973 mass
selective detector. The products were separated using WCOT fused silica column coated with VF-23 and identified using NIST
search engine, version of 2.0 f.

UU/VIS spectra were obtained using a HPLC system and a UV6000LP PDA-detector
Results and discussion
A trial resulted in specific CH4 emission rate of 153 ± 14 nmol CH4 g-1 wax h-1 in air when exposed to UV-radiation. The GCMS
analysis of the leaf wax revealed（C 29H60）and 15-nonacosanone（C 29H58O）as the predominant compounds accompanied by linear
C13-C15 aldehydes, 2 -pentadecanone and 2 -hexadecanone in low concentrations. The surface lipids have only a weak UV
absorption tailing into the 300-330 nm region. However, the photolytic products of isolated surface wax as well as the secondary
products were identified by GCMS analysis.
In sum, methane formation from leaf surface wax is a two step process initiated by a photolytic rearrangement reaction of the
major wax component, i.e. 15-nonacosanone followed by an α-cleavage of the generated ketone. In the latter reaction thepresence of concurrent path ways become significant for the yield of methane as only the Norrish I route leads to methane. The
ratio of the Norrish I and II routes can be estimated using the two products 1 -tridecene（Norrish II）and tetradecane（Norrish I）.
This ratio C14H30/C13H26 has been estimated to 0,021, and hence the methane route is a minor, but nevertheless significant process.
Mass spectrometry was found to be an excellent analytical tool for rationalization of the photolytic pathways

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 35: Environment I

PTh-059

11:10 － 12:20

Application of HPLC/MS in determination of heroin metabolites in saliva
an urine samples

Marijana Curcic2, Vesna Milovanovic3, Biljana Ciric4, Marina Pijanovic5, Vesna Kilibarda4,
Slavica Vucinic4, Nobutake Sato1, Takeshi Nakano6, Biljana Antonijevi2
1
4

Nihon Waters, Tokyo, Japan, 2University of Belgrade, Belgrade, Serbia, 3Serbian Chemicals Agency, Belgrade, Serbia,
Poison Control Center, Belgrade, Serbia, 5Medilab Polyclinic, Cacak, Serbia, 6Osaka University, Osaka, Japan

Keywords:
saliva； urine； heroin； metabolite； glucuronide
Novel aspects:
Developing accurate, simple, cheap and suitable method in determination of heroin metabolites in saliva an urine with HPLC/MS
Abstract:
Introduction
Urine is a common, and saliva represents an alternative specimen for substances of abuse determination in toxicology. In thisstudy, one step was to optimize a method for saliva and urine specimen preparation for heroin metabolites, codeine,
6 -monoacetylmorphine（ 6 -mam）and morphine determination by high performance liquid chromatography-mass spectrometry（HPLC/MS）, and then this method was applied on saliva and urine samples taken from the patients. As a preliminary analysis,
test strips for opiates identification in patients` urine were used. Saliva and urine samples from patients whose preliminary test
was“positive“ were taken for further analysis.
Methods
Sample preparation
Both, saliva and urine specimens was prepared using liquid/liquid extraction of codeine, 6 -mam and morphineby mixture ofchloroform and isopropanol（ 9 ： 1 ； v/v）.
HPLCMS analysis
Extracts were analyzed by HPLC-ESI-MS technique： at Waters Alliance ® system, the separation column Waters Spherisorb® 5 μm, ODS2, 4.6 × 100 mm was used； mobile phase： ammonium acetate ： acetonitrile（80：20； v/v）, mobile phase flow rate 0.3 mL/
min, autosampler temperature 20°
C； injection volume 50 μL； Mass detection range： 100400 m/z, centroid mode, interscan delay 0.1
s, scantime0.5 s, splitless, four voltage values： 70, 60, 50 i 38 V, ES+, source temperature 150°C , desolvation temperature 430°C, gas
flow for desolvation 362 L/h and at cone 135 L/h, capillary voltage 3 kV - mass spectrometer Waters Micromass® ZQ™（Waters
Corporation, Milford, MA, USA）.
Calibration and optimization were done using morphine standard（ion 286）10 mg/L at flow rate 10 mL/min. Regression andcorrelation analyses were performed with the probability level of 0.05. Mass spectra were analyzed by software Waters
MassLynx™（Waters Corporation, Milford, MA, USA）.
Results and Discussion
Calibrations for each analyzed substance in both specimens were done in the concentration range from 0.1 to 1 mg/L and thecoefficients of correlation were above 0.99. Recoveries for morphine and codeine determination in saliva was 99 % , while for 6 mam it was 94 % and recoveries for morphine, codeine and 6 -mam for determination in urine were 103 % , 101 % and 93 % ,respectively. Limits of detection and quantification of a proposed method were 0.01 mg/L and 0.05 mg/L, respectively for both
sample types.
Applying the proposed method on real samples of patients（n=10）where positive reaction was obtained on test strips wedetermined concentrations of heroin metabolites in selected samples in following ranges： Concentration of codeine in the salivaof the heroin abusers ranged from 0.05 to 5.33, for morphine between 0.05 and 5.33 and for 6 -mam between 0.01 and 0.68 mg/L.
Concentration of codeine in the urine samples of the same patients ranged from 0.22to 5.74, for morphine between 0.15 and 6.32
and for 6 -mam between 0.05 and1.78 mg/L.
A proposed HPLC/MS method for codeine, 6 -mam and morphine determination in saliva and urine samples is accurate, simple,
cheap and suitable for routine analysis and monitoring of heroin abuse.

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 35: Environment I

PTh-060

13:30 － 14:40

Analysis method of polybrominateddiphenylether using GC-MS and GCMS/MS coupled with automated identification and quantification systemwith a database

Katsuhiro Nakagawa, Tomoaki Kondo, Kouki Tanaka, Yuki Sakamoto, Shuichi Kawana,Haruhiko Miyagawa
Shimadzu Co., Kyoto, Japan

Keywords:
GC/MS, GC-MS/MS, polybrominateddiphenylether
Novel aspects:
For analysis of BDEs using GC/MS or GC-MS/MS, the novel method using AIQS-DB and the conventional（isotope dilution method）
was developed for easy operation and reduction of standards.
Abstract:
There are 209 isomers of polybrominated diphenyl ether（BDEs）with each one showing different level of toxicity and detection
frequency in environmental samples. GC-MS is commonly used for the analysis of BDEs.
A common method of BDEs analysis requires the expensive standards and instrument of gas chromatograph/ double focusing
high resolution magnetic sector mass spectrometer（GC-HRMS）. Moreover, the handling and maintenance of GC-HRMS
instruments is a time-consuming and labor-intensive work. This situation is critical problem especially for environmentallaboratories in under developing countries.
Kadokami et al.［ 1 ］developed a novel automated identification and semi-quantification system with a database（AIQS-DB）
which allows an automatic identification and semi-quantitation of 1,000 pollutants without standard sample analysis. The databaseincludes retention indices, mass spectra, and internal calibration curves for 1,000 pollutants. The pollutants are identified using themass spectrum and retention time predicted by retention index and retention times of n-alkanes. Semi-quantitation is performed
using internal calibration curve.
We developed a method for BDEs analysis. For reduction of the necessary authentic standard, AIQS-DB was applied to BDEs
with less toxicity and detection frequency, while the conventional method（isotope dilution method）was applied to BDEs with ahigher toxicity and detection frequency in order to obtain precise quantitation results. For easy handling and maintenance, single
quadrupole mass spectrometer of GC-MS was used.
The developed method was applied to sediment samples. BDEs were detected and semi-quantitation results were obtained.
However, several samples with heavy matrix could not detected BDEs due to peak overlapping. GC-MS was not applied to heavy
matrix sample analysis such as sediment sample. Gas chromatography triple quadrupole mass spectrometry（GC-MS/MS）was
applied to improve the separation of BDEs from heavy matrix, ensuring easy handling and maintenance. Using GC-MS/MS, BDEs
were successfully identified and determined, corresponding to the results by the conventional method by HR-GCMS.
1. Novel gas chromatography-mass spectrometry database for automatic identification and quantification of micropollutants,
Kiwao Kadokami, Kyoko Tanada, Katsuyuki Taneda, Katsyhiro Nakagawa, J. Chromatogr A, 1089 pp 219-226, 2005.

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 35: Environment I

PTh-061

11:10 － 12:20

LC-HRMS investigation of the human in vitro metabolism of brominatedflame retardants using APPI and ESI.

CHARLOTTE MARTEAU1, SYLVIE CHEVOLLEAU1, ISABELLE JOUANIN1, ELISABETH PERDU1,GEORGES DE SOUSA2, ROGER RAHMANI2, DANIEL ZALKO1, LAURENT DEBRAUWER1
1

INRA, UMR 1331, Toxalim/AXIOM/Metatoul, Toulouse, FRANCE, 2INRA, UMR 1331, Toxalim, Sophia Antipolis, FRANCE

Keywords:
Brominated flame retardants, APPI, High resolution mass spectrometry, metabolism
Novel aspects:
Complementary use of ESI and APPI for complete characterisation of BFRs formed in vitro. HRMS provides discrimination between
isobaric hydroxy-polybrominated diphenylethers. New information about metabolic fate of BFRs in human.
Abstract:
Brominated flame retardants（BFRs）are widely used in the production of polymers and plastics in order to prevent fires inelectronic and domestic goods. These compounds, mainly represented by tetrabromobisphenol-A （TBBPA） andpolybromodiphenyl ethers（PBDEs）, are suspected to act as endocrine disruptors［1,2］and their toxicity is of growing concern.
However, although the occurrence of BFRs has been shown in almost all environmental compartments, limited data is available
about their fate, in particular in humans.
GC-MS is widely used nowadays for the analysis of PBDEs, though not perfectly suited for the highest MW PBDEs（octa- to decaBDEs）, mainly due to thermal degradation problems. For TBBPA, a derivatisation step is required. In this work, LC-MS methods
based on ESI and APPI associated with high resolution ion analysis, were developed for the analysis of BFRs metabolites, which
we expected to be moderately polar to polar molecules. These methods were applied to the study of in vitro metabolism of BFRs
after incubation with human primary cultures of hepatocytes. HRMS full scan analysis was found to be useful not only for theidentification of known metabolites, but also for the detection of non targeted metabolites, which were identified by MS/MS
experiments.
First, the efficiency of ESI, APCI and APPI was compared for the analysis of TBBPA, PBDEs and their metabolites formed after
incubation with hepatocytes. ESI was found to be well suited for the analysis of TBBPA and their metabolites, as well as forhydroxylated metabolites of PBDEs. We have previously shown that LC-MS using Atmospheric Pressure Photo Ionization（APPI）
could constitute a possible alternative for the analysis of PBDEs［ 3 ］. In the continuation of our work, the use of APPI wasassessed for the analysis of PBDE metabolites with particular emphasis on hydroxylated PBDEs.
After the optimization of APPI ionization parameters（nature and% of dopant, heated nebulizer and transfer capillarytemperatures, LC mobile phase composition and flow rate）, a high resolution acquisition was set up on a LTQ-Orbitrap
instrument operating in the full scan mode at a resolution of 60,000, which was necessary to get a total resolution of e.g. the m/z
498,7 ions formed from both OH-tetra-BDE［M-H］- ions and OH-penta-BDE［M-H-HBr］- ions.
The developed methods were then applied to the study of TBBPA and BDE-47 metabolism after in vitro incubations with humanhepatocytes. Absolute quantification of metabolites was achieved by using radiolabelled molecules. LC-ESI-HRMS allowed theidentification of conjugated forms of TBBPA, namely glucuronide, sulphate and doubly conjugated metabolites. For BDE-47, LCAPPI-HRMS allowed not only the identification of expected metabolites such as hydroxylated PBDEs, but also the characterizationof other metabolites, such as a tetra-BDE dihydrodiol and conjugated metabolites. For the latter metabolites, identification wascompleted by the use of ESI, allowing the access to the m/z of quasi-molecular ions.
Results obtained in vitro using human primary hepatocyte cultures as well as other human cell lines demonstrate that human cells
can biotransform TBBPA into conjugated metabolites and BDE-47 into hydroxylated, dihydrodiol and conjugated metabolites. The
potential of these metabolites to be used as biomarkers of exposure in human samples will further be evaluated within the frameof ongoing impregnation studies.
References［ 1 ］Law RJ, Herzke D, Harrad S, Morris S, Bersuder P, Allchin CR（2008）Chemosphere 73：223-241［ 2 ］Antignac JP, Cariou R, Zalko D, Berrebi A, Cravedi JP, Maume D, Marchand P, Monteau F, Riu A, Andre F, Le Bizec B（2009）
Environ. Pollut. 157：164-173［ 3 ］Debrauwer L, Riu A, Jouahri M, Rathahao E, Jouanin I, Antignac JP, Cariou R, Le Bizec B, Zalko D（2005）J. Chromatogr. A
1082：98-109

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 35: Environment I

PTh-062

13:30 － 14:40

Information of the behavior of the persistent organic pollutants in the sea
around Japan

Masahiro Tsurukawa1, Motoharu Suzuki2, Yuki Haga1, Chisato Matsumura1, Takeshi Nakano3,
Masayuki Kunugi4

HYOGO Prefectural Institute of Environmental Sciences, Kobe, Japan, 2Kobe University, Kobe, Japan, 3Osaka University,
Osaka, Japan, 4Tokyo University of Science, Chiba, Japan

1

Keywords:
POPs, HCHs, enantioselective analysis, Japan Sea
Novel aspects:
Pollution levels of POPs in sea water and air over the ocean were investigated. Also isomer and enantiomer analysis wereconducted to estimate the source and pollutant pathway of POPs.
Abstract:
Although a large amount of Persistent Organic Pollutants（POPs）have been used in Asian countries such as China or Korea, thestate of contamination has not been clarified in Japan Sea surrounded by these countries. In this study, pollution levels of POPs in
sea water and air over the ocean were investigated by using voluntary sampling of passenger ships. Also isomer and enantiomer
analysis were conducted to estimate the source and pollutant pathway of POPs.
The sea water samples（approx. 50L）and air samples（approx.108m3）were taken by the passenger ship（NYK cruses CO.,LTD ,ASKA- 2 ）equipped with concentrating device between 2009 and 2011.Also in downstream site of Chang Jiang, water samples（approx.6L）were taken in 2010 and 2011.
Identification and quantification of POPs were performed using a gas chromatograph,（HP6890N Agilent）/high-resolution mass
spectrometer（800D,JEOL Ltd.）equipped with HT- 8 PCB capillary column（60m*0.25mm id. Kanto Chemical）and BGB172 capillary column（30m*0.25mm id, BGB Analytik AG）for enantioselective analysis
In this result, the highest concentration of Hexachlorocyclohexanes（HCHs）was in the sea around the northern Hokkaido, 880pg/L.
DDTs was higher in Tsushima straits than in the north of Japan Sea. On the other hand, it is several times higher concentration in
Chang Jiang than in around the Sea of Japan, 1300&#65374；2600pg/L（HCH）.
From the view point of isomer pattern, the ratios（ α/γ ）of Sea waters were apparently different according to the areas. In
general, lowerα/γratios were observed in low-latitude region. Especially in downstream site of Chang Jiang, it was the lowest
value,1.4±0.1, and 1.9±0.6 in the seas around Korea,
4.2±0.6 in southwestern Sakhalin. These results seem to reflect the usage of γ-HCH（Lindane）in China and indicate that HCHs
was discharged to the marginal seas of the region through rivers in considerable amounts and transported to Japan Sea via
oceans.
Dechlorane Plus（DP）is a chlorinated FR additive introduced as a replacement for Dechlorane, or Mirex.
In the past, there are few data of DP in the environment. In this study the concentration of DP in the sea water was investigated
for the first time. The range of DP is 0.7&#65374；14pg/L in the sea around Japan and 5 &#65374；16pg/L in Chang Jiang.
As for chiral analyis, it is regarded that enantioenrichment indicates that it was released some time ago and has since been
subjected to recycling from water or soil. EF value of α-HCH was close to racemic in southwest Sakhalin（0.5±0.02）,downstreamsite of Chang Jiang（0.51±0.02）. In Chang Jiang and the northern Hokkaido, enantiomer fractions（EFs）of alpha-HCH was nearly0.5（racemic）. It suggests that those areas were affected by relatively-recent pollutant source.
In Japan Sea, as latitude become higher, EF values tend to become lower. Since 2005’s survey , EF values was 0.46±0.05 in middle
Japan Sea, 0.44±0.03 in northern Japan Sea（ around Hokkaido）,0.46±0.01（pacific sideboard of Tohoku area）. These results
indicate that southwest Sakhalin, downstream site of Chang Jiang and Tsushima Straits area polluted by relatively new HCHs.
From comprehensive viewpoint, there is a possibility of contamination in Japan Sea, which is caused by the transportation fromother countries and polluted area by HCH was found in around Hokkaido.

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 35: Environment I

PTh-063

11:10 － 12:20

LC/ESI(-)-MS/MS analyses of the biotransformation products of dibenzo-pdioxin by Sphingobium sp. strain KK2Allyn H Maeda, Emi Aoki, Robert A Kanaly
Yokohama City University, Yokohama, Japan

Keywords:
LC/ESI（-）-MS/MS, dibenzo-p-dioxin, bacteria
Novel aspects:
LC/ESI（-）-MS/MS was utilized to determine biotransformation products of dibenzo-p-dioxin by a bacterium.
Abstract:
Biaryl ether environmental pollutants such as dibenzo-p-dioxin, dibenzofuran and their halogenated congeners are considered to
be environmentally-persistent pollutants that originate from incineration processes of domestic and industrial waste. There ismuch interest to know the ultimate environmental fates of these compounds and this includes the contributions of soil
microorganisms to their biotransformation. A soil bacterium, Sphingobium sp. strain KK22 was recently isolated from ahydrocarbon-degrading microbial consortium and was found to biotransform dibenzo-p-dioxin following induction on the 3 -ring
polycyclic aromatic hydrocarbon phenanthrene. Strain KK22 was exposed to 50 mg/L dibenzo-p-dioxin in 50-ml size microcosms
and was sampled multiple times over 48 hours. Liquid chromatography electrospray ionization tandem mass spectrometry coupledwith UV detection in negative ionization mode（UV-LC/ESI（-）-MS/MS）was conducted by full scan analyses on acidified
microcosm organic extracts and revealed multiple putative ions of interest that were not detected in biotic and abiotic controls.
Further investigation by CID product ion scanning showed that least two biotransformation products corresponding to thedeprotonated molecular ions［M - H］- = 251 and［M - H］- = 267 were present. Fragmentation analyses revealed that thesecompounds appeared to be ring-opened structures derived from initial angular dioxygenation of the dibenzo-p-dioxin molecule bystrain KK22 and they were tentatively identified as 6 -（ 2 -hydroxyphenoxy）- 6 -oxo-hex- 4 -enoic acid and a singly-hydroxylated
derivative respectively. If confirmed, these chemicals shall represent previously unreported metabolites from dibenzo-p-dioxin by abacterium. Further investigation is continuing to determine the nature of dibenzo-p-dioxin transformation by strain KK22 throughthe utilization of LC/ESI（-）-MS/MS.

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 35: Environment I

PTh-064

13:30 － 14:40

Analysis of Sea-Dumped Chemical Warfare Agents from Sediment
Samples Taken at in Baltic Sea

Ullastiina Hakala, Martin Söderström, Olli Kostiainen, Jenni Taipalus, Paula Vanninen
VERIFIN/Department of Chemistry/University of Helsinki, Finland

Keywords:
chemical warfare agents, Baltic sea, environmental analysis, GC-EI/MS, LC-APCI/MS/MS
Novel aspects:
Novel information of CWA contamination of the Baltic sea.
Abstract:
After the Second World War, a large amount of chemical warfare agents（CWAs）were dumped at the Baltic Sea. For example, inthe Bornholm dumpsite located within Danish economic zone, it is estimated that over 500,000 shells and containers containing
over 11,000 tons of chemical warfare agents were dumped in 1947 by the Soviet Military Authority in Germany.
In this study, sediment samples were collected during different sampling cruises conducted in the Bornholm dumpsite or around it
between 2006 and 2011. The target chemicals were mustard, tabun, Clark I & II, Adamsite, Lewisite I & II andα-chloroacetophenone as well as arsenic oil containing Clark I, triphenylarsine, phenyldichloroarsine and trichloroarsine, and relateddegradation products. After an appropriate sample preparation procedure, the samples were analysed using GC-EI/MS withselected ion monitoring（SIM）as such and after derivatisation with propanethiol, and LC-APCI/MS/MS with selected reaction
monitoring（SRM）as such and after oxidation using hydrogen peroxide.
Analysed sediment samples showed considerable spreading of arsenic-containing chemicals within the dumpsite as well as
between the dumpsite and the Bornholm Island. The highest found sediment concentrations have been over 16,000 ng/g ofdegradation products of Clark I and 39,000 ng/g of triphenylarsine within the primary dumpsite.［ 1 ］
Baltic Sea contains also unexplored areas, e.g. Gotland and Gdansk deep, that have been claimed as dumping sites of CWAs. The
Chemical Munitions Search and Assessment（CHEMSEA）project has been established to carry out investigation at the official andunofficial dumping areas in the Gotland and Gdansk deep. The main focus of the project is to locate the dumped CWAs andsample the surrounding environment to assess the possible threat. The outcomes of the project will also include
recommendations on operating procedures, and guidelines to employ in those areas, to be used by maritime administrations ofrespective countries, as well as a contingency plan for risk management. The CHEMSEA project is a transnational collaboration
including project partners from five EU countries and a number of associated organizations, including governmental agencies andinternational organizations. This project is part of the Baltic Sea Region Program and is partly financed by the European Union. The
project was started in the fall of 2011 and will continue until early 2014.［ 2 ］

References［ 1 ］T. Missiaen, M. Söderström, I. Popescu and P. Vanninen, Sci. Total Environ., 408（2010）3536-3553.
［ 2 ］J. Jeldowski and T. P. Long, Mar. Technol. Soc. J., 46（2012）28-36.

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 35: Environment I

PTh-065

11:10 － 12:20

Online SPE LC-MSMS for screening and quantifying anti-cancer drugs andmetabolites in hospital’s wastewaters and rivers

Mikael Levi2, Claude Villard3, Therese Schembri3, Anja Gruening1, Stephane Moreau2, Pierre Boissery4,
Daniel Lafitte2

Shimadzu Europa GmbH, Duisburg, Germany, 2Shimadzu France SAS, Marne la Vallee, France, 3Aix Marseille Universite,
Marseille , France, 4Agence de l’Eau Rhone Mediterranee & Corse, Marseille, France

1

Keywords:
cancer drugs, waste water, LC-MSMS
Novel aspects:
The main difficulty is the wide panel of chemical structures to be separated using a single rapid automated method
Abstract:
Introduction :Over 50 cytotoxic chemotherapies are used in hospitals. The main anti-cancer drugs used in cancer chemotherapy can beclassified into several categories： cytotoxic, the most represented, but also hormones, immune response modifiers andantibodies. Most cytotoxic agents used in cancer chemotherapy interact with DNA or its precursors. Very few studies areevaluating the future of these drugs in wastewater. Potential risks associated with these discharges are poorly understood andrequire study work and research to better understand the hazards, exposure characterization and assessment risks to humanhealth and the environment. The purpose of the study is to establish an analytical methodology to screen most of the anti-cancer
drugs currently used in hospital waste waters.
Methods :The goal was to develop a simultaneous analysis of a large number of anticancer drugs from water samples with a limited amountof sample. These samples are processed by on-line solid phase extraction（SPE）to isolate and concentrate the different
cytotoxics. Following extraction, compounds are transferred to an UHPLC column for separation. Detection is performed using
Multiple Reaction Monitoring mode on an ultrafast triple quadrupole mass spectrometer. Special attention was given to theorthogonal selectivity and to the working pressure compatibility of the extraction and analytical columns.
Preliminary results:
The UHPLC system（NEXERA, Shimadzu Corp.）is equipped with an online SPE cartridge which, thanks to the innovative switchingvalves configuration, is isolated from the high pressure LC. This allowed us to combine on-line SPE with UHPLC. The first step of
the project was to optimize the MRM transitions for each molecule of the chosen list and to choose the best mobile phasecomposition compromise for optimum sensitivity. Once that process done for the 25 selected drugs, several SPE sorbent for
extraction of a wide range of different chemical class compounds were tested. The same study was conducted on the choice of
the analytical column trying to combine several retention mechanisms. The final stage of development of the method was devoted
to optimization of the sensitivity to reach the lowest limit of quantification and to study of the dynamic range for each molecule.
Finally the methodology was validated with real wastewater sample to assess extraction and ionization recovery as well as
ruggedness of the system.

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 35: Environment I

PTh-066

13:30 － 14:40

Identification of chlorinated aromatics as impurity of chlorinated paraffinsby GC-HRMS or GC-HR-Tof-MS

Takumi Takasuga1, Takeshi Nakano2, Yasuyuki Shibata3

1
Shimadzu Techno-Research Inc., Kyoto, Japan, 2Osaka University, Osaka, Japan, 3National Institute for Environmental
Studies, Tsukuba, Japan

Keywords:
POPs, Chlorinated Paraffins, Impurities, Identification, PCB
Novel aspects:
Identified high levels of POPs in technical CPs and products by GC-HRMS or GC-HR-Tof-MS
Abstract:
Chlorinated paraffins（CPs）are industrial products used as metal-working fluids and flame retardants for plastic materials. Shortchain CPs（SCCPs）seems to persist in the environment and bioaccumulate in biota, and are under review by the Stockholm
Convention on persistent organic pollutants.
We identified high levels of POPs such as PCBs, PCBz, PCNs and HCHs from air sampler materials. After detailed investigations
these POPs are concluded as impurities of technical chlorinated paraffins（CPs）, which used for rubber materials as flame
retardant at 2 - 6 % contents. The exact mass spectrum and accurate mass of these chemicals are fixed theoretical mass with few
ppm errors. The profile of PCBs congeners are close to technical PCBs, which suggest chlorination of biphenyls as impurities ofparaffins. The homologue profiles of PCBs are relatively lower chlorination pattern with some specific congeners.
These technical CPs are imported from China. The production of total CPs in China has continued to increase, reaching 600,000
tones in 2007. Although the huge production and use of CPs in China could imply potential contamination of various media, there
is little information on exposure to SCCPs. In our previous study, the analytical methods for SCCP were investigated in detail andapplied for dietary samples. Preliminary evidence on the significant increase of SCCP in food sample Beijing in 2009 warrants urgent
investigations to refine dietary intake estimates by targeting food types and source identification.
On the other hand, the finding high levels of legacy POPs as impurities in technical CPs are more serious for POPs inventories
around the world.

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 35: Environment I

PTh-068

13:30 － 14:40

Determination of absolute configuration of PCB atropisomer and analysisof enantiomeric excess in the human sample

Takeshi Nakano1, Chisato Matsumura2, Masahiro Tsurukawa2, Toshihiro Okuno2, Mitsunobu Toda1,
Tadashi Mori1, Yoshihisa Inoue1
1

Osaka University, Suita, Japan, 2Hyogo prefectural Institute of Environmental Sciences

Keywords:
PCB atropisomer, human sample, circular dichroism, RI-CC 2 /TZVPP calculations, enantiomeric excess
Novel aspects:
Absolute configuration of PCB-183（aS and aR）were determined, aS-PCB-183 and aR-PCB-183 in serum are determined enantioselectively.
Abstract:
The electronic circular dichroism（CD）spectra of a pair of enantiomeric 2,2’,3,4,4’,5’,6-heptachlorobiphenyls（PCB-183）were
investigated for the first time experimentally and theoretically. Geometrical optimization at the DFT-D 3 -B-LYP/TZVP level revealed
that the two phenyl planes of PCB-183 are nearly orthogonal（89°）. Due to the sterically large chlorine atoms, PCB-183 becomes
atropisomer. Thus, optical resolution was performed by chiral HPLC（column： OJ-H）, affording enantiomerically pure first elute（100% ee）and optically enriched second elute（74% ee）. Their experimental UV-vis and CD spectra in n-hexane were comparedwith those obtained by theoretical calculations at the RI-CC 2 /aug-TZVPP level.
Determination of absolute configuration is crucial in the structure elucidation and their biological activities of chiral compounds,
and it is also true for PCB atropisomer. However, the determination of absolute configuration of PCB has never been performed.
Here absolute configuration of PCB-183（aS and aR）were determined, aS-PCB-183 and aR-PCB-183 in human sample are determinedenantio-selectively.
Sample collection： The serum and adipose tissue samples were obtained after receipt of written informed consent. After about
10ml of blood were centrifuged by 3000 rpm, serum samples were obtained and stored in -20°C until analysis.
Extraction and cleanup： About 2 g of serum was added cleanup spike solution, 2.5ml of diethyl ether, and 5 ml of ethanol, thenextracted with 10ml of hexane twice. Hexane extracts were passed through 1 g of florisil/ 1 g of silica gel double layer column（Supelco Inc., U.S.）, eluted with 15ml of 15 % diethyl ether/hexane. Eluate was concentrated to 0.1ml under gentle stream ofnitrogen, and then added syringe spike solution1).
GC and LC conditions for chiral separation
To determine absolute configuration of each PCB atropisomers, the enantioselective separation of PCB atropisomer was
performed using HPLC（TOSOH CO-8020, SHIMADZU LC-10AT）with CHIRALCEL（DAICEL）OJ-H（4.6mmIDx150mm）. The adsorbent
is cellulose tris（ 4 -methylbenzoate）silica gel coating type, particle size 5 μm, sample loop ： 20μL, column temp ： 38°C, n-hexane
was used as elution solvent with flow rate 0.3mL/min, UV 291nm.
PCB-183 in human sample was enantio-selectively determined by GC/HRMS.（JEOL JMS-800D）using BGB-172 column（20 % tertbutyldimethylsilylated beta-cyclodextrinin methylphenylcyanopropylpolysiloxane, 30m length x 0.25mmID, 0.25μm Film Thickness,
BGB Analytik AG）. Carrier gas was helium, and injector and transfer line temperature were 230°C and 245°C. 1 μl of samples
were injected splitless at an initial temperature of 120°C, 4 °C/min to 180°C, 1 °C/min to 230°C, and held for 10minutes. The ion
source was operated in the electron-impact mode（EI, 38eV, 250°C）.
Experimental data and theoretical calculation results
The concentrations is calculated as epsilon（209）=73000. Experimental CD spectra of aR/M are roughly similar in comparison withtheoretical calculation of aS/P.（aS： axial S）. In experimental CD spectra, comparison between fraction A（100%ee）and fraction
B（74%ee）multiply 1.35 with inversion of sign were in excellent agreement. The first elution peak on BGB-172 column by GC/MS is
assigned as aS/P. The first elution peak on OJ-H column by LC/MS was also assigned as aS/P.
1 ）Chisato Matsumura et al, Organohalogen compounds, 69, 275-278, 2007

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 35: Environment I

PTh-069

11:10 － 12:20

Analysis of natural organic dyestuffs extracted from textiles.

Ivan Viden, David Kohout, Jiri Kosina, Josef Chudoba
Institute of Chemical Technology, Prague, Czech Republic

Keywords:
masspec, dyestuffs, natural, historical, textiles
Novel aspects:
During the first phase of colour fading of textiles the aglycone concentration is increasing because of the O-glycosidic bondbreakdown.The moment O-glycosidic bonds are completely hydrolysed, aglycone concentrations begin sinking.
Abstract:
Study of the fading of natural dyes in（historical）textiles is rather complicated because of low concentration of investigatedchromophores plus a big surplus of matrice elements（textile fibres and degradation products）and because of the lack ofsignificant portions of the testing material.
Most suitable method is extraction/soft hydrolysis by mixture of methanol formic acid（95 ： 5 ）followed by LC-MS analysis. Mostof the previous papers were concentrated on the analysis of chromophores themselves（aglycones）while glycosylated
chromophores were not the principal target. The main reason was that the extraction by mixture of 37% HCl/MeOH/H 2 O（ 2：1：
1 ）would hydrolyse O-glycosylated chromophores. Therefore, it is not possible to study the concentration changes ofO-glycosides along with their artificial aging.
The overall goal of this work is to devise efficient techniques for extracting dyes from textile samples without simultaneously
decomposing some of the dye components. Moreover, this type of extraction is more suitable for MS-analyzers, as HCl is acorrosion agent and, at the same time, formic acid can be readily evaporized after the extraction.
Silk textiles were dyed by mordant dyes（anthraquinones）extracted from the madder roots and exposed to two types of artificial
aging（thermal and photooxidative）, before and after the aging experiment. Analyses of the samples were carried out by
extraction, 30 minutes-sonification at 60 °C followed by evaporation of the solvents in a N2-stream. Separation of analytes was
carried out by gradient reverse phase chromatography on C18-column. Mobile phases werewater and/or acetonitrile both with 0,1 % of formic acid. ESI in negative mode and HRMS detection using ORBITRAP technology
were used for the analysis. Monitoring analytes were rubyretric acid（alizarin primeveroside）, alizarin glucoside, alizarin, lucidin
primeveroside, lucidin glucoside, lucidin, galiosin（pseudopurpurin proveroside）, rubiadin primeveroside, rubiadin, purpurin.
Conclusions
During the first phase of colour fading of the analysed textiles the concentration of the aglycone itself（not bonded to the sugar
moiety）is increasing because of the O-glycosidic bond breakdown（which is the most vulnerable to a fission）. The moment all
O-glycosidic bonds are completely hydrolysed, aglycone concentrations begin to sink. O-primeverosides first change onto
O-glykosides of aglycones. The fotooxidation is more destructive then thermal conditions.
The most stable are O-glycosides and O-primeverosides of alizarin, which at the same time are the most concentrated
chromophores in madder. Lucidin, rubiadin and pseudopurpurin are more degraded than alizarin. These facts lead to search firstin historical textile for aglycons of chromophores, these are more stable.
The single MS spectra of glycosylated aglycones are always accompanied by typical daugther ions of the aglycones［M-162,053］for O-glucosides and［M-294,095］- for O-primeverosides occuring in the ESI ionizator.

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 35: Environment I

PTh-070

13:30 － 14:40

Atmospheric pressure ionisation and ion mobility novel techniques for the
analysis of the POPs on the Stockholm convention including dioxins

Bert van Bavel1, Jody Dunstan2, Peter Hancock2, Rhys Jones2, Ingrid Ericson1, Jessika Hagberg1
1

MTM Research Center, Örebro University, Sweden, 2Waters Corporation, Manchester, Great Britian

Keywords:
APGC, ion mobility, POPs, dioxins, Stockholm Convention
Novel aspects:
First time data on APGC and dioxins and ion mobility
Abstract:
Within the UNEP program ’Assessment of Existing Capacity and Capacity Building Needs to Analyze POPs in Developing Countries’
several activities were undertaken during the period from 2006 to 2010. The program is focused on the development of analytical
capacity for the POPs under the Stockholm Convention including several pesticides（DDT, chlordane, toxaphene）and industrial（by）-products（dioxins, PCBs）. Recently brominated flame retardants（BDE）and an organic fluor compound（PFOS）wereadded to the convention. One of the conclusions of the program was that it is quite a challenge to analyze all POPs in the sample
types proposed for the global monitoring program（GMP）. This is especially through for countries in developing countries.
Traditionally low resolution GC/MS is used for the analysis of the chlorinated POPs, mostly operating in the EI mode or NCI modefor specific compounds including toxaphene and the brominated BDEs. For dioxins high resolution GC/MS systems are often
required to avoid inferences and to achieve the low LODs needed. One of the difficulties to develop a universal method for the
Stockholm convention POPs is that different ionization techniques are needed for different POPs.
Preliminary results show that APGC might be a way forward to the development of a universal detection system based on mass
spectrometry. Atmospheric pressure ionisation under charge transfer conditions often results in very soft ionisation compared to
EI or even NCI conditions. For many of the POPs on the Stockholm convention only the molecular ion is formed under these
conditions. By avoiding fragmentation the sensitivity in the MRM or SIR mode is enhanced. Especially for compounds known toshow extensive fragmentation（for example Aldrin, Dieldrin）under EI conditions, the results using APGC showed nearly exclusively
the molecular ion. Sensitivity was tested for dioxins using the APGC ion source with dry N 2 on a Waters Xevo TQ-S in the MRM
mode. The results were surprising and the sensitivity was comparable with high resolution GC/MS, the standard for dioxin
analysis. Injection of a 1 /10 dilution of calibration standard CSL（Wellington Labs, Ontario, Canada）of concentration range 10-100
fg/ul resulted in S/N from 10 to 65.
Another problem of the isomer specific dioxin analysis is the co-elution of non-toxic PCDD/DF congeners with the 17 toxic isomers
to be analysed according to the TEQ concept. Currently no commercial column available is able to separate all congeners, andconfirmation analysis has to be performed on a different GC column. Ion mobility might be able to a solution to this problem, byseparating ’critical’ PCDD or PCDF congeners in an ion mobility cell. Normally only used for larger（bio）molecules recent
developments have also shown a potential to separate smaller molecules, thus adding an additional MS separation dimension.
Preliminary results using ion mobility to separate ’critical’ dioxin and furans pairs will be presented.

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 35: Environment I

PTh-071

11:10 － 12:20

Mixed-mode Liquid Chromatography/Tandem Mass Spectrometry Analysis
of Glyphosate and Its Application in Exposure Study

Chunyan Hao1, Christopher B Edge2, David Morse1, Dean G Thompson3, Jeff E Houlahan2

Ontario Ministry of the Environment, 2Biology Department, University of New Brunswick, 3Great Lakes Forestry Center,
Canadian Forest Service

1

Keywords:
glyphosate herbicide, LC/MS-MS, mixed-mode liquid chromatography, multiple reaction monitoring（MRM）, direct aqueous
injection（DAI）
Novel aspects:
A direct aqueous injection mixed-mode liquid chromatography/tandem mass spectrometry method was developed and applied
to a large scale whole ecosystem glyphosate exposure experiment for the first time.
Abstract:
Glyphosate-based herbicides are the most commonly used herbicides in Canadian agriculture and silviculture. However, it is quite
challenging to monitor glyphosate and its related compounds in the environment due to their high polarity, high water solubility,
low volatility, and the lack of chromophore and fluorophore in the molecules. Tedious and time-consuming derivatization is often
required for the analysis. Taking advantage of the multimode（reversed-phase and weak anion-exchange combined）separation
mechanism offered by the Acclaim WAX- 1 column, a direct aqueous injection（DAI）mixed-mode liquid chromatography/tandem
mass spectrometry（LC/MS-MS）method was developed for the determination of glyphosate, aminomethylphosphonic acid
（AMPA）and glufosinate. The analysis was carried out by using an electrospray ionization（ESI）source in negative ionization andmultiple reaction monitoring（MRM）scan mode. Two MRM channels were monitored for each target compound to achieve
positive identification. Aqueous environmental samples were directly injected and analyzed in 12 minutes with no sample
concentration or derivatization steps. Isotope labelled internal standards 13 C, 15 N-glyphosate and 13 C, 15 N-AMPA were used tocompensate matrix effects during the measurement. The instrument detection limits（IDLs）for glyphosate, AMPA and glufosinate
were 1, 2 and 0.9 μg/L, respectively.
The method was applied to determine glyphosate residue concentrations for a large scale whole ecosystem experiment designed
to access potential ecotoxicological effects of glyphosate based herbicides in small wetland systems. Each naturalized small
wetland was split in half with impermeable plastic barriers so that one side of the wetland would be treated with formulated
glyphosate herbicides（VisionMax or Roundup WeatherMax）and the other side would serve as untreated control. The herbicide
formulation was applied at different rates to yield two different aqueous exposure concentrations, one was the relevant level ineither agriculture environment（210 μg a.e./L）or forestry environment（550 μg a.e./L）and the other was the predicted worst casescenario exposure level（2,880 μg a.e./L）. Herbicide treatments were applied to wetlands in replicates（N = 5 or 6 ）either alone
or in combination with nutrient additions. The concentrations of glyphosate at specific periods after treatments were monitoredby the LC/MS-MS method for both the treated side and the control side of each wetland. Selected samples were also analyzed byanother gas chromatography method using nitrogen-phosphorous detection（GC-NPD）after derivativzation of glyphosate and
AMPA for comparison purpose. The results demonstrated that the novel mixed-mode LC/MS-MS method was effective andreliable for the analysis of glyphosate based commercial herbicide formulations in various surface water samples. This method
would provide a quick, easy and accurate approach for the determination of glyphosate and its related compounds in theenvironment to satisfy the needs for emergency response, water quality monitoring and regulation enforcement.

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 35: Environment I

PTh-072

13:30 － 14:40

Desorption atmospheric pressure photoionization-mass spectrometry forthe determination of chemical composition of atmospheric aerosols

Evgeny Parshintsev1, Ingrid Lamazere1,2, Jose Ruiz-Jimenez1, Tiina Kauppila1, Kari Hartonen1,
Tapio Kotiaho1, Marja-Liisa Riekkola1
1

University of Helsinki, Helsinki, Finland, 2IUT Paul Sabatier, Castres, France

Keywords:
DAPPI-MS, atmospheric aerosols, filter samples, organic compounds, semiquantification
Novel aspects:
DAPPI-MS was applied for the first time to ambient atmospheric aerosol samples with possibility of semiquantification using
tandem mass spectrometry
Abstract:
The Earth’s climate is a highly dynamic and complex system in which aerosols have been increasingly recognized to play a key
role. Because of the complexity, quantification of the climate effects of aerosols is still uncertain and is a challenging aspect inclimate research. Qualitative and quantitative information about the compounds in aerosol particles is essential if we are tocomprehend their role and effects in the atmosphere. To date, numerous analytical techniques, both on-line and off-line, havebeen developed in order to determine size-dependent chemical composition of ambient aerosol particles 1,2. They all, however,
have different disadvantages, including complicated sample preparation, which may alter the original chemical composition, lowtime resolution or, e.g. in aerosol mass spectrometry, mixed spectrum of many compounds present in the particle which is difficult3
offers an alternative to the existing
to interpret. Desorption atmospheric pressure photoionization-mass spectrometry（DAPPI-MS）
on-line techniques. DAPPI-MS has been developed for the mass spectrometric analysis of polar and non-polar compounds directlyfrom a variety of surfaces. In DAPPI, the analytes are desorbed from the sample surface using heated solvent flow, after which thegaseous analytes are ionized via photon initiated gas-phase reactions. In this research, semi-on-line analysis, based on filter
sampling of all-size ambient aerosols, and DAPPI-MS with consecutive DAPPI-MS 2 was developed. This approach allows theverification of structures of compounds found in aerosols by tandem mass spectrometry. The DAPPI-MS system gave linear
response over the calibration range from 20 to 400 ng applied on filter for over 30 studied compounds（acids, aldehydes andamines）. This allowed semiquantitative analysis of the compounds from the filter samples. Since no sample preparation was
required and since the sampling system can be easily integrated to DAPPI-MS analysis, the method developed herein can beconsidered highly promising in the field of atmospheric research.
1
Pratt et al., Mass Spec. Rev. 2012, 31, 17-48
2
Duarte et al., TrAC 2011, 30, 1659-1671
3
Haapala et al., Anal. Chem. 2007, 79（20）, 78677872

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 36: Advances in Ion Mobility Mass SpectrometryPTh-073

11:10 － 12:20

Evaluating multiplex fragmentation and ion mobility separations toimprove the quality of rapid LCMS peptide mapping analyses forbiotherapeutic proteins.

KATSUTOSHI NAGASE1, KENJI HIROSE1, Scott Berger2, Henry Shion2, St John Skilton2, Weibin Chen2
1

Waters, Tokyo, Japan, 2Waters Corporation, Milford, MA

Keywords:
Ion mobility, peptide mapping, biotherapeutic
Novel aspects:
Ion mobility gas phase separations and multiplexed fragmentation acquisition improve peptide assignment quality for rapid LCMS
peptide mapping of biotheraputics.
Abstract:
Introduction
LCMS Peptide mapping is a fundamental technique for defining the primary structure of biotherapeutic proteins. Improvements inseparations, mass detection, and informatics have reduced acquisitions to an hour, and data processing from days to hours. Suchassays are invaluable for biotherapeutic characterization and identification of product variants, but lack the throughput required to
become effective screening tools supporting several early（clone screening, QbD）and late（Formulations, Stability）development
activities. Modern TOF analyzers have enabled the collection of rapid accurate mass peptide mapping data, but accurate mass
identifications are ambiguous for some peptide assignments, and constitute insufficient evidence for confident identification of
new variants. Here, we investigate how multiplexed fragmentation and ion mobility LCMS can provide more confident, rapid map
assignments.
Methods
Analytical-scale UPLC-QTof MS methods were developed for rapid（5, 10 min gradient）, and typical（90 min）length peptide mapsof a biotherapeutic IgG 1 monoclonal antibody. Accurate mass precursor and multiplexed fragmentation data were acquired using
LCMSE acquisition methodology. For conventional 90 min acquisitions, the precursor/ fragmentation（MS/MSE）duty cycle was 1
sec, equally divided, reduced to 0.4 sec cycle time during rapid analyses. This enabled sufficient data points across ion peaks tofacilitate precursor identification and quantification, and establish chromatographic linkage of precursors and their fragments. In
selected experiments, the integral ion mobility functionality was enabled, permitting additional ion separation prior to the collision
cell, with other parameters unchanged. Data processing was accomplished with a developmental version of BiopharmaLynx
software.
Preliminary Data
Initial results indicate that rapid monoclonal antibody peptide maps using 10 minute gradients are capable of yielding highcoverage（>90 %）peptide maps with readily interpretable MSE multiplex fragmentation patterns that can be used to validate
accurate mass assignment of peptides. Multiple cases of peptide coelution（peptides with chromatographic profiles having apex
elution within 1 / 7 th of a chromatographic peak width at half height）were observable, resulting in the generation of chimericmultiplex fragmentation spectra. This chimeracy does not preclude using such ions in a confirmatory role（e.g. a quality metric of
minimum number or percentage of accurate mass b/y ions to validate an accurate precursor mass assignment）. Parallel studies
conducted with the ion mobility functionality enabled showed the elimination of fragment ion chimeracy, as the resulting dataprocessing required not only that MSE fragment ions share chromatographic retention profiles with their precursor ions, but alsothat they exhibit common ion mobility drift time with those precursors. The 10 min gradient studies indicated that Ion mobility
improved data clarity, but did not appear to be essential for fragment ion validation of accurate mass assignments. Therefore, wehave undertaken a second round of antibody peptide map analyses using an even shorter 5 min gradient elution. Results of theshorter experiment indicate that coverage was not negatively affected by the shorter gradient, and that coelution/chimeracy
issues were more prominent. We are currently evaluating the results of the ion mobility enabled analyses at this shorter gradient
length. Further work is ongoing with both rapid peptide mapping workflows applied to additional biotherapeutics to factor in map
complexity to these studies, and assess the dynamic range and quantitative reproducibility of the delivered results.

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 36: Advances in Ion Mobility Mass SpectrometryPTh-074

13:30 － 14:40

Conformational and functional analysis for quality control of protein drugs
using ion mobility mass spectrometry

Haruhiko Kamada1,2, Masaki Inoue1, Yasuhiro Abe1, Kazuya Nagano1, Kenji Hirose3, Yasuo Tsutsumi1,2,
Shin-ichi Tsunoda1,2
1

NIBIO, Osaka, Japan, 2Osaka University, Osaka, Japan, 3Nihon Waters KK, Tokyo, Japan

Keywords:
antibody, conformation analysis
Novel aspects:
Protein structural differences in the liquid phase can translate into measurable conformational differences apparent in the gas
phase mobility of protein ions.
Abstract:
Background： A Conformational change of biopharmaceuticals affects pharmacological potency and property of final drug product.
A new category of mass spectrometer has been developed with capability to fractionate biomolecules in gas phase by size,
shape, and charge（via an ion mobility separation）prior to MS detection. Ionmobility mass spectrometry（IM-MS）, which has thecapability to fractionate molecules in a gas phase by size, shape, and charge prior to MS detection, is a useful device to predictthe conformational change of biomacromolecule. This capability can be exploited by researchers to simplify analysis of complex
samples, or profile structural hetetogeneities within a protein preparation. This study can show direct application to the analysis
of protein structural heterogeneity and will provide the strategy of quality analysis of biopharmaceutics.
Methods： A Synapt HDMS system（Waters）was operated in mobility-TOF mode for all analysis. Mass Lynx was used for
instrument control and data processing. Trastuzumab （Herceptin） and Eternercept （Enbrel） were used as model
biopharmaceuticals and prepared in phosphate buffer to the right consistency. Etanercept in phosphate buffered saline, pH 7.2,
was heated at 95 degree from 2 min and 10 min and afterward cooled to 4 degree. Trastuzumab was gradually heated from 60 to
90 degree over a period of 15 min and then cooled to 4 degree. The bioactivity of Trastuzumab was evaluated with bioassay using
BT-474 cells. BT-474 cells in exponential growth were seeded on day 1 in 96-well plates at 1 x 10 4 cells per well. They wereallowed to adhere over night and then treated with non- or heat-treated Trastuzumab at indicated doses. After 5 days of
incubation, WST- 8 proliferation assay was performed as supplier protocol.
Results： A different mobility shift was detected between the denatured antibody drug and the non-treated sample by IM-MS
analysis. IM-MS data processing involved the generation of three-dimensional m/z versus drift time versus intensity plot scalled
Driftgram. Driftgrams of each different denatured condition were different in each samples. Significant differences can be observedby comparing the ion mobility data for the 28+ charge state, which is the strongest signal in driftgram. In addition, the extent of
this shift depended on the denaturating conditions. Furthermore, the bioactivity of Trastuzumab was correlated with the degree ofchange in denaturating conditions. The control sample was shown the cytotoxicity to this cell line, but heat-denatured samples
were decreased the cytotoxicity. While we have to perform further investigation, conformational study using IM-MS might beuseful to evaluate the quality check of protein drugs.
Conclusions： Ion mobility separations provide an additional separation dimension that permits resolution of underlying protein
structural distributions within individual charges states of a protein electrospray mass spectrum. Protein structural differences in
the liquid phase can translate into measurable conformational differences apparent in the gas phase mobility of protein ions.
Scientists can use this capability to study the stability, refolding, and underlying structural heterogeneity of biotherapeutic proteins.
Such structural experiments can be accomplished by MS infusion studies requiring only minutes of data acquisition.

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 36: Advances in Ion Mobility Mass SpectrometryPTh-075

11:10 － 12:20

Effects of hydrophobic oligopeptides on the aggregation process of
Parkinson`s disease target protein alpha-Synuclein

Kathrin Lindner1, Christiaan Karreman1, Hanne Gerding1, Marcel Leist1, Michael Gross2, David Clemmer3,
Michael Przybylski1
1
3

University of Konstanz, Konstanz, Germany/Baden-Württemberg, 2Washington University St. Louis, St. Louis, USA/MO,
Indiana University, Bloomington, USA/IN

Keywords:
Parkinson`s disease, alpha-Synuclein, oligomerization,
Novel aspects:
Based on the sequence of the central amyloidogenic domain hydrophobic oligopeptides were used in order to accelerate or
prevent the aggregation process of the Parkinson`s disease key protein alpha-Synuclein.
Abstract:
Synopsis
The intrinsically disordered protein alpha-Synuclein（αSyn）is known to be implicated in both idiopathic and inherited forms of
Parkinson´s disease（PD）. The pathological hallmark of sporadic PD is the formation of intracyoplasmic fibrillar deposits in
dopaminergic neurons（Lewy bodies）in the substantia nigra. The amyloid fibrils are formed by conformational changes from α-helices to β-sheets. Accordingly, one strategy to overcome the disease would be to find a drug that could stabilize the α-helicalconformations and/or destabilize the β-sheet conformations of the amyloidogenic protein1. The 140 amino-acid αSyn consists ofthree main domains：（i）, an N-terminal region（ 1 -60）containing several imperfect KTKEGV repeats；（ii）, a hydrophobic nonamyloid component of Alzheimer`s disease（NAC）region（61-95）； and（iii）, a highly negatively charged C-terminal region（96-140）.
The hydrophobic NAC domain has a critical role for aggregation, especially the hydrophobic amino acid stretch Val71-Val82.
However, how αSyn is involved in the pathogenesis of neurodegenerative disease is not understood at present. Here we report
the influence of synthetic hydrophobic oligopeptides, so called β-sheet breaking peptides, on the aggregation process of the
Parkinson`s key protein αSyn.
Methods
Recombinant expressions of full-length αSyn and αSyn polypeptides were performed using the E. coli expression system BL 21（DE 3 ）
［pLys］strain and the T 7 RNA polymerase system. Six different hydrophobic oligopeptides were manually synthesizedaccording to Fmoc-strategy on TGR-PHB resins. HPLC purification of αSyn and αSyn peptides were performed with Thermo-3000
semi preparative system on a Vydac-C 4 column（250x4.6 mm）with a linear gradient（0.1 % trifluoroacetic acid with 0 -80 %acetonitrile）at a flow rate of 1 mL/min and the purity was verified by ESI-MS.
Purified αSyn wt, βSyn, oligopeptides or mixture of αSyn or βSyn and oligopeptides were dissolved in 20 mM Na2HPO4 pH 7.5 atconcentration of 30 µM and incubated at 37°C with agitation for several days. Electrophoretic separations were performed by TrisTricine PAGE with 12-15% on a Mini Protean- 3 cell and stained with Coomassie brilliant blue and/or silver nitrate.
Preliminary data
Application of IMS-MS to oligomerization-aggregation mixtures of αSyn in vitro recently provided the first identification of specificautoproteolytic truncation and degradation products. In particular, a highly aggregating fragment was identified by cleavage
between Val71 and Thr72 in the central aggregation domain of αSyn2.
The in vitro oligomerization of αSyn was investigated by incubation at 37°C in sodium phosphate buffer（pH 7.5）for up to 25 days.
The formation of oligomers and αSyn species with molecular weights lower than that of full-length αSyn were observed withslowly increasing abundances over two weeks. Comparative in vitro studies of the non-aggregating brain protein, beta-Synuclein（βSyn）, which lacks the central amyloidogenic domain（residues 72-83）, showed neither oligomerization-aggregation nor any
autoproteolytic cleavage within 21 days of incubation. Focused on the sequence of the central amyloidogenic domain and inconsideration of the fact that proline preclude β-sheet structure we synthesized six hydrophobic oligopeptides in order toaccelerate or prevent the aggregation process of the Parkinson`s key protein αSyn. The interactions were studied by gelelectrophoretic separation, immunoblotting, circulardichroism and IMS-MS.

1
Soto C, Kindy MS, Baumann M, Frangione B. Inhibition of Alzheimer’s amyloidosis by peptides that prevent β-sheet conformation.
Biochem. Biophys. Res. Commun. 1996；226： 672680.
2
Vlad et al. Autoproteolytic Fragments are Intermediates in the Oligomerization/
Aggregation of the Parkinson’s Disease Protein Alpha-Synuclein as
Revealed by Ion Mobility Mass Spectrometry. ChemBioChem,2011； 12： 2740-2744

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 36: Advances in Ion Mobility Mass SpectrometryPTh-076

13:30 － 14:40

Analysis of different conformers of carbonic anhydrase2 using ion mobility
coupled with electrospray ionization

KENJI HIROSE1, TAIJI KAWASE1, YOSHIAKI NABUCHI2, MITSUO TAKAYAMA2
1

Nihon Waters K.K., Osaka, Japan, 2Yokohama City University, Kanagawa, Japan

Keywords:
ion-mobility-spectrometry, conformation analysis, ESI
Novel aspects:
The presence of five different discrete conformers of CA 2 was confirmed by using ion mobility coupled with electrosprayionization.
Abstract:［Introduction］
Ion mobility spectrometry（IMS）coupled with electrospray ionization（ESI）has been recognized as a useful method for the
analysis of conformation of proteins, because ESI generates gas-phase protein ions reflecting the conformations in solution and
IMS separates gas-phase protein ions according to different collision cross sections corresponding to shape or conformations evenat the same m/z values. Here we analyzed IMS driftgrams for a distribution of charge states of the gas-phase carbonic anhydrase 2（CA 2 ） ions produced from ESI processes. The driftgram obtained suggested the presence of several different stableconformations of CA 2 ions. The conformations of CA 2 in gas-phase and liquid-phase have been discussed on the basis of the ESI
processes such as 1. charge equilibrium state of CA 2 in buffer solution, 2. charge separation in high electric field, 3. charged
droplet formation, 4. desolvation, and 5. adiabatic expansion in orifice region.
［Method］
Mass spectra were acquired on a SYNAPT HDMS quadrupole IMS orthogonal acceleration TOF mass spectrometer and MassLynx
data processor（Waters Corp., Milford, MA）. A protein solution prepared at pH3.6 was infused into the ESI source at a flow rateof 20 microL/min. An electrocapillary voltage and sampling cone voltage were 2.8 kV and 30 V, respectively. A source temperatureand desolvation temperature were 100ºC and 200ºC, respectively. For ion mobility measurement, nitrogen was used as a buffergas, and IMS cell pressure was maintained at 0.5 mbar. The IMS wave velocity was 300 m/s and the wave pulse height was 7.9 V.
CA 2 was purchased from Sigma（St. Louis, MO）.
［Results and discussion］
The positive ion ESI mass spectrum of CA 2 obtained from the solution at a relatively acidic condition of pH3.6 showed a
distribution of charge states 8 + to 32+, which has a bimodal pattern constituting of 11+ and 17+ corresponding to local maximum
peaks and a local minimum peak of 15+. The distribution pattern indicating a conformational state of CA 2 suggested thepresence of both extended and folded conformations in solution. At pH3.6, the resulting highest charge state of 32+ wasconsistence of the number of basic amino acid residues（ 9 Arg, 16Lys, 11His）, so that the conformations of CA 2 ion aredetermined by the combination of protonation on the basic residues. To obtain the information about gas-phase conformations ofCA 2 ions, the IMS experiments were performed upon all the charge state CA 2 ions from m/z 908.1（32+）to m/z 3629.4（ 8 +）.
The resulting drift time distributions from the charge states 32+ to 26+ showed singlet patterns suggesting the presence of single
conformation I of CA 2 ion. The peak corresponding to the conformation I was observed from 32+ to 17+. The drift time
distributions from 25+ to 20+ showed doublet patterns suggesting the presence of different two conformations I and II. The peakcorresponding to the conformation II was observed from 25+ to 14+. The drift time distributions for 18+ and 17+ showed quartet
patterns suggesting the presence of four different conformations I, II, III and IV. The conformations, III and IV were observed from19+ to 13+, and 18+ to 13+, respectively. The drift time distributions for 12+ and 11+ did not show any splitting patterns but
broaden peaks, while those for 10+ and 8 + showed singlet sharp peaks suggesting the presence of a folded native conformation
V of CA 2 ion.
References
1 ）Y. Nabuchi, N. Murao, Y. Asoh and M. Takayama, Anal. Chem., 79, 8342（2007）.
2 ）Y. Nabuchi, K. Hirose and M. Takayama, Anal. Chem., 82, 8890-8896（2010）.

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 36: Advances in Ion Mobility Mass SpectrometryPTh-077

11:10 － 12:20

The Accuracy and Reproducibility of Calibration in Trapped Ion Mobility
Spectrometry

Victor Fursey, Mark Ridgeway, Desmond Kaplan, Melvin A Park
Bruker Daltonics, Billerica, MA, USA

Keywords:
Trapped Ion Mobility Spectrometry, Calibration stability, Calibration accuracy, Comparison with conventional IMS
Novel aspects:
First quantification of TIMS accuracy and stability for providing data directly comparable to conventional IMS with nitrogen driftgas.
Abstract:
Introduction
Trapped Ion Mobility Spectrometry（TIMS）is a recent addition in the field of mobility selective analyzers. In TIMS, ions are radiallyconfined in a quadrupolar ion funnel by RF voltages while axially trapped by the balance of the frictional force in a gas flow andthe electric force generated by a DC electric field（Vtunnel）that varies as a function of position within the TIMS device. Reducing theDC electric field strength over time allows trapped ions to sequentially elute from low to high mobilities. By calibrating a list of
known reduced mobility（K 0 ）values vs. 1 /V tunnel , K 0 can be determined from V tunnel and compared directly with results fromconventional IMS systems.
Methods
A list of compounds varying in mass, charge, and compound class with known K0 values was compiled from available literature aswell as experimentally determined K0 values acquired using a custom built two meter conventional IMS system with nitrogen driftgas. These compounds where then analyzed using a research prototype TIMS-Q-TOF and the K0 from three different methods ofcalibration compared versus traditional IMS data. In addition the stability of the TIMS device was studied by monitoring the elution
potential of four peaks of known K0 over a 10 hour period to determine the stability of the calibration.
Results:
TIMS analysis of the 622（CAS NO.： 186817-57- 2 ）, 922（CAS NO.： 58943-98- 9 ）, 1222（CAS NO.： 16059-16- 8 ）, and 1522（CAS NO.：
3830-74- 8 ）m/z peaks from Agilent low concentration tune mix（G1969-85000）were used to investigate the stability of calibration
over a ten hour period. A variance of less than 2 % of the value of V tunnel for a given peak was observed. Data from these calibrants
were then used to test three approaches for conversion of elution potential to K 0. In the first approach a simple calibration ismade from a single run of low concentration tune mix. A linear plot is then fitted of 1 /Vtunnel vs. K0. Unknown K0 values can thenbe determined if pressure, temperature, and gas composition remain fixed. This simple approach also assumes external factors
such as post TIMS transit times are identical for all ions. While extensive study of post TIMS transit times hasn’t been completed,transit times on the order of 10% of the analysis time are typical and are subject to both the mass-to-charge ratio and K0 of ions.
To reduce the effect of post TIMS transit times a second approach in which a delay is added into the scan, and calibration carriedout at five different delay times to allow extrapolation back to zero delay. While this second approach reduces the impact of post
TIMS times, it unfortunately has the limitation of only being valid for analysis of species over the same scan time and same scan
voltage range. A third calibration is therefore proposed and being tested in which five different scan rates are used for thecalibrants and unknowns. By using this approach both the post TIMS times and the effect of variable scan speeds are taken into
account and should provide a highly accurate calibration method for TIMS analysis.

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 36: Advances in Ion Mobility Mass SpectrometryPTh-078

13:30 － 14:40

Enhancement of Nanowire Formation by Mixing Multiple Modified
Amyloid Peptides: Mechanism and Application

Hiroki Sakai1, Ken Watanabe1, Christian Bleiholder2, Thomas Wyttenbach2, Michael T Bowers2,
Kazuyasu Sakaguchi1

Department of Chemistry, Faculty of Science, Hokkaido University, Sapporo, Japan, 2Department of Chemistry andBiochemistry, University of California, Santa Barbara, California, USA

1

Keywords:
amyloid fibrils, peptide self-assembly, ion-mobility spectrometry, oligomer structure, functional nanowires
Novel aspects:
Formation of extended oligomer structure is drastically enhanced in our mixed peptide system that gives extremely long resulting
fibrils.
Abstract:
Protein misfolding and aggregation that result in the formation of amyloid fibrils are associated with many diseases, such as
Alzheimer’s disease and transmissible spongiform encephalophaties. Amyloid fibrils have rigid filamentous structures that are
several nanometers wide and a few micrometers long and are also formed from a variety of peptides in vitro. Thus, amyloidforming peptides have been focused on as potential materials to form functional nanowires used in nanotechnology.
We have previously developed a method to control fibrillation to effectively obtain functionalized nanowires using StructureControllable Amyloid Peptides（SCAPs）, wherein various three-amino-acid residue units are attached at the N-termini of amyloid
peptide. We also found that mixing multiple SCAPs with different units of Lys 3（K 3 -TTR）and Glu（E33 -TTR）residues drasticallyenhances the fibrillation and gives extremely long fibrils that are far over 40 µm long. Various functionalized nanowires have beensuccessfully prepared through the effective formation of probed fibrils formed from mixed SCAPs. In the current study, we utilized
the ion-mobility spectrometry-mass spectrometry（IMS-MS）method to characterize the mechanism of mixing SCAPs on the
fibrillation enhancement.
K 3 -TTR, E 3 -TTR and their 1 ： 1 mixture were electrosprayed from acidic aqueous solutions. Ions pass through a drift cell forcollision with helium buffer gas and their mass-selected collision cross-section are directly measured on a home-built instrument.
In the mass spectra of K3-TTR and E3-TTR there were some oligomer peaks up to trimer. On the other hand, the 1 ： 1 mixture
had multiple oligomeric species up to pentamer. The peak position of the oligomeric species of the mixture was centered
between those of K3 -TTR and E 3 -TTR in the high-resolution mass spectra. Therefore, the formation of heterooligomers is morefavorable than those of the homooligomers. Interestingly, collision cross-section analysis revealed a dramatic structural difference
between the homo- and hetero-oligomers. The oligomers of the 1 ： 1 mixture dominantly form extended fibrillar structure evenat the dimer level whilst species with this structure are absent or minor in K3-TTR and E 3 -TTR. These results show that mixing
SCAPs controls the self-assembly ability of soluble peptides to form extended structure, and thus should also influence theformation of the extremely long fibrils.
In summary, it is suggested that mixing multiple SCAPs controls the structure of resulting fibrils. The length in particular should becontrolled through altering self-assembly property of the peptide oligomers. Elucidating the relationship between the structures ofoligomers and resulting fibrils will therefore give an important insight to tailor functionalized fibrillar assembly.

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 36: Advances in Ion Mobility Mass SpectrometryPTh-079

11:10 － 12:20

Internal energy of ions in travelling wave ion mobility spectrometry:dependence on instrument parameters and ion properties

Denis Morsa, Valerie Gabelica, Edwin A De Pauw
Mass Spectrometry Laboratory, University of Liege

Keywords:
Ion temperature, T-wave, Ion mobility
Novel aspects:
This work addresses the quantification of the internal energy of ions inside TWIG mobility cells on commercial instruments andshow how this energy evolves with ion mass and physical properties.
Abstract:
Ion mobility spectrometry（IMS）separates ions according to their tridimensional shape in the gas phase. It relies on electric fields
that drag ions inside a cell filled with gas； the larger are the ions, the greater is the resistance and the longer is their travelling
time. This technique can be coupled with mass spectrometry（MS）in order to achieve a two-dimensional separation. Currently
available commercial IMS-MS instruments are based on the travelling wave technology. T-waves involve high electric fields, which
induce fast ion velocities inside the cell. Based on these properties, Shvartsburg and Smith 1 arose potential ion heating due toinelastic collision with the gas. We experimentally proved this point by probing the fragmentation of p-methoxybenzyl
pyridiniumions on the first-generation SYNAPT spectrometer（Waters, Manchester, UK）.2 We present new data acquired on the
SYNAPTG 2 HDMS, and heating value for systems of different mass, size and number of vibrational degrees of freedom.
Experiments were carried out both on a Synapt HDMS and Synapt G 2 HDMS（Waters, Manchester,UK）. The product ion spectra（ratio between parent and fragment）and ion mobility spectra of three different benzylpyridinium ions as well as small proteins
were recorded as a function of the wave height, wave speed, gas pressure, and the nature of the gas（He, N2, Ar, CO2）. The
arrival time distribution of the fragment was deconvoluted to distinguish fragments formed before, inside, or after the mobility cell.
Matching calculations based on integrated RRKM or Arhénius equations with experiments, we obtained the vibrational internal
effective temperature Teff,vib of the parent ion.
We studied the heating of the p-methoxybenzyl pyridiniumion in both the Synapt G 1 and G 2 for ideal conditions of separation.
On the Synapt G1, we found a temperature Teff,vib= 555 ± 2 K（wave height = 9 V, wave speed = 600 m/s, p（N2）in IMS = 0.55mbar）. On the Synapt G2, the parameters were different because of the mobility cell architecture（wave height = 35 V, wave
speed = 1000 m/s, p（N 2 ）in IMS = 2.3 mbar） and we found T eff,vib = 700 K. This value lies between those obtained forp-chlorobenzylpyridinium（720 K）and p-tertbutylbenzylpyridinium（630 K）. For leucine-enkephalin, we obtained Teff,vib   =420 K, whichis compatible with the estimation of Merenbloom et al.3 These results indicate a dependence of Teff,vib   on the ion size. The relevant
physical parameters could be the ion collision cross section（CCS）and/or the number of vibrational degrees of freedom（DOF）.
On both instruments, we found a correlation between the ion speed v = KE = K0N0E/N and the ion temperature： the amount oftransferred energy increases with ion speed. This translates into an influence of the wave height（WH, influencing the field E）andgas pressure（p, influencing the gas number density N）on Teff,vib. The influence of the wave speed（s）on Teff,vib behaves as if theeffective electric field Eeff undergone by ions decreases when v becomes higher than KE. Thismeans that it becomes harder forions to keep up with the field changes as they pass from one wave to the next one.
is the length of the cell, td is the drift time）
We found that Teff,vib   could be predicted from the apparent ion drift velocity vd = L/t（Ldon the SYNAPT G 2 whereas a direct correlation between the drift speed v and Teff,vib could be established on the SYNAPT G1.
1. Shvartsburg A. and al.. Analytical chemistry80,9689-99（2008）.
2. Morsa D and al... Analytical chemistry83,5775-82（2011）.
3. Merenbloom, S.I. and al.Journal of the American Society for Mass Spectrometry23, 553-62（2012）.

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 36: Advances in Ion Mobility Mass SpectrometryPTh-080

13:30 － 14:40

An ion mobility mass spectrometer with an axial sector TOF analyzer

Alexey A Sysoev, Alexander A Sysoev, Sergey S Poteshin, Denis M Chernyshev
National Research Nuclear University MEPhI, Moscow, Russia

Keywords:
ion mobility spectrometry, time-of-flight mass spectrometry, atmosphere pressure ionization
Novel aspects:
Combining high resolving power IMS and orthogonal acceleration sector TOF mass analyzer can allow compactness and betterion sampling efficiency for orthogonal accelerator comparing existing reflectron based designs.
Abstract:
Recently developed ion mobility mass spectrometer is discussed. The instrument includes atmosphere pressure ionization ion
sources, high resolution drift tube ion mobility spectrometer, differential pumping interface and axial sector TOF mass analyzer
with helical path of ions. Data collection is performed using fast ADC based integrating transient recorder that allows real time
data collection.
High resolving power IMS used here was previously described as a part of IMS/MS with triple quadrupole analyzer［ 1 ］and an
instrument with Faraday plate detector［ 2 ］. Novelty of described approach is based on combining high resolving power IMS andorthogonal acceleration sector TOF mass analyzer. Main advantages of mentioned approach are compactness and better ion
sampling efficiency for orthogonal accelerator comparing existing reflectron based designs.
The instrument was tested in positive ion mode using IMS standards（2,6-di-tert-butylpyridine, tetrapropylammonium iodide,tetrapentylammonium iodide, tetraoctylammonium bromide）and antibiotics（amoxicillin, ampicillin, lomefloxacin, ofloxacin）.
Both methanol and acetonitrile were used as solvents.
Primary tests has shown that the combination of the IMS and an orthogonal acceleration axial sector time-of-flight mass analyzer
can allow obtaining 100 mobility resolving power and 2000 mass resolving power. Mobility / mass distribution data collection time
was varied between 3 and 100 seconds. For mentioned parameters nM and sub-nM range limits of detection were shown forstudied compounds in electrosprayed liquid samples.
1. Alexey Sysoev, Alexey Adamov, Jyrki Viidanoja, Raimo A. Ketola, Risto Kostiainen, Tapio Kotiaho, Development of an ion
mobility spectrometer for use in an atmospheric pressure ionization ion mobility spectrometer / mass spectrometer instrument forfast screening analysis, Rapid Commun. Mass Spectrom. 2004, 18, 3131-3139.
2. Alexey Adamov, Timo Mauriala, Victor Teplov, Jaakko Laakia, Christian Schack Pederson, Tapio Kotiaho, Alexey A. Sysoev,
Characterization of a High Resolution Drift Tube Ion Mobility Spectrometer with a Multi-Ion Source Platform, International Journalof Mass Spectrometry, 298, 2010, 24-29.

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 36: Advances in Ion Mobility Mass SpectrometryPTh-081

11:10 － 12:20

Energy-resolved ion-mobility tandem mass spectrometry: a new tool for
probing gas-phase isomerization and fragmentation of small molecules

Yayoi Hongo1, Takemichi Nakamura1, Takae Takeuchi2
1

RIKEN, Wako, Japan, 2Nara Women’s University, Nara, Japan

Keywords:
Energy-resolved ion-mobility tandem mass spectrometry（ER-IMS/MS 2 ）； fragmentation； isomerization； small molecules；
structural characterization
Novel aspects:
Energy-resolved ion-mobility tandem mass spectrometry（ER-IMS/MS 2 ）was shown to be a useful tool for characterizing small
molecules’ ion isomerization and fragmentation in mass spectrometers.
Abstract:
We introduce energy-resolved ion-mobility tandem mass spectrometry（ER-IMS/MS2）as a means to detect structural changes insmall molecules that may occur in the gas-phase inside mass spectrometers. The ER-IMS/MS2 approach enabled us to detect
structural changes（or isomerization）of gas-phase ions prior to and/or in parallel to fragmentation that give m/z changes. A few
examples have shown that compound specific isomerization is detectable by ER-IMS/MS 2； valuable information for structuralcharacterization of small molecules can be obtained.
In the past decade, IMS of mass-resolved ions has become widely recognized and applied as a means to characterize gas-phase
3 D-structures of a wide range of large biomolecules. On the other hand, unlike many large biomolecules in the native states, ionsderived from diverse small molecules（<2,000 Da）are not necessary to have well defined stable 3 D structures in the gas-phase. Incase a certain level of conformational flexibility exists, simple correlation between peaks separated by IMS and isomeric ion
structures may not be established.
Tandem mass spectrometry（MS2）has been one of the most powerful and versatile means to probe structure of gas-phase ionsthrough fragmentation chemistry. One obvious limitation of MS2 is, however, its limited ability to detect isomerization of ions in the
fragmentation process. Traveling-wave ion mobility spectrometry（TWIMS）offers us unique opportunities to looking at not only
mobility of mass-resolved ions but also fragmentation process of the mass-resolved ions, simultaneously. We took advantage ofthis to develop ER-IMS/MS 2 strategy for characterization of small molecules. By linking structural （shape） change andfragmentation chemistry/energetics observed in ER-MS 2 experiments, both isomerization of precursor ions upon collisional
activation and generation of isomeric product ions can be investigated in conjunction with computational chemistry.
A Synapt G 2 instrument（Waters, Manchester）was used for all the experiments. Small-molecule samples were introduced into
the ESI source with infusion mode or LC/MS mode and precursor ions of interest were selected at Q1. Data was acquired with
“IMS-On“ mode（nitrogen in the IMS device）at various collision energy（CE）settings in the 1 st（pre-IMS）and/or 2 nd（postIMS）collision cell, both filled with argon.
Among the various analytes tested including bioactive natural products and their derivatives, some showed clear signature of CEdependent isomerization of precursor ions. Some showed CE-dependent generation of isomeric product ions, as well. For
example, protonated molecule of erythromycin appeared to pops off one of the sugar moiety with no noticeable structural change
in the aglycone. The following dehydration reaction appeared to give isomeric product ions. Both CE-dependent isomerization of
precursor ions and isomer-dependent fragmentation processes were observed in case deprotonated molecule of folic acid wasselected as the precursor.
As isomerization of gas-phase ions are structure specific chemistry in general, ER-IMS/MS2 strategy is a promising tool for MSbased structural characterization of small organic molecules.

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 36: Advances in Ion Mobility Mass SpectrometryPTh-082

13:30 － 14:40

Separation of Peptide Sequence Isomers by Capillary Electrophoresis
Coupled with Ion Mobility Mass Spectrometer

Yury E Glazyrin1,2, Gleb G Mironov2, Robert N Ben2, Maxim V Berezovski2
1

Krasnoyarsk State Medical University, Krasnoyarsk, Russia, 2University of Ottawa, Ottawa, Canada

Keywords:
Ion Mobility, Capillary Electrophoresis, Peptide Sequence Isomers, Separation
Novel aspects:
Peptide sequence isomers were separated by Capillary Electrophoresis coupled with Ion Mobility Mass Spectrometer
Abstract:
Sequence inversions are a major class of isomers of proteolytic peptides which are important for protein identification. Using mass
spectrometry（MS）for separation of the equal mass isomers is limited. Capillary electrophoresis（CE）was proven as high-power
technique for separation of very close molecules by their size or shape and could be effective for isomer separation from the
mixtures. Ion mobility separation（IMS）device coupled to mass spectrometer is the powerful tool for detection and separation ofisomeric peptides. But it is still a problem to separate isomers with the short reversed sections of the overall sequence.
In this study we compared two techniques for separation of three synthetic peptides with the short reversed AVPI sections：
AVPIGGGAVPIG（Direct-Direct, DD）, AVPIGGGIPVAG（Direct-Reverse, DR）and IPVAGGGIPVAG（Reverse-Reverse, RR）. The
alanine residues were methylated. In liquid phase the separation was made by CE with the ultraviolet（UV）optical detection. A
mobility evaluation of ionized isomeric molecules in gas phase was made by IMS with tandem MS（MSMS）detection. The
experiments with CE-IMS-MSMS instruments connected online were carried out to compare the separation capabilities of thesetechniques.
The CE-UV experiments showed separation of the peptides from mixtures up to 20 % of peak height above the baseline. The
separation occurred in acidic CE buffers only and increased with the lowering of the pH value. The DD peptide migrated incapillary more slowly whereas the DR and RR peptides moved faster and were not separated from each other under all tested
conditions.
The mobility of the single charged positive ions was measured by IMS-MSMS separately for all peptides. The difference between
DD and DR was 1.6 %, between DD and RR was 5.9 %, between DR and RR was 4.2 %. The most different DD and RR peptides wereseparated from the mixture by IMS-MSMS at 70 % of peak height above the baseline.
Online combination of the methods showed the overlay of the CE migration time and the IMS mobility parameters measured in
one experiment with MSMS detection of the separated components.
The difference in ion mobility of the isomers in gas phase as well as in solution depends on their charge state and charge
distribution that affects on folding. In highly acidic solution the differences in the right part of the sequence cause changes in thefolding. Whereas the left part of the sequence has greater effect on mobility of single protonated peptides in gas phase.

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 37: Challenges in High Resolution and High Accuracy Mass Measurement Mass
SpectrometryPTh-083

11:10 － 12:20

Challenges in high resolution and accuracy mass measurement for drug
discovery and development using FT ICR and Orbitrap mass
spectrometers

Zenzaburo Tozuka
Osaka University

Keywords:
FTICRMS, OrbitrapMS, AMS, MIST, Microdose
Novel aspects:
high sensitive, accurate structure elcidation of comprehensive drug metabolites using nanoLC-LTQ orbitrap, FT ICR MS and AMS tosolve safety testing of drug metabolites in microdose clinical study
Abstract:
I challenged in high resolution and accuracy measurement of LTQ FT ICR MS during development in Bremen to solve theestimated two different fragment pathways of FK228（Istodax）having same mass but different exact mass and different
composition of atoms 1 ）. On the base of their composition analysis by FT ICR MS, I could elucidate the structure of in vitro 14
metabolites and in vivo 8 metabolites of FK228 using SRM datadependent exclusion MSn measurements 2 ）. Challenges to
elucidate structure of 9 metabolites of Nicardipine for microdose clinical study（MDCS） 3 ）and determine the 12 biomarkers ofbreast cancer by shot gan analysis in combination with their antibody analysis using brest cancer tissues（International Patent）
and in target proteome analysis of CYP 3 A 4 promoter factors binding their DNA segments（ISSX abstract）, and CYP 3 A 4 Raloxifene active metabolites adduct 4 ）were succeeded by two LTQ FT ICR MS in our laboratory. According to safety testing ofdrug metabolites（MIST）guidance, the quantitative and qualitative analysis of comprehensive metabolites of 14C-tolbutamide（TB）
without standard samples was succeeded by UPLC-RI-LTQ Orbitrap MS 5 ）. MIST items of 14C-TB and 14C-acetoaminophen 6 ）in
MDCS were conducted by AMS and nanoLC-LTQ Orbitrap that was possible to elucidate the structure of metabolites using MDCS
samples（urine and plasma）in addition to their quantitative analysis.
References
1 ）Z. Tozuka et al., LTQ FT MS accurate mass and SRM data dependent exclusion MSn measurements for structure determinationof FK228 and its metabolites, J Mass Spectrom Soc Jpn. 53 89-99（2005）,2 ）Z. Tozuka et al., Strategy for structural elucidation of drugs and drug metabolites using（MS）n fragmentation in anelectrospray ion trap, J Mass Spectrom. 38 793-808（2003）,
3 ）N. Yamane et al., Microdose clinical trial： Quantitative determination of nicardipine and prediction of metabolites in humanplasma, Drug Metab Pharmacokinet. 4 （ 4 ）389-403（2009）,
4 ）H. Yukinaga et al., Identification of Cytochrome P450 3 A 4 Modification Site with Reactive Metabolite Using Linear Ion TrapFourier Transform Mass Spectrometry, Chem.Res. Toxicol., 20（10）, 1373-1378（2007）,
5 ）Z. Tozuka et al., Comprehensive Quantitative and Qualitative Liquid Chromatography Radioisotope Mass Spectrometry
Analysis for Safety Testing of Tolbutamide Metabolites Without Standard Samples, J.Pharmaceu. Scie. 100, 4024-4036（2011）,
6 ）Z. Tozuka et al., Microdose Study of 14C-Acetaminophen With Accelerator Mass Spectrometry to Examine Pharmacokinetics ofParent Drug and Metabolites in Healthy Subjects, Clin Pharmacol Ther. 88, 824-830（2010）

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 37: Challenges in High Resolution and High Accuracy Mass Measurement Mass
SpectrometryPTh-085

11:10 － 12:20

Silicon speciation in petroleum products using a multi-technical approachby Mass Spectrometry for a better understanding of catalyst poisoning

Fabien Chainet2, Jeremie Ponthus1,2, Charles-Philippe Lienemann2, Marion Courtiade2,
Olivier François X Donard3
1

IFP Energies Nouvelles, 2IFP Energies Nouvelles, Solaize, France, 3LCABIE-IPREM, UMR 5254, CNRS UPPA, Pau, France

Keywords:
silicon speciation, catalyst poisoining
Novel aspects:
Due to the thermal degradation of PDMS in the presence of hydrocarbons, more than 100 silicon species were highlighted andcould act as a poison on hydrotreatment catalysts
Abstract:
Silicon speciation is of considerable importance in the oil and gas industry due to the negative effect of its relative species on theperformance of hydrotreatment catalysts. In petroleum products, silicon mainly comes from the use of antifoaming such aspolydimethylsiloxanes（PDMS）to avoid emulsions in the different processes such as cokefaction, visbreaking, steamcracking or
distillation and to enhance the crude oil recovery from the reservoir. PDMS, consisting in a structural unit of（CH 3 ）2 -Si-O-, has
excellent properties of low surface tension and initial great thermal stability. Due to the high temperature applied in thermal
cracking processes, PDMS degrades around 300 °C and mainly generates cyclic siloxanes（Dn ）with also many different silicon
species in petroleum products that can affect the performance of catalysts.
In the oil and gas industry, the possible reactivity of hydrocarbon radicals with PDMS degradation products, was never reported
and the chemical nature of these related silicon species must be determined. Up to now, these issues have been well identified
but only addressed through the total silicon determination. These results have displayed total concentration levels ranging fromµg.kg -1 of Si up to some mg.kg -1 of Si in petroleum products measured by elemental techniques. Moreover, the complexity of
petroleum matrices, the wide variety of compounds concerned combined with possible contamination problems occurring during
silicon analysis has hampered the development of speciation studies.
A very innovative and complete multi-technical approach based on MS techniques was developed and applied to identify andquantify silicon species. PDMS degradation samples, produced under thermalcracking of hydrocarbons on a pilot plant and several
naphtha and gasolines coming from different refining processes were investigated. For low molecular weight silicon compounds,
gas chromatography（GC）hyphenated to MS in single ion monitoring（SIM）was performed in gasolines. Cyclic siloxanes wereconfirmed as the major degradation products with trace of linear siloxanes. However, suspected unknown silicon compounds
cannot be detected as their structures and fragmentations patterns remained unknown. To overcome this challenge, FT-ICR/MS
was performed in positive electrospray mode（ESI）and allowed the characterization by their raw formula of more than 50 new
silicon compounds with several unsaturations in naphtha and gasoline samples. To obtain a quantification and a structural
identification of these compounds, GC-ICP/MS providing a specific detection combined to GC-GC/MS addressing the chemical
structure were carried out both in PDMS degradation samples and in naphtha and gasolines. Finally, MS/MS experiments werealso carried out to confirm the suspected chemical structures.
More than one hundred silicon species were highlighted in this work. Silicon compounds present in PDMS degradation samples
and in gasoline were compared and confirmed the correct representativity of the evaluated conditions of PDMS degradation
samples. The presence of PDMS, intermediate polymers and low molecular weight silicon compounds could explain the
distribution of silicon amount in the different cuts of petroleum products and probably the poisoning effect on hydrotreatment
catalysts.

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 37: Challenges in High Resolution and High Accuracy Mass Measurement Mass
SpectrometryPTh-086

13:30 － 14:40

Development and validation of high resolution liquid chromatographytime of flight method of Bacopaside-I and metabolites forpharmacokinetic study

Sontaya Sookying2, Kornkanok Ingkaninan*2, Dumrongsak Pekthong3, Sarawut Oo-puthinan3, JIE XING1,
Zhaoqi Zhan1

Shimadzu (Asia Pacific) Pte Ltd, Singapore, 2Department of Pharmaceutical Chemistry and Pharmacognosy, Faculty of
Pharmaceutical Sciences, Naresuan University, Phitsanulok 65000, Thailand, 3Department of Pharmacy Practice, Facultyof Pharmaceutical Sciences, Naresuan University, Phitsanulok 65000, Thailand
1

Keywords:
Bacopaside-I, quantification, validation, metabolite, pharmacokinetics
Novel aspects:
Quantitative analysis of Bacopaside I by high resolution LC-MS（TOF）method and metabolite identification.
Abstract:*Corresponding author

Introduction
Bacopa monnieri (L.) Wettst.（Brahmi）is an Ayurvedic medicine plant used for centuries as a memory enhancer. There have been
several studies on biological effects of this plant for a therapeutic potential in treatment and prevention of neurological diseases
and improvement of cognitive processes. Bacopaside I（C46H74O20S）, a dammarane-type triterpenoid saponin, is one of the main
active components of the plant and, therefore, it has been produced and used in biological and therapeutic studies. Various
analytical method such as HPTLC, ELISA and HPLC methods were used to determine bacopasides I and other active components
in the plant extracts as well as biological samples. However, so far LC/MS method for quantitative analysis of Bacopaside I in
biological samples has not been reported. We report for the first time the development and validation of a high sensitivity LC-MS（TOF）method, aiming for pharmacokinetic study of bacopasides I and identification of its metabolites produced in vitro and invivo.

Experimental
Rat urine and feces were used as the biological matrix for method development and validation of bacopaside I quantitative
analysis. Liquid-liquid extraction method was employed in sample extraction and purification. The LC-MS method used is based onhigh resolution TOF which is suitable for both quantitative analysis of Bacopaside I in biological samples and qualitative analysis
to identify any metabolites formed. An LCMS-IT-TOF（Shimadzu Corporation, Japan）has been used in this study
Results and Discussion
This study was carried out in three stages, development and optimization of a high resolution LC/MS（TOF）method, validation
trials and identification of metabolites of bacopaside I from various in vitro hydrolysis samples using a MetID Solution program. A
gradient elution method using a Kinetex C18 column（1.7 um, 50 mmL x 2.1mmID）was developed and optimized. It was found thatboth target compounds（Bacopaside I and an internal standard, Digitoxin）showed very strong mass peaks（m/z 977.4387 and763.4243）in negative ESI mode. A high resolution MS and MS/MS method were established to obtain best sensitivity and highest
reliability of analysis.

The performance of the method has been evaluated systemically using extract samples prepared from rat urine and feces. The
linearity（r 2 ）was 0.9985 for a range of from 4.8 ng/mL to 380 ng/mL. The specificity and matrix effect were studied and it was
found that the method specificity was highly reliable due to the use of accurate mass（target m/z +/- 50 ppm）on the highresolution TOF mass spectrometer. Matrix effect was not obvious under the LC/MS condition. The LOQ of the method was lower
than 4.8 ng/mL. The reproducibility at this concentration level（spiked into urine）was 8.7 %（RSD of concentration, n= 6 ）. The
recoveries of bacopaside I spiked into urine were 91.1 % and 107.1 % for three concentration levels（50, 80 and 100 ng/mL）.
However, the recovery of feces extract samples was found lower than that of urine samples at 40~70%. Further study to improve
the recovery of bacopaside I in feces samples is undergoing. The method has been applied to in vitro hydrolysis samples. Some
metabolites were found from the acid hydrolysis samples by using MetID solution program, which is based on an editablebiotransformation database.
Conclusions
A high resolution LC-TOF method for quantitative analysis of bacopaside I in biological samples has been established on LCMS-ITTOF. The method can be used for pharmacokinetics study and it is also capable of identification of metabolites of bacopaside I byusing a MetID soution

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 37: Challenges in High Resolution and High Accuracy Mass Measurement Mass
SpectrometryPTh-087

11:10 － 12:20

Characterization of Humic and Fulvic acid fractions separated by size
exclusion chromatography and application of 15T FT-ICR MS

Min-Hui Son, Yoon-Seok Chang
POSTECH, Pohang, Korea

Keywords:
Humic and Fulvic acids, Size Exclusion Chromatography（SEC）, FT-ICR MS
Novel aspects:
Humic and fulvic acid fractions separated by SEC was analyzed with 15T FT-ICR MS and this is the first study that humic and fulvic
acid fractions were examined in Korea.
Abstract:
Understanding the characterization of molecular level of humic and fulvic acid, which has enormous complexity and highmolecular weights, is still very difficult in spite of the application of high resolution mass spectrometry or various ionization
techniques. For this reason, separation techniques, such as size exclusion chromatography（SEC）or capillary electrophoresis（CE）,
and ultra high resolution mass spectrometry（FT-ICR MS）have been attempted to the characterization of humic and fulvic
compounds in many studies. In spite of their important physicochemical properties, however, they were not completely
characterized at molecular level due to the tremendous complexity of their structures. In this study, humic substances including
humic and fulvic acid were separated by size exclusion chromatography（SEC）and analyzed with negative ion mode electrosprayionization（ESI）ultrahigh resolution mass spectrometry（15T FT-ICR MS）. Suwannee river humic acid（SRHA）and fulvic acid（SRFA）
standards obtained from IHSS（International Humic Substance Society）were used for molecular weight distributions. In order tobetter interpretation of mass spectra, humic and fulvic acid unfractionated were also analyzed. Size exclusion chromatography has
been widely used as separation technique, but 15T FT-CR MS is not common as conventional high mass spectrometry.

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 37: Challenges in High Resolution and High Accuracy Mass Measurement Mass
SpectrometryPTh-088

13:30 － 14:40

Dependence of Field Evaporation in Three-Dimensional Atom Probe on
the Crystallographic Orientation

Mahito Shimizu, Masato Morita, Yuya Hanaoka, Masanori Owari
The University of Tokyo, Tokyo, Japan

Keywords:
three-dimensional atom probe, field evaporation, crystallographic orientation, time-of-flight, high mass resolution
Novel aspects:
We newly discussed field evaporation dependence on the crystallographic orientation of the sample surface for improving threedimensional atom probe tomography.
Abstract:
Understanding of relationship between material property and microstructure is one of the main topics of materials science. For
achievement of this challenge, knowledge of materials structure is required at the sub-nm scale. Historically, spatial resolution ofavailable microstructural analysis techniques was insufficient for elucidation of the role of materials elements to materials
properties. More recently, however, the spatial resolution limits have been amazingly improved by the advent of microstructural
analysis techniques such as three-dimensional atom probe（ 3 DAP）.
3 DAP is a field ionization microscope equipped with a time-of-flight mass spectrometer（TOF-MS）and a position-sensitive
detector（PSD）. The samples used in 3 DAP are usually shaped into sharp needle geometry whose tip radii are approximately 100
nm. In 3 DAP, when strong electric field is applied to the tip of a sharp needle sample by high standing voltage（DC）and a voltage
pulse or a laser pulse, the atom which has the smallest number of nearest neighbor atoms ionizes in sequence. Then, the positiveion is removed from the surface of specimen and travels to a PSD accelerated by the electric field. This ionization is called fieldevaporation. The elemental identity of the ion is determined with a TOF-MS. The lateral position of each atom in the sample isdetermined from the position arrived on a PSD. The depth coordinate from the tip of the sharp needle sample is determined fromthe position in the field evaporation sequence. A sub-nm scale three-dimensional image of the specimen is reconstructed fromthese data in the virtual space by the computer algorithm.
In order to improve analysis accuracy of 3 DAP tomography, knowledge about influential factor affecting field evaporation isneeded. In this study, we noted the effect of crystal structure on field evaporation. As samples, Tungsten（W）, which had bodycentered cubic lattice, was used. W needles with different crystallographic orientations were analyzed by 3 DAP. In the case of
the sample with［110］orientation, field evaporated ions were mostly W3+ and W4+. On the other hand, in the case of the［100］sample, field evaporated ions were mostly W3+, W4+ and W5+. It was revealed that field evaporated ions are dependent on the
crystallographic orientation of the sample surface. In addition, it was found that the accuracy of 3 DAP tomography was also
dependent on the crystallographic orientation of the sample surface. In the atomic scale three-dimensional image of the［110］sample, crystal planes could be observed regardless of depth. On the other hand, in the atomic scale three-dimensional image ofthe［100］sample, crystal planes could be observed only at the surface.
These two facts, both field evaporated ions and 3 DAP tomography accuracy were dependent on the crystallographic orientation,
were very interesting. By considering the relationship between these two facts, we will discuss how the crystallographic
orientation of sample surface affects the field evaporation mechanism in 3 DAP.

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 37: Challenges in High Resolution and High Accuracy Mass Measurement Mass
SpectrometryPTh-089

11:10 － 12:20

Theoretical Verification of a Strategy for Determining Elemental
Composition Based on Ultrahigh-resolution Mass Spectrometric Data

Tatsuhiko Nagao1, Daichi Yukihira1, Yoshinori Fujimura1, Kazunori Saito2, Daisuke Miura1,
Hiroyuki Wariishi1
1

Kyushu University, Fukuoka, Japan, 2Bruker Daltonics K.K., Yokohama, Japan

Keywords:
elemental composition, isotopic fine structure, ultrahigh-resolution, FT-ICR-MS, heuristic rule
Novel aspects:
This study highlighted potential superiority of using ultrahigh-resolution mass spectrometry and additional constraint rules for
identifying unique correct elemental composition independent of standard compounds.
Abstract:
Background
Identifying metabolites with mass spectrometric data requires comparisons with spectral databases or reference standard spectra,
despite the fact that a large proportion of biological metabolites are not commercially available. Thus, a metabolite identification
method independent of standard compounds is indispensable technique for metabolomics.
Fourier transform-ion cyclotron resonance-mass spectrometry（FT-ICR-MS）has high mass accuracy and ultrahigh mass resolving
power. Previously, we demonstrated the possibility of determining the elemental composition of metabolites using the peak area
ratio of 13C, 15N, 18O, and 34S isotopic fine structure observed by FT-ICR-MS independent of standard compounds. In this study, we
represented an algorithm to calculate elemental composition from mass spectrometric data automatically. Then, we validated thealgorithm through simulations using in silico-generated 20,258 mass spectra to understand the effect of error size of isotopic peaks
on elemental compositions obtained via the algorithm.
Results
To prepare in silico mass spectra of metabolites, molecular formula of metabolites was needed. First, 20,258 kinds of different
elemental compositions comprising only C, H, O, N, P and S elements were selected from the original dataset including molecularweight and molecular formula of metabolites downloaded from http：//www.metabolome.jp/. To replicate mass spectrum of FTICR-MS in silico, theoretical mass and theoretical area ratio of isotopic peaks to monoisotopic peak were re-calculated for eachentry. Theoretical monoisotopic masses were treated with 0.1 - 1 ppm random error to replicate the measured mass accuracy of
FT-ICR-MS. Theoretical relative isotopic area was replicated by an opposite procedure of estimating the number of element. Then,random errors were added to relative isotopic areas with varying maximum errors from +10% to -25%. To compensate the random
errors in isotopic area, estimated numbers for each element are enlarged by ±25 % safety factor, thereby generating ranged
numbers of element. In the simulation of determining elemental composition, deprotonated peak（［M-H］- ）was taken into
consideration for the negative ion mode, and proton- and alkali metal-adducted peaks（［M+H］+ ,［M+Na］+ ,［M+K］+ ）for the
positive ion mode. Simulations were performed for 100 times to average number of estimated elemental compositions.
Since isotopic area is fluctuant because of the space charge, the relationship between isotopic area error and the number of
estimated elemental composition was investigated. Both in the simulation of negative and positive ion modes, the percentage of
metabolites identified as a unique correct elemental composition was decreased from 61 % to 45 % and from 58 % to 38 % withdiminishing maximum area error from +10% to -25%, respectively. Thus, approximately 50% of 20,258 metabolites were generated
more than 2 candidates. These results indicated that using 13C, 15N, 18O and 34S isotopic fine structure is insufficient for limiting
elemental composition as few as possible. To expand the percentage of metabolites identified as unique correct elemental
composition, we added another constraint rules to the algorithm. First, since we found that either C4（48）or H3NP（48.00031）was
replaced with a complement one between a correct elemental composition and incorrect ones, we used oxygen/phosphor ratio（O/P ratio）to remove such incorrect candidates. The O/P ratio in KEGG compounds was investigated thoroughly； thereby the
O/P ratio was elucidated as greater than 2. By applying the O/P ratio, the percentage of metabolites led unique correct elemental
compositions was increased to 60 % . Moreover, the valence check rule introduced by T. Kind and O. Fiehn（2007）was also
effectively increased the percentage of metabolites obtained unique correct candidate up to 90%.

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 37: Challenges in High Resolution and High Accuracy Mass Measurement Mass
SpectrometryPTh-090

13:30 － 14:40

Analysis of Wedelia trilobata flower extract using Liquid ChromatographyOrbitrap Mass Spectrometry

Jhih-Yang Huang1, Ean-Tun Alex Liaw1, Chien-Kuo Han2, Ming-Feng Chen3, Jean Chih-Chun Huang1

National Pingtung University of Science & Technology, Pingtung, Taiwan, 2Asia University, Taichung, Taiwan, 3National
Cheng Kung University, Tainan, Taiwan

1

Keywords:
Wedelia trilobata； LC-Obitrap-MS； antioxidant activity； phenolics； flavonoids
Novel aspects:
By using the advanced instrument such as Orbitrap mass spectrometer for accurate mass and high-resolution measurement, one
can expect for simplified procedures, reduced analysis times and increased confidence in results.
Abstract:
Wedelia trilobata, categorized in the Heliantheae tribe of the Asteraceae family, is a plant widespread in subtropical countries
such as Brazil, India, Burma, Ceylon, China and Japan. This kind of herbal plants has been reported in traditional Chinese medicine
to show anti-inflammatory properties and used as a detoxifying agent for liver protection from toxicity. To fully understand theactive compounds and their functionality mechanisms, systematic analyses are in need to provide more scientific evidence. In thisstudy, we attempted to analyze the components in the ethanolic extract of the Wedelia trilobata flowers using liquid
chromatography coupled with electrospray ionization/Orbitrap mass spectrometry（LC-Orbitrap-MS）, which is recently developed
for accurate mass and high-resolution measurement. By using this advanced instrument, simplified procedures can be expected,
as well as reduced analysis times and increased confidence in results for challenging analysis such as low level components incomplex mixtures. In the meantime, we also assessed the antioxidant activity in terms of DPPH（2,2-diphenyl- 1 -picrylhydrazyl）
scavenging capacity and the total phenolic and flavonoid contents of the extract using spectrophotometric methods. Our MS
results suggest that the ethanolic extract may contain some phenolic compounds such as luteolin and quercetin and the phenolicgulcosidic conjugates. On the other hand, the results of antioxidant activity show that the extract at a concentration of 20 μg/mL
could scavenge 50% DPPH radicals, while that for the concentration of the luteolin standard solution was 6.0 μg/mL. One milligramof the extract was found to have total phenolic compounds of 96.2 μg gallic acid equivalents and total flavonoids of 24.5 μg
quercetin equivalents.

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 37: Challenges in High Resolution and High Accuracy Mass Measurement Mass
SpectrometryPTh-091

11:10 － 12:20

Relevance of Gas and Pressure for Thermalization of Cluster Ions in the
Hexapole Ion Trap of a FT-ICR Mass Spectrometer

Jürgen H Gross, Doris Lang, Iris Mitsch
Heidelberg University, Heidelberg, Germany

Keywords:
cluster ions, mass calibration, ESI, MALDI, FT-ICR-MS
Novel aspects:
Proper selection of the buffer gas pressure is relevant for the mass range covered by cluster ion peaks of intensities useful formass calibration on certain hybrid FT-ICR instruments.
Abstract:
Introduction
Cluster ions are quite useful to establish mass calibration of instruments equipped with desorption/ionization sources over a widem/z range. In our laboratory, we are routinely using the abundant［argininen+H］+ and［argininenH］- cluster ions［1,2］for externalmass calibration in positive-ion and negative-ion ESI mode, respectively. Likewise,［CsnIn-1］+ and［Csn-1In］- cluster ions can be used
for calibration purposes in MALDI operation. The caesium salt cluster ions are generated from caesium triiodide, CsI3, in 2 -［（ 2 E）
- 3 -（ 4 -tert-butylphenyl）- 2 -methylprop- 2 -enylidene］malonitrile（DCTB）matrix［ 3 ］.
In hybrid quadrupole Fourier transform ion cyclotron resonance（FT-ICR）mass spectrometers, however, multiple ion-guiding andion-trapping events occur prior to mass analysis. During a recent implementation of a liquid injection field desorption/ionization（LIFDI）ion source on this instrument, the enormous relevance of the type and pressure of the buffer gas collisions in the
instrument’s accumulation RF-hexapole ion trap were recognized, because the selection of these settings was decisive for thedetection of the easy-to-fragment molecular ions of hydrocarbon species［ 4 ］. Thus, we studied the effect of argon versus helium
buffer gas and their pressure on cluster ion distributions intended for mass calibration in ESI and MALDI modes.
Experimental
The experiments were performed on a Bruker Apex-Qe FT-ICR mass spectrometer（Bruker Daltonik GmbH, Bremen, Germany）
equipped with a 9.4 T superconducting magnet. The analyzer of this instrument is a quadrupole-ICR hybrid comprising a RF-only
hexapole（h 1 ）, a selection RF/DC quadrupole（Q）, a second RF-only hexapole（h 2 ）, and a high voltage ion transfer flight tube
to bring ions from h 2 through the magnetic field gradient into the ICR cell. Typically, h 2 is used for ion accumulation prior to ICR
analysis. In tandem MS mode, h 2 is also employed for collision-induced dissociation（CID）and subsequent accumulation of theresulting fragment ions.
Ions were accumulated in the collision hexapole for 0.1 to 0.2 s and then transferred into the ICR cell. The standard buffer gas
argon and the lighter alternative helium were admitted to h 2 at various pressures. Ions were excited and detected using standard
settings as established in ESI and MALDI mode. Data acquisition was performed in broadband mode with 512k to 1 M data points.
Typically, 32 to 64 transients were accumulated for one magnitude mode spectrum. External mass calibration was performed on
the cluster ions. The instrument was controlled by the Bruker ApexControl software（V 3.0.0）and data analysis was performed
using the Bruker DataAnalysis software（V.4.0）.
Results and Discussion
Argon buffer gas served better for cluster ion thermalization in h 2 than helium, a trend that is opposed to expectations derived
from previous LIFDI experiments. The pressure of buffer gas is more relevant for the cluster ion distribution, in particular for［CsnIn1］+ and［Csn1In］cluster ions. Abundances of cluster ions of intermediate m/z of about 1500 to 3000 were increased uponraising the buffer gas flow to the maximum（2.0 l/s）while high-mass clusters beyond m/z 5000 benefited from medium gas flow（1.0l/s）into h2. Thus, proper selection of the buffer gas pressure is relevant for the mass range covered by cluster ion peaks ofintensities that are useful for calibration.
References
1. Soga, T.； Kakazu, Y.； Robert,M.； Tomita, M.； Nishioka, T. Electrophoresis2004,25, 1964-1972.
2. Schug, K.； Lindner, W., Int. J. Mass Spectrom.2004,235, 213-222.
3. Lou, X.； van Dongen, J.L.J.； Meijer, E.W., J. Am. Soc. Mass Spectrom. 2010,21, 1223-1226.
4. Linden, H.B.； Gross, J.H., Rapid Commun. Mass Spectrom.2012,26, 336-344.

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 38: Mass Spectrometry for Metabolic Diseases

PTh-092

13:30 － 14:40

A quantitative analytical method for determining the levels of glucosedependent insulinotropic polypeptides GIP1-42 and GIP3-42 in humanplasma using LC-MS/MS/MS

Atsushi Miyachi1, Nana Morimoto1, Yuichiro Yamada2, Ken-ichi Harada3

SANWA KAGAKU KENKYUSHO CO.,Mie,Japan, 2Akita University School of Medicine, Akita, Japan, 3Meijo University,
Nagoya, Japan

1

Keywords:
GIP, Asp-N, LC-MS/MS/MS, diabetes, plasma
Novel aspects:
Proteolysis of GIP by Asp-N endopeptidase, not trypsin, is useful for a stable simultaneous quantification of GIP 1 -42 and GIP 3 -42
in human plasma using LC-MS/MS/MS.
Abstract:[Purpose]
Glucose-dependent insulinotropic polypeptide（GIP）is a 42-amino acid peptide hormone released from duodenal K-cells after theabsorption of glucose or fat. Similar to its structurally related peptide glucagon-like peptide- 1 （GLP- 1 ）, which is released fromintestinal L-cells, GIP is considered to be an incretin hormone. In addition to its incretin effect, GIP is considered to increase theactivity of lipoprotein lipase（LPL）, which is a key enzyme of lipid metabolism, as well as to promote the glucose absorption of
adipose cells in the presence of insulin； it is also associated with bone metabolism. However, some in vivo roles of GIP are still
unknown. Therefore, the quantitative evaluation of GIP secretion and active GIP levels is important. Active GIP 1-42 as well as GLP- 1
is degraded rapidly by dipeptidyl peptidase- 4 （DPP- 4 ）to yield GIP3-42, which is inactive. Therefore, the concentration of active
GIP1-42 in peripheral plasma is very low.
Current methodologies used for peptide and protein quantification include immunoassays and mass spectrometric techniques.
Immunoassays, though sensitive, lack the necessary selectivity for recognizing the distinction between peptides, their recombinant
forms, metabolites, and posttranslational products. In many cases, suitable antibodies for each protein analyte may not be
available. Suitable antibodies for active GIP1-42 are more unlikely to be obtained. Additionally, it is noted that the plasma GLP- 1
levels measured using different immunoassay kits are not consistent. The pretreatment of plasma samples is reported to beimportant. To this end, analysis by liquid chromatography-mass spectrometry（LC-MS）is useful. Although there have been several
reports about the quantification of GIP using LC-MS（/MS）, there is no report on the sensitive quantification of GIP1-42 and GIP3-42
in a clinical trial using only small volume of plasma. In this study, we used an LC-MS/MS/MS approach for the simultaneous
absolute quantification of GIP1-42 and GIP3-42 in human plasma.
[Method]
Synthetic standard GIP peptides were spiked into charcoal stripped human plasma. Stable isotope（15N/13C）-labeled GIP1-42 and
GIP3-42 were used as internal standards. Peptide fragments of GIP1-8 and GIP3-8 obtained by digestion using Asp-N endopeptidase
were selected as analytes. Since both the fragmented peptides did not contain methionine, which is oxidized easily, the sample
stability during and after extraction was expected to be good. Protein precipitation（PP）was performed during sample
pretreatment before the digestion, and solid phase extraction（SPE）was performed after the digestion. Samples were injected
onto a capillary column（C18； 150 mm x 75 µm I.D.）, and GIP1-8, GIP3-8, and internal standard peptides were detected by hybrid
triple quadrupole/linear ion trap mass spectrometry（QqQ/LIT-MS）operated in positive electrospray ionization mode.
[Results and Discussion]
The lower limit of quantification（LLOQ）of the reported method was 1 pM for GIP1-42 and 10 pM for GIP3-42 using 200 µL of humanplasma, which were similar to the LLOQ of the GIP obtained by an immunoassay. The accuracy and precision values indicatedgood reliability of the analytical method. We applied this method to a clinical trial for diabetic subjects treated with DPP- 4 andalpha-glucosidase inhibitors. Our results suggested that the described methodology successfully determined the efficacy of DPP- 4
inhibitor alone, alpha-glucosidase inhibitor alone, and in combination, based on the plasma levels of GIP1-42 and GIP3-42.

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 38: Mass Spectrometry for Metabolic Diseases

PTh-093

11:10 － 12:20

Metabolomic Analysis of Autism Spectrum Disorders Using Saliva

KEIJI GAMOH, KAZUTAKA SUGAI
Kochi University, Kochi, Japan

Keywords:
Autism, Metabolomics, Saliva, LC/MS
Novel aspects:
Demonstration of the metabolomic analysis of saliva and the comparison of the metabolites between autism spectrum disorders
and controls based on the liquid chromatography/mass spectrometry（LC/MS）
Abstract:
Objectives:
Elucidation of autism spectrum disorders based on the metabolomic analysis of biological substrates in saliva.
Development of liquid chromatography/mass spectrometric analytical methods for the metabolites in saliva using a reversedphase separation mode and an electrospray ionization mode.
Investigation of comparison of the concentrations of metabolites between the autism spectrum disorders and controls.
Autism spectrum disorders (ASD) are a behaviorally defined group of neurodevelopment disorders characterized by impairments
in social interaction and communication, and repetitive, overly focused behaviors. While the syndrome has been shown to behighly heritable, various theories have been presented suggesting both genetic and environmental factors, such as dietary or
chemical exposures. Analytical chemical investigation of autism spectrum disorders based on liquid chromatography/mass
spectrometric method has been proceeded. Our interests were targeted to the concentration of biological metabolites in saliva
of autism spectrum disorders. In the present study we demonstrated the metabolomic analysis of saliva and the investigation ofcomparison of the metabolites between autism spectrum disorders and controls using a reversed-phase separation mode andan electrospray ionization mode of the liquid chromatography/mass spectrometry (LC/MS).

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 38: Mass Spectrometry for Metabolic Diseases

PTh-094

13:30 － 14:40

Affintiy - Mass Spectrometry: A Proteome Signature for Intra-Uterine
Growth Restriction Reveals Pathological Protein Glycosylation Abberationsin Umbilical Cord Blood

Manja Wölter1, Claudia Röwer1, Cornelia Koy1, Ulrich Pecks2, Michael O Glocker1
1

Proteome Center Rostock, 2Womens Clinic, University of Aachen

Keywords:
Affinity-MS, cord blood serum profiling, proteome signature, apolipoprotein C-III, protein glycosylation
Novel aspects:
Multiparametric MS data analysis of a proteome signature for IUGR describes deglycosylated Apo C-III derivatives as key markers
for the first time
Abstract:
Intrauterine Growth Restriction（IUGR）affects about 3 to 8 % of all pregnancies. It is defined as a condition in that the fetus does
not reach its genetically given growth potential, resulting in low birth weight. IUGR is an important cause of perinatal morbidity
and mortality, thus contributing substantially to medically indicated preterm birth. The pathogenesis of IUGR still remains unclear.
Analysis of complex gestation-related patho-physiological conditions like IUGR requires methods that can cope with thecomplexity of the samples. We here describe mass spectrometric profiling of umbilical cord blood serum proteins to identify
disease-related protein alterations with impact in diagnostics and even prognostics of gestation-associated diseases.
Cord blood samples from 30 newborns（15 from normal pregnancies and 15 from IUGR cases）were taken post-natally from theumbilical cord vein. Serum was prepared using standardized protocols. Samples were processed using affinity-beads. After elution,
protein solutions were spotted directly onto stainless steel MALDI targets and mixed with ferulic acid, each in duplicate. Protein
mixtures were analyzed with a Reflex III MALDI ToF mass spectrometer equipped with the SCOUT source and delayed extraction
in linear positive ion mode, using an acceleration voltage of 20 kV. Spectra were recorded in a mass range from 4 -25 kDa,
accumulating 900 shots per spectrum. Spectra evaluation was performed with the ClinProTools 2.2 software and in-housebiostatistic analysis tools.
Protein mixtures of cord blood samples were subjected to affinity fractionation in order to remove highly abundant proteins. Afteraffintiy enrichment, MALDI mass spectra showed on average more than 60 protein ion signals between m/z 4000 and 25,000. The six
best differentiating ion signals were at m/z 8205, 8766, 13,945, 15,129, 15,308, and 16,001, forming a proteome signature with which wewere able to distinguish between IUGR and inconspicuous controls. Of note, apolipoprotein C-III0（m/z 8766, lacking glycosylation）
was found more abundant in the IUGR samples, irrespective of gestational age, whereas other apo C-III forms with different
glycosylation structures were not. Apo C-III is involved in triglyceride metabolism, which itself is discussed to be of importance in
IUGR pathogenesis.
To assign ion signals to specific proteins, samples were subjected to SDS PAGE followed by PMF. We identified apolipoprotein
C-III together with other apolipoproteins（apo A-I, A-IV）, fetuin A, and others. Identification of apo C-III was confirmed by MS/MS
analysis. The MS/MS spectrum of the precursor ion at m/z 1196.61 showed a characteristic fragmention signal at m/z 638.37 from a
D-G cleavage, together with other specific fragment ion signals, confirming the presence of the amino acid sequence 41-51from
apolipoprotein C-III.
Furthermore, apo C-III concentrations in serum were measured by ELISA and show that protein abundance differences between
IUGR and control cord blood samples can be determined reliably using an affinity-enrichment procedure combined with MALDIMS
profiling. Mass spectrometry-based multiparametric analysis of cord blood samples is capable of differentiating individual samples
from the IUGR group from those of the control group with high confidence.
In conclusion, we suggest（i）apolipoprotein C-III0 as a key-marker of the IUGR proteome signature, and, that（ii）subtle alterationsin glycosylation need to be considered for understanding the pathomechanisms in IUGR.

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 38: Mass Spectrometry for Metabolic Diseases

PTh-095

11:10 － 12:20

Automated Analysis of 25-hydroxyvitamin D2 and D3 with APCI-MS/MS
Coupled with ZIVAK ONH-200BD Sample Preparation System

MURAT CELIK, HASAN OZGENZIVAK TECHNOLOGIES, KOCAELI, TURKEY

Keywords:
25-hydroxyvitamin-D3, 25-hydroxyvitamin-D2, automated sample preparation, tandem mass, Zivak
Novel aspects:
The first automated MS analysis of 25-hydroxyvitamin D 2 and D 3 was made with ZIVAK ONH-200BD automated sample
preparation system without any human control for reducing user errors.
Abstract:
Introduction
The measurement of serum concentrations of 25-hydroxy vitamin D 2 and D 3 is the most accurate way to determine vitamin D
status in human body. 25-hydroxy metabolites of vitamin D, plays a critical role in controlling calcium and phosphate levels in the
body. Quantitation of these metabolites is very important because of their clinical significance in variety of disorders like many
chronic conditions and latency diseases, osteomalacia and rickets disease in children.
Because of their importance, analysis methods of 25-hydroxy metabolites must be reliable and sensitive. The conventional
methods based on HPLC-UV has disadvantages for sensitivity, specificity and analysis time. On the other hand, the methods basedon immunoassay has some specifity problems. As a result of these conditions, interest in LC-MS/MS methods growing during
recent years.
Methods
A reliable, rapid and sensitive LC-MS/MS method with automated sample preparation system for analysis of 25-hydroxy
metabolites was developed. Standarts, calibrators, controls and reagents of ZIVAK 25-hydroxyvitamin D 2 -D 3 LC-MS/MS Analysis
Kit kindly provided by ZIVAK Technologies, Turkey. All sample preparation steps below was made by ZIVAK ONH-200BD
automated sample preparation system without any human control for reducing user errors in sample preparation.
500 ul of serum samples pipetted into sample preparation vials. 400 ul of Reagent 1 including salt solution in appropriate
concentration added and mixed serially by vortex unit of sample preparation system. Then, 400 uL of deuterated 25-hydroxyvitaminD 3 internal standard mixture in organic solvent added mixed serially by vortex unit of sample preparation system. Aftercentrifugation at 5.000 rpm for 5 min, 50 ul of upper phase injected to LC-MS/MS system. Deionized water and methanol with 0,1%
formic acid were used as mobile phases for gradient elution. 50x4.6 mm reversed-phase HPLC column used for chromatographic
separation. Analysis run time was 6 minutes. Confirmation and quantification ions for all analytes and internal standards
determined in APCI（+）scan mode. ZIVAK TANDEM GOLD LC-MS/MS system used for analysis.
Preliminary Data
Recovery for analytes were between 97%-105%. Linearity from 2,5 to 400 ug/L for both analyte. Correlation coefficients were 0,9989
and 0,9995 for 25-hydroxyvitamnin D 2 and D 3 respectively. Limit of detection for 25-hydroxyvitamin D 2 was determined as 1 ug/L
while limit of detections for 25-hydroxyvitamin D 3 was 0.8 ug/L. Coefficients of variability values for 25-hydroxyvitamin D 2 and D 3
were 2,4 and 2,7% for intra-assay and 3,4-3,6% for inter-assay. No interferences was found for these analytes.
The NIST SRM 2972 sample was measured with this automated system and the results were found in median range for bothanalyte.

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 38: Mass Spectrometry for Metabolic Diseases

PTh-096

13:30 － 14:40

A LC/MS/MS Method Enables Simultaneous Detection of Diagnostic
Biomarkers of Alkaptonuria, Ornithine Carbamoyltransferase Deficiency
and Neuroblastoma Disease

Chien-Chen Lai1, Wei-Yi Hsu2, Fuu-Jen Tsai2

1
National Chung Hsing University, Taichung, Taiwan, 2Department of Medical Research, China Medical University
Hospital, Taichung, Taiwan

Keywords:
LC/MS/MS； Alkaptonuria； Ornithine Carbamoyltransferase Deficiency； Neuroblastoma； Creatinine； Screening
Novel aspects:
Creatinine and the metabolites in urine from patients with alkaptonuria, OCTD, or neuroblastoma were quantified in one-run using
LC/MS/MS. The level of creatinine was to normalize the urine sample differences.
Abstract:
In this study, a liquid chromatography-tandem mass spectrometry（LC/MS/MS）method was developed for simultaneousquantification of diagnostic markers of neuroblastoma, ornithine carbamoyltransferase deficiency, and alkaptonuria in urine,
namely homovanillic acid（HVA）/vanillylmandelic acid（VMA）, orotic acid（OA）, and homogentisic acid（HGA）. After sample
preparation, which involved only the dilution procedure, samples were quantified by LC/MS/MS. Full-scan MS/MS mode enabled
the urinary markers to be quantified with a high degree of specificity and sensitivity. Rather than using a separate enzymatic
method to normalize the concentration of creatinine in urine, we quantified the level of creatinine in urine in one LC/MS run. The
limits of detection were 10 µg L-1 for HGA, 25 µg L-1 for HVA/VMA, and 50 µg L-1 for OA with a single-to-noise ratio of 3 ； the limitsof quantification were 50 µg L-1 for HVA and HGA, 100 µg L-1 for VMA, and 250 µg L-1 for OA. Multiple calibration curves exhibited
consistent linearity and reproducibility. The linear dynamic range of quantification of the analytes covered 2 to 3 orders ofmagnitude, depending on the analyte. The relative standard deviation of the developed LC/MS/MS method was less than 4 % for
the intra-day validation and 10% for the inter-day validation. The results show that our LC/MS/MS technique is a highly sensitiveand rapid method for screening for urine biomarkers that are diagnostic of three rare metabolic diseases.

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 38: Mass Spectrometry for Metabolic Diseases

PTh-097

11:10 － 12:20

Development of a high sensitive quantitation method for serum C-peptide
by isotope-dilution mass spectrometry

Tomoya Kinumi, Mari Goto, Akiko Takatsu

National Institute of Advanced Industrial Science and Technology, Tsukuba, Japan

Keywords:
Isotope-dilution mass spectrometry, C-peptide, Immunoaffinity purification
Novel aspects:
We first established the precise measuremt of low concentration of serum C-peptide by mass spectrometry using combination ofchemical modification and immunoaffinity purification.
Abstract:
Introduction
Human C-peptide is a 31-mer peptide which is secreted into the blood from beta-cell in the pancreas where pro-insulin is cleaved
into insulin and C-peptide during post-translational processing. Human C-peptide concentrations in blood plasma（reference
interval 0.5-10.0 ng/mL）reflect the level of beta-cell function associated with insulin resistance, and indicate insulin secretory
failure. Measuring C-peptide concentration in plasma provides a guide for therapy in diabetes. Because the quantitation of plasma
C-peptide has been performed by an immunochemical technique, the accuracy strongly depends on that of the standard material.
To establish a precise quantitation technique for blood C-peptide covering the range of the reference interval, we have developedchemical modification and immunoaffinity purification technique for the determination of serum C-peptide by isotope-dilution
mass spectrometry.
Experiment
Two concentrations of serum sample were prepared by adding the certified reference material of C-peptide（NMIJ CRM 6901-a）tohuman serum to be 0.3 and 10 ng/mL. The isotopically labeled C-peptide, D8-Val2-C-peptide was used for the internal standard. A
solid phase extraction was performed using Oasis HLB and MCX（Waters）, and a immunoaffinity capture was performed using a
Magnosphere immobilized magnetic beads（JSR）with an anti-C-peptide antibody. AQC（ 6 -Aminoquinolyl-N-hydroxysuccinimidyl
carbamate）reagent was used for N-terminal modification of the peptide. An LC-MS/MS analysis was conducted by Prominence
20A HPLC（Shimadzu）with a reversed-phase C18 column and a TSQ Quantum Discovery tandem quadrupole mass spectrometer（Thermofisher Scientific）equipped with electrospray ion source in selected reaction monitoring（SRM）mode.
Results and Discussion
We first examined the high concentration（10 ng/mL）of serum sample by a two-step solid phase extraction using Oasis HLB andMCX prior to LC-MS/MS. The deviation of the quantitative results was more than 10 %. To improve the sensitivity and reduce thedeviation, the enriched sample by the solid phase extraction was modified with AQC reagent. The AQC-modification increased 10fold in the signal intensity and reduced the deviation on the measurement to less than 5 %. In case of the low concentration（0.3ng/mL）of a serum sample, we could not observe the signal of the AQC-modified C-peptide using solid phase extraction for the
sample enrichment. However, we successfully quantified AQC-modified C-peptide with 10 % of the deviation by an immunoaffinity
purification using anti-C-peptide antibody-immobilized magnetic beads instead of the solid phase extraction prior to the AQCmodification. In conclusion, we have successfully developed the quantitation method for serum C-peptide in the range of 0.3-10
ng/mL with the deviation of 5 -10 % by combination of the immunoaffinity purification and the chemical modification by AQC
reagent.

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 38: Mass Spectrometry for Metabolic Diseases

PTh-098

13:30 － 14:40

Label-Free Screening for Epigenetic Drug Discovery Using Mass
Spectrometry to Monitor Protein and Nucleic Acid Modification Events

Jennifer Rossi, Peter Rye, Lauren Frick, Hisashi Iwase, Masahiro Maeda, William LaMarr
Agilent Technologies

Keywords:
epigenetics, label-free, mass spectrometry, histone, methylation
Novel aspects:
This research demonstrates a novel and high-throughput means of analyzing epigenetic targets, often difficult to investigate during
the drug development process.
Abstract:
Epigenetics, the study of changes in gene expression caused by mechanisms other than deviations in DNA sequence, is rapidly
emerging as a field with tremendous drug development potential across a broad range of therapeutic areas. Epigenetic discovery
via traditional screening technologies is complicated by the ability of these enzymes to impart multiple modification events onto
singular substrates resulting in complicated and dynamic enzyme kinetic environments. In this study, we investigated the use ofmass spectrometry as a universal platform for the detection and quantification of multiple protein and nucleic acid modification
events（i.e. histone acetylation status, histone methylation status, DNA methylation status）. The use of mass spectrometry
facilitates direct detection of native enzyme substrate and product pairs, and allows for rapid assay development of a wide arrayof chemical analytes（peptides, oligonucleotides, whole proteins, etc）. The label-free methodology mass spectrometry provides
can enable targeted screening across a large collection of epigenetic processes and represents a ubiquitous approach to thiscomplex research area.

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 38: Mass Spectrometry for Metabolic Diseases

PTh-099

11:10 － 12:20

Comprehensive analysis of human proteome in hippocampus tissue from
Alzheimer disease patient.

hyekyeong Min1, Hyung Joo Lee2, Se-Young Kim1, Gun Wook Park1, In Jung Ji1, Kyung-Hoon Kwon1,
Jong Shin Yoo1, Young Mok Park1, Jin Young Kim1

Korea Basic Science Institute,Ochang-eup,Cheongwon-gun,Chungbuk,Korea, 2Yonsei Proteome Research Center, Yonsei
University, Seoul, Korea

1

Keywords:
alzheimer hippocampus proteome ETD analysis
Novel aspects:
A total of 5337 different proteins were confidently identified in control samples and 4990 in AD（alzheimer disease）patients,
respectively.
Abstract:
Alzheimer’s disease is a progressive neurodegenerative disorder and the most common form of dementia. The disease isconfirmed by the presence of neuritic plaques and neurofibrillary tangles in the cerebral cortex at autopsy, but the accuracy ofantemortem diagnosis, especially at the early stages of the disease, is not ideal. Thus, there is a substantial need for the discovery
and validation of diagnostic biomarkers. We report a global proteomic analysis of hippocampus tissue from normal human and
Alzheimer patient. Proteins were separated into soluble and membrane fractions. Proteins were digested with trypsin, andresulted peptides were further separated into 12 fractions by off gel isoelectric focusing method. Each samples were analyzed by
UPLC coupled with reversed phase LC-ESI-MS/MS analysis and we applied both collision induced dissociation（CID）and electrontransfer dissociation（ETD）based fragmentation. Analytical column was home-made microcapillary column consisting of C18（Aqua； particle size 3 μm）packed into i.d. 75 μm, o.d. 360 μm silica tubing. For protein identification, the MS/MS data wereanalyzed using the IPI human database with the protein discoverer software. A total of 5337 different proteins were confidently
identified in control samples and 4990 in AD（alzheimer disease）patients, respectively.

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 38: Mass Spectrometry for Metabolic Diseases

PTh-100

13:30 － 14:40

Rapid and sensitive determination of the intermediates of advanced
glycation end products in human nail by UPLC-ESI-TOF-MS

Jun Zhe MIN1, Makoto Yamamoto1, Tatsuya Higashi2, Kenichiro Todoroki1, Koichi Inoue1,
Toshimasa Toyo’oka1

School of Pharmaceutical Sciences, University of Shizuoka, Shizuoka, Japan, 2Faculty of Pharmaceutical Sciences, Tokyo
University of Science, Japan

1

Keywords:
Human nail, 4,5-Dimethyl-1,2-phenylenediamine； 3 -Deoxyglucosone； Methylglyoxal； Glyoxal
Novel aspects:
As we know, for the first time, these three kinds of the dicarbonyl intermediates of AGEs, which were 3 -DG, MG and GO, werefirst found in human nail samples.
Abstract:
The resolution of the intermediate Advanced Glycation End products（AGEs）in human nail was carried out by the combination of
4,5-dimethyl-1,2-phenylenediamine（DMPD）derivatives and ultra performance liquid chromatography with electrospray ionization
time-of-flight mass spectrometry（UPLC-ESI-TOF-MS）. The reaction of the reagent with 3 -Deoxyglucosone（ 3 -DG）, methylglyoxal（MG）and glyoxal（GO）effectively proceeds at 60°C for 2 hr. The resulting derivatives were efficiently separated by a gradient）using a reversed-phase ACQUITY UPLC BEH C18（1.7
program（a mixture of H2O and CH3CN containing 0.1% formic acid（HCOOH）
mm, 50 mm × 2.1 mm i.d.）column and sensitively detected by TOF-MS. The detection limits（S/N= 5 ）of the TOF-MS were 10-50
fmol. A good linearity was achieved from the calibration curves, which was obtained by plotting the peak area ratios of theanalytes relative to the internal standard（IS）, i.e., 2,3-hexanedione, versus the injected amounts of 3 -DG, MG and GO（r2> 0.999）,
and the intra-day and inter-day assay precisions were less than 6.89 % . The derivatives of the compounds in human nail weresuccessfully identified by the proposed procedure. As we know, for the first time, these three kinds of dicarbonyl intermediates in
the formation of AGEs, which were 3 -DG, MG and GO, were first found in human nail samples. Using these methods, the amountsof compounds in the nails of healthy volunteers and diabetic patients were determined. When comparing the index from thediabetic patients to those from healthy volunteers, there is no significant difference in the content of the MG and GO in the nails.
However, a statistically significant（P<0.001）correlation was observed between the 3 -DG concentrations. Therefore, because theproposed method provides a good mass accuracy and the trace detection of the dicarbonyl intermediates of AGEs in human nails,
this analytical technique could be a noninvasive technique to assist in the diagnosis and assessment of disease activity in diabetic
patients. Here we present a novel sensitive, simple method for the simultaneous determination of dicarbonyl compounds inhuman nails.

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 38: Mass Spectrometry for Metabolic Diseases

PTh-101

11:10 － 12:20

Determination of flavonoids in infected plants using LC-MS/MS

Jong Sung Jin1, Hae Gyeong Kim1, Mee Sung Lee1, Mi Jin Kim1, Sung Chul Shin2
1

Korea Basic Science Institute, Busan, Korea, 2Gyeongsang National University, Jinju, Korea

Keywords:
Flavonoids, LC-MS/MS, Infected plants, Metabolomics
Novel aspects:
The flavonoid content in the infected plant with the disease development decreased inithially after infection but then gradually
increased before finally progressively decreasing.
Abstract:
Flavonoids play an important role as pre-infection inhibitors, providing plants with a certain degree of basic resistance against
pathogenic microorganisms. It was confirmed by LC-MS/MS that as procedure of disease.
In this work we identified the flavonoids of the plants and compared their characteristic chromatographic fingerprints with
infected one using liquid chromatography electrospray ionization mass spectrometry（LC-MS/MS）.

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 38: Mass Spectrometry for Metabolic Diseases

PTh-102

13:30 － 14:40

Development of methods to identify previously unknown secondary
metabolites from myxobacteria based on a comprehensive metabolic
profiling workflow

Thomas Hoffmann2,3, Daniel Krug2,3, Clive Seymour1, Gabriela Zurek4, Aiko Barsch4, Rolf Mueller2,3

1
Bruker BioSciences Pty. Ltd., Preston, Australia, 2Pharmaceutical Biotechnology, Saarland University, Saarbruecken,
Germany, 3Helmholtz-Institute for Pharmaceutical Research Saarland (HIPS), Saarbruecken, Germany, 4Bruker Daltonik
GmbH, Bremen, Germany

Keywords:
non-targeted metabolomics, identification of secondary metabolites, high resolution MS and MS/MS
Novel aspects:
Characterization of complex samples by combining targeted and non-targeted LC-TOF-MS based Metabolomics workflows - anefficient de-replication strategy.
Abstract:
Introduction:
The extraction of relevant information from complex data sets remains an important bottleneck in（microbial）metabolomics
research. Compared to a focused, targeted approach this is even more important in non-targeted metabolomics, where the goalis the identification of all compounds produced by a particular bacterial strain. Many of these compounds are part of primarymetabolism and therefore out of scope when research concentrates on secondary metabolites. Other metabolites may be very
common for a certain genus of bacteria and already well known but the large subset of compounds that are“really new“ is hard
to identify.
By combining targeted and non-targeted metabolomic approaches, including searches against in-house and freely available databases, it is possible to narrow down and identify numerous features derived from HPLC-high resolution MS/MS measurements.
Methods:
6 biological replicates each of myxobacterial（Sorangium cellulosum）extracts and growth medium blanks were separated bygradient elution at 600 ul/min and 45 degree C using a C18 column on an RSLC system（Dionex）. ESI-MS measurements were
performed using positive ionization on a maXis QTOF（Bruker Daltonik）with a repetition rate of 2 Hz for MS and 3 Hz for MS/
MS. Targeted screening was carried out using precise extracted ion chromatograms（XICs）, retention time and isotope patternevaluation in the TargetAnalysis software . Data pre-processing, statistical interpretation and creation of a Scheduled precursor list
（SPL）for targeted MS/MS experiments was performed with the ProfileAnalysis software.
Results:
The automated feature finding algorithm detected 2000 - 5000 features within one HPLC-MS chromatogram on a myxobacterial
extract. In a first step these features were subjected to a search against an in-house database using accurate mass, isotope
pattern fit, and retention time in order to identify known compounds. Non-targeted metabolite profiling, using statistical methods
such as ANOVA, t-test as well as PCA analysis, identified 176 features related to the growth of the bacteria by comparing
myxobacterial extracts to blank samples（growth medium）. These features were automatically added to a scheduled precursor
list（SPL）to focus fragmentation experiments on the relevant subset of compounds within the complex mixture. This enabledfragmentation of low abundant features which might have been missed during an automatic precursor selection without
predefined compounds of interest. High resolution full scan MS and MS/MS spectra were used to identify target metabolites byqueries in open source libraries（e.g. METLIN）. Remaining unidentified features were subsequently compared to an in-house
database to identify putative derivatives of known compounds. Calculation of sum formulae suggests the presence of new
ambruticin derivatives. 63 out of 176 filtered features from myxobacterial extracts could be identified based on MS/MS data. The
applied strategy effectively supported the de-replication, i.e. identification of already known compounds in complex metabolite
extracts, thereby facilitating the screening for“new“ bacterial secondary metabolites.

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 38: Mass Spectrometry for Metabolic Diseases

PTh-103

11:10 － 12:20

Use of proton-affinitive derivatization in HPLC-positive-ESI-MS analysis of
biologically important carboxylic acids

Hajime Kato, Shin Mitsuzuka, Kowa Yamashita, Mitsuteru Numazawa
Tohoku Pharmaceutical University, Sendai, Japan

Keywords:
lipidomics, proton affinity derivatization, bile acids, eicosanoids, fatty acids
Novel aspects:
Picolyl esters as proton affinity tag for analysis of carboxylic acids using HPLC-ESI-MS were investigated.
Abstract:
Introduction
Bile acids, fatty acids and eicosanoids are biologically important as biomarker for diagnosis of various diseases or inborn error of
metabolism. However, these substances generally show poor sensitivity in HPLC-ESI-MS analyses with their naked molecules.
Introduction of proton-affinitive or permanently charged tags into carboxyl moieties of such molecules proved to be advantageous
to improve detection sensitivity of these molecules toward ESI in HPLC-MS. During the course of our studies on the analysis ofhydroxy steroids by HPLC-ESI-MS, we have found that the introduction of the proton-affinitive tags（picolinate： Pi derivative）
enabled to determine estrone, estradiol, testosterone, dihydrotestosterone, dehydroepiandrosterone, pregnenolone, cortisol,
corticosterone and aldosterone molecules with the limit of quantification（LOQ）of approximately 0.5- 1 pg/ml human serum byselected reaction monitoring（SRM）in a positive mode. The aim of this study is to develop proton-affinitive Pi tags introducted bysimple derivatization for HPLC-ESI-MS analyses of biologically important carboxylic acids.
MethodDerivatization： Carboxylic acids（bile acids, fatty acids and eicosanoids）were treated with 2 -bromomethylpyridine or 2 （bromomethyl）- 6 -methylpyridine in CH 3 CN in the presence of diisopropylethyamine. The derivatives thus obtained were
purified by solid-phase extraction using ODS cartridge using 90% CH3CN-H2O as an eluting solvent. Identification： The structuresof derivatives were confirmed by high resolution ESI-MS and 1H-NMR spectroscopy. HPLC-MS： Finnigan TSQ Vantage AM（ESIpositive）； column： X-Bridge（C18, 150 mm x 2 mm I.D., 5 mm, Waters）； mobile phase： CH3CN-H2O-CH3COOH（65 ： 35 ： 0.1 v/v/v）； flow rate： 0.2 ml/min.
Preliminary data
In the case of hydroxysteroids, we selected picolinic acid and its related compounds as proton-affinity tags in our previous report.
In this case, esterification of hydroxysteroids with picolinic acid was performed by mixed anhydride method based on the use of
2 -methyl- 6 -nitrobenzoicanhydride with more than 70 % yields. In this report, we selected 2 -bromomethypyridine or
2 -bromomethyl- 6 -methylpyridine as proton-affinity tags for derivatization of carboxylic acids. In itial efforts were focused on thederivatization of bile acids as reference standards. Treatment of bile acids（cholic acid, chenodeoxylcholic acid, ursodeoxycholicacid, deoxycholic acid and lithocholic acid） with 2 -bromomethylpyridine in CH 3 CN solution in the presence ofdiisopropylethylamine gave the expected picolyl ester of carboxylic acids. The picolyl ester of each bile acid provided well-shaped
HPLC peak without formation of by-products. The positive-ESI mass spectra of the picolyl ester of these bile acids werecharacterized by the appearance of protonated molecule as base peaks. SRM utilizing the inherent transition for each picolyl esterof bile acid enabled to determine these bile acids with extremely high sensitivity. Quantitation of these bile acids utilizing stable
isotope labeled internal standards was also investigated. An application of the present derivatization method to fatty acids andsome eicosanoids which include carboxyl group were also discussed.

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 38: Mass Spectrometry for Metabolic Diseases

PTh-104

13:30 － 14:40

Comprehensive CID with accurate mass measurement for analyte
identification in metabolomics and natural products: HPLC-TOF-MS
analysis with database searching

Joe Binkley, Binkley Joe, Kevin Siek, Li Zhang
LECO Corporation

Keywords:
Metabolomics, LC/MS, MS/MS, Databases
Novel aspects:
Comprehensive fragmentation with deconvolution and the mass accuracy and resolving power of the TOF MS enables analytecharacterization in metabolomics and natural products analysis using traditional MS/MS databases.
Abstract:
Tandem mass spectrometry（MS/MS）has become the benchmark for analyte identification using mass spectrometry with thegeneration fragment ions occurring through varied mechanisms. Among these, collision induced dissociation is by far the mostcommon. The other prominent tool for characterization purposes in mass spectrometry is accurate mass analysis. This has been
the domain of FTMS and magnetic sector systems but time of flight systems have made significant inroads in the past decade. The
growing presence of hybrid instruments which provide both MS/MS and accurate mass information has created even more
opportunities. An alternative to these approaches is post source collision induced dissociation on a high performance time offlight system. When fragmentation occurs in a controlled pressure region and high resolving power and mass accuracy is
combined with analyte deconvolution software which generates precursor and fragment ion spectra there is a powerful synergy.
In this study post source, deconvoluted fragment ion spectra are generated for analytes detected in complex natural productsamples. The spectra are searched against common databases for MS/MS spectra including Metlin and the quality of the hits andthe spectra are compared. The results show that across a range of signal-to-noise levels and m/z ranges the post source fragment
ion spectra provide spectra comparable to library spectra and high integrity hits. Much of this is attributable to the high massaccuracy observed in both the precursor and product on spectra（>45,000 resolving power and better than 2 ppm massaccuracy）. In addition, the post source dissociation provides superior relative isotope abundance measurements for precursor
and product ions which facilitates analyte identification. Specific examples of the utiilty of this approach in metabolomics andnatural product characterization are discussed.

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 38: Mass Spectrometry for Metabolic Diseases

PTh-105

11:10 － 12:20

Acylcarnitine analysis by ESI-MS/MS with smaller amount of sample andrapid analytical time

Hironori Kobayashi1, Yuichi Mushimoto1, Yuki Hasegawa1, Kenji Yamada1, Jamiyan Purevsuren1,
Tomoo Takahashi1, Toshikazu Minohata2, Junko Iida2, Yamaguchi Seiji1
1

Shimane University, Izumo, Japan, 2Shimadzu Corporation, Kyoto, Japan

Keywords:
newborn screening, acylcarnitine, amino acids, rapid analysis
Novel aspects:
Rapid and small-volume-injection analysis of amino acids and acylcarnitines by ESI-MS/MS
Abstract:
Introduction
After late 1990’s, analysis of acylcarnitines and amino acids by MS/MS has become popular in newborn screening. This screening
made it possible to examine more than 20 diseases at once including disorders of amino acid metabolism, organic acidemia, andfatty acid oxidation disorders. Currently, a non-derivatization method is predominant. Current method of analysis requires 5 -20 mlof injection volume, and requires over two minutes at one analysis, approximately 72,000 samples a year. Because a sample isextracted from dried blood spot on filter paper in newborn screening, smaller volume of injection is preferred for lessmaintenance. We developed the method of more rapid and smaller injection volume by using Nexera MP and LCMS-8030 triple
quadrupole mass spectrometer（ShimadzuCorporation）.
Methods
A 3 mm punch of blood spot was processed using a NeoBase Non-derivatized MSMS Kit（PerkinElmer）and analyzed by multiple
reaction monitoring（MRM）. Dried blood spot of known concentration in each analyte were used. Samples were measured by
Nexera MP and LCMS-8030 equipped with an ESI source positively charged. Flow rates of mobile phase provided in Kit was 0.1 ml/
min. Injection volume was changed in 10 µl, 5 µl, 2 µl, and 1 µl, respectively.
Results
Sensitivity was sufficient even in 1 µl of injection volume, as compared with original injection volume of 10 ml. Accuracies andprecision in intra- and inter-day assay were within 10%. An analysis cycle of 74 seconds per sample was achieved in this situation.
Conclusion
Combination of Nexera MP and LCMS-8030 achieved higher throughput, more than 100,000 samples a year, than current screening.

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 38: Mass Spectrometry for Metabolic Diseases

PTh-106

13:30 － 14:40

Simultaneous determination of acylcarnitines and amino acids for inborn
error of metabolism using UPLC/MS/MS

Haruka Oda1, Yasuhiro Maeda1, Tetsuya Ito1, Yuji Hotta1, Yoko Nakajima1, Sayaka Kato1, Shinji Saito1,
Naruji Sugiyama2, Kazunori Kimura1
1

Nagoya City University, Nagoya, Japan, 2Aichi-Gakuin University, Nagoya Japan

Keywords:
acylcarnitine, newborn screening, amino acids, UPLC MS/MS, metabolic disease
Novel aspects:
We developed detail determinaton method of acylcarnitines and amino acids without delivatization for metabolic disease by
UPLC/MS/MS. This method enables acute diagnosis of metabolic disease.
Abstract:
Background: Tandem mass spectrometry（MS/MS）analysis of acylcarnitines and amino acids has been used to screen newborns
for organic acidemias, fatty acid oxidation defects and amino acid metabolism abnormality. Although this method provides results
in a few minutes, the method cannot separate acylcarnitine isomers and amino acid isomers. Moreover, the peak intensity ofanalyte by MS/MS would be unstable by ion suppression to detect many kinds of acylcarnitines and amino acids at the same
retention time in the chromatography. We previously reported determination method of acylcarnitine isomers from free carnitine（C 0 ）to octanoylcarnitine（C 8 ）by HPLC/MS/MS. Simultaneous analysis of both short-chain acylcarnitine and long-chain
acylcarnitine by HPLC was very difficult as the polarity of these acylcarnitines is different greatly. The determination method of C 0
to octadecanoylcarnitine（C18）was developed by derivatizing acylcarnitines. However, the derivatization reagent is atypical andderivatization reaction takes trouble. We investigated the simultaneous determination method of acylcarnitines（C 0 -C18）and
amino acids（Val, Leu, Ile, Met, Phe, Orn, Cit, Arg, Tyr）without derivatization by ultra high performance liquid chromatography（UPLC）-MS/MS. A more accurate diagnosis of inborn error of metabolism is enabled by this analytical method.
Method: A mixture of 5 micro L of 28 acylcarnitines, 9 amino acids, corresponding labeled acylcarnitines and labeled amino acids
was injected onto Acquity UPLC BEH C18 column（2.1 x 150 mm, Waters）and eluted at a flow rate of 0.3 mL/min using a step
gradient alternating between 0.08 % aqueous ion-pairing reagent（IPCC-MS3, GL sciences）and 0.3 % formic acid in methanol.
Acylcarnitines and amino acids were analyzed in positive ion multiple reaction monitoring（MRM）mode of Quattro Premier XE
triple quadrupole mass spectrometer（Waters）.
Acylcarnitnes and amino acids were extracted from serum of patients with inborn error of metabolism using an OASIS MCX solid
phase extraction cartridge（Waters）. The extracted solution was analyzed by above method.
Results: All acylcarnitine isomers were separated in 35 min and all amino acids were analyzed in 13 min using UPLC. The limits ofquantifications（LOQs）were 0.05 micro mol/L for acylcarnitines and 1.0 micro mol/L for amino acid, respectively. The precision
data for serum sample showed that intra-day coefficients of variance（CVs）at three concentrations were less than 15 % .
Acylcarnitines and amino acids which were not the marker in screening were specifically detected in analysis of the serum
samples of patients with inborn error of metabolism. For example, methylmalonylcarnitne（C 4 DC）and 3 -methylcrotonylcarnitine（C 5 ： 1 ）were accumulated in the serum of patients with methylmalonic acidemia（marker； propionylcarnitine（C 3 ））and
holocarboxylase synthetase deficiency（marker； 3 -hydroxyisovalerylcarnitine（C 5 OH））, respectively.
Discussion: We developed simultaneous determination method for acylcarnitines （C 0 -C18） and amino acids without
derivatization. The analytical time is shorter by using UPLC and the quantification of low concentration is possible because
analysis in MRM mode is very sensitive. The separation of analyte by UPLC column would achieve stability of the individual peaksensitivity by MS/MS. Therefore, concentration of near the cut-off value is determined precisely and the decrease of the pseudo
positive is expected. The acylcarnitines and amino acids profile obtained by this method enabled a correct diagnosis. This method
can apply to monitoring of the effect of treatment by observing a change of acylcarnitines and amino acids.

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 39: MS Informatics for Quantitation

PTh-107

11:10 － 12:20

An Attempt to Quantitative Analysis for Clinical Proteomics by Nano LCnano-ESI-SRM-MS Using Stable Isotope-labeled Iodoacetanilide as well as
N-Ethylmaleimide

Sadamu Kurono1,2, Yuka Kaneko1,2, Shuji Matsuura1, Satomi Niwayama3

1
Osaka University, Osaka, Japan, 2Wako Pure Chemical Industries, Ltd., Osaka, Japan, 3Texas Tech University, Lubbock,
TX

Keywords:
Quantification, Stable isotope, Iodoacetanilide, N-ethylmaleimide
Novel aspects:
A combination of stable isotope-labeled and -unlabeled iodoacetanilides or N-ethylmaleimides and LC-ESI-SRM-MS offers a new
method of absolute quantification for clinical proteomics.
Abstract:
Quantitative analysis of proteins is essential to clinical proteomics. We have developed a methodology for relative quantificationof proteins by modifying cysteine residues with stable isotope（ 13 C）-labeled iodoacetanilide（ 13 C 7 -IAA）or -unlabelediodoacetanilide（IAA）as well as stable isotope（d）-labeled N-ethylmaleimide（d5-NEM）or -unlabeled N-ethylmaleimide（NEM）,
followed by nano LC-nano-ESI-mass spectrometry. In addition to the combination of the above labeling, the selected reaction
monitoring（SRM）technique, accomplished by specifying the precursor ion and specifically monitoring its few most intense andmost reliable CID product ions, was also applied to identification and absolute quantification by the above mass spectrometer forclinical proteomics. We examined the possibility for absolute quantification of commercial proteins such as bovine serum albumin（BSA）and some candidate proteins for breast cancer biomarkers from nipple discharge of breast cancer patients.
For absolute quantification of BSA, we selected two tryptic peptides for SRM and obtained their synthetic peptides. The synthetic
peptide solution adjusted at around pH 9.0 or 7.0 and reduced with dithiothreitol or TCEP（Bond-Breaker TCEP Solution, Neutral
pH, Thermo Fisher Scientific）for IAA/13C7-IAA or NEM/d5-NEM modification, as the case may be, was divided into two solutions.
One of the solutions was incubated with an excess amount of 100 mM IAA DMSO or NEM solution, while the other with 100
mM 13C7-IAA DMSO or d5-NEM solution. The IAA- or NEM-modified solution was adjusted to the following five concentrations：
1000, 500, 100, 50, and 10 fmol/µL, while the 13C7-IAA- or d5-NEM-modified solution was adjusted to 100 fmol/µL. Each IAA- or NEMmodified solution mixed with 13C7-IAA- or d 5-NEM-modified solution was loaded into nano LC（UltiMate 3000, Thermo Scientific
DIONEX）-nano-ESI-Ion Trap MS（HCTultra, Bruker Daltonics）system and a standard curve was created by the SRM technique.
For absolute quantification of commercial proteins we used three kinds of proteins： BSA, ovalbumin, and α-lactalbumin. In thecase of BSA, we selected two tryptic peptides for the SRM analysis, which were observed at a high intensity on MS. Two clustersof peaks representing IAA- and 13 C 7 -IAA-modified tryptic peptides were observed to be 7 Da apart due to the presence ofseven 13C atoms and detected with the same retention time, which means that the isotope effects by the introduction of IAA
and 13C7-IAA do not exist in this system. One of the above two peptides, LCVLHEK, was custom-synthesized, and modified with
IAA or 13C7-IAA. Five IAA-modified peptide solutions with different concentration（1000, 500, 100, 50, and 10 fmol/µL）were prepared,and each solution was mixed with a constant concentration of 13C7-IAA-modified peptide（100 fmol/µL）. The SRM conditions wereas follows： IAA-modified LCVLHEK （m/z 487.8 doubly-charged precursor ion >m/z 333.2, 625.4 singly-charged productions）, 13C7-IAA-modified LCVLHEK（m/z 491.3（same as above）>m/z 340.2, 625.4（same as above））, and the chromatogram area
ratio of m/z 333.2/340.2（Cys-containing）and m/z 625.4/625.4（non-Cys-containing）. The standard curve based on the results ofabove five mixtures had R2 value that was close to 1 （0.995）. The relative standard deviation（RSD）values indicated repeatability
were calculated from three replicated SRM analyses for each mixture and the RSD ranged from 3.9 % to 17.0 % . The R 2 values
obtained from other commercial proteins also showed those close to 1, and the RSD values were similar to those of BSA. The
similar result was also obtained using NEM/d 5 -NEM and from other two proteins. Therefore the combination of the IAA
and 13 C 7 -IAA modification in addition to SRM was found to be an effective tool for absolute quantification for proteins.
Furthermore we are also trying to apply this technique to absolute quantification of some candidate proteins for breast cancer
biomarker.

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 39: MS Informatics for Quantitation

PTh-108

13:30 － 14:40

Multivariate analysis software to support large scale MRM study

Atsushi Ogiwara1, Hisae Anyoji1, Jun Watanabe2, Junko Iida2, Mitsuhiro Kanazawa1
1

Reifycs, Inc., Tokyo, Japan, 2Shimadzu Corporation, Kyoto, Japan

Keywords:
MRM, Multivariate Analysis, Alignment, Quantification, Qualification
Novel aspects:
Alignment functionality in Signpost MS software improves the capability of quantification and qualification of multi-sample MRM
data.
Abstract:
MRM analysis is widely used in various areas including metabolomics for the sake of quantification and qualification. In order tocarry out such analyses, a number of MRM transitions for a large number of samples must be processed. Software tools have been
desired to treat such data with user-friendly interfaces to conduct from data capturing to multivariate analysis. We havedeveloped integrated multivariate analyses software named Signpost™ MS, which can treat various kinds of MS instruments data.
Previously, this software is mainly used for the analyses of scan data, where data are represented as a series of spectra. Here wehave expanded the functionality of the software to support MRM data.
One of the important functions of this software is“alignment“. In the case of scan data derived from LC-MS, there are threefundamental axes： 1 ）separation axis as retention time, 2 ）spectrum axis as m/z, and 3 ）quantity axis as ion intensity. Thoughmass values are quite solid, retention time by liquid chromatography fluctuates and time axes should be aligned between
measurements. Once time axes are aligned, now corresponding peaks in different measurement can be observed at the same
position of the aligned time axis.
In the case of MRM data, where the dimension of spectrum axis is reduced to single or several transitions, still we provide thealignment function to overlay peak shapes by adjusting time axes.
When we measure an analyte of high concentration, it might not be difficult to detect the peak derived from the analyte. However
the lower the analyte concentration, the peak height becomes the lower, and the detection of the peak becomes the moredifficult. In such a case, we cannot use intensity to detect peaks. However, if we can predict the location of the peak on time axis,
we can detect the peak.
Alignment function is also useful in another case where several candidate peaks can be observed due to interference peaks ornoise. In such a case, location information of time axis may be a decisive factor.
Alignment is conducted based on the similarity of shapes of chromatogram. Thus it would be hard to align chromatograms ofpoor shapes, due to the weak signal response of the analyte. However, in such cases we can use other transition information thatis measured simultaneously. For example, internal standard signals can be used for quantification measurement, and other analytetransition signals can be used for MRM profiling.
Once we have capture peaks, then they will be presented visually to validate authenticity of the selected results. We also
introduce some basic rules to evaluate the validity of peak shapes.
The software begins by loading raw data from an LC-MS experiment. In the case of MRM assay, loaded data are rearranged
according to transitions. Grouping information of samples should be given by users. For samples belonging to the same sample
group, time axes are aligned each other by using common transition data. Then peaks are detected and presented visually forvalidation. Results of the peak shape validity evaluate on are also presented. Conformed peaks are arranged into a table to beanalyzed by multivariate analyses like PCA or cluster analyses.
In this study, we show novel use of MRM data in the context of metabolomics biomarker discovery using an LCMS-8030 triple
quadrupole mass spectrometer integrated with Nexera UHPLC system（Shimadzu Corporation, Kyoto, Japan）.

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 39: MS Informatics for Quantitation

PTh-109

11:10 － 12:20

High Pressure Acid Dissolution of alpha-alumna for trace elements
determination by ICP-MS

Jung Ki Suh, Chang Soo Kim

Korea Research Institute for Standards and Science, 267 Gajeong Ro, Yuseong Gu, Daejeon 305-340, Rep. of Korea

Keywords:
alpha-alumna, high pressure acid dissolution, ICP-MS, JCRM R 031, metal impurities
Novel aspects:
This presentation describes alpha-alumna sample can be easily dissolved by simple acid treatment
Abstract:
High purity α-alumna has important uses in the electronics and ceramics industries. The properties of α-alumna are muchaffected by the level of impurities present, and hence the determination of trace elements in this material is of great practicalimportance. It is well known that the dissolution of α-alumna sample for analysis of trace impurities is very difficult.
This presentation describes α-alumna sample can be easily dissolved by simple acid treatment. Granule and wafer type of α-alumina sample were pulverized with vibratory micro mill device（FRITSCH, GmHB）. And then, an enough amount of α-alumina
powder sample（ 1 g）was decomposed with 5 mL of H2SO4 and 15 mL of deionized water in 50 mL size of Teflon vessel of highpressure acid digestion device by heating in drying oven for 16 h at 230°C. After cooling, the decomposed sample was diluted with
deionized water and then metal impurities were analyzed by ICP-MS.
The elements studied include Co, Cu, Cr, Ga, Fe, Mg, Mn, Na, Si, and Zn. The limit of Quantification（LOQ）for each element in
ICP-MS was presented.
For the method validation, the metal impurities in the certified reference material of alumina（JCRM R 031, 032, 033）were analyzed
by the same method described in this paper and compared with certified values.

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 40: Environment II

PTh-110

13:30 － 14:40

High-Speed Survey Method for Photo-degradation Products ofPharmaceuticals Using UV-LED Lighting Device and DART-TOF Mass
Spectrometer

Toshio Tashima1, Kaori Asano2, Takeyuki Suzuki2, Tsuyoshi Matsuzaki2, Takeshi Nakano3
1
3

GeneStem Co. Ltd., Osaka, Japan, 2The Institute of Scientific and Industrial Research, Osaka University, Osaka, Japan,
Research Center for Environmental Preservation, Osaka University, Osaka, Japan

Keywords:
PPCPs, Photo-degradation, DART-MS, UV-LED, Pharmaceuticals
Novel aspects:
This highest-speed and simplest method will become extremely useful to survey photodegradation products of pharmaceuticals
and personal care products（PPCPs）in environment and drug-development research fields.
Abstract:
Over the past decade, attention as organic pollutants in aquatic environments has been gradually increasing to pharmaceuticals
and personal care products（PPCPs）. A large amount of pharmaceuticals have been released continuously into aquatic
environments for more than a century. Thus, there have been many reports about the occurrence of pharmaceutical products inriver water, seawater, and wastewater.
GC-MS and LC-MS or LC-MS/MS are used as the most common techniques for determination of pharmaceuticals in aquatic
environments. The selected ion monitoring（SIM）, the selected reaction monitoring（SRM）and the multiple reaction monitoring（MRM）methods are utilized as the technique for determination of pharmaceuticals in aquatic environment. When thesetechniques are used for determination of pharmaceuticals, it is required to obtain the information about the specific ions of target
pharmaceuticals prior to starting analyses of them. This indicates that these convenient techniques become useless entirely indetermination of unknown compounds.
Photo-degradation is suggested to play an important role in elimination of some of PPCPs from surface waters. In fact, the fate ofpharmaceuticals has been recognized by solar and ultraviolet irradiation to them. However, there seems to be also a few reportson the determination of photo-degradation products of pharmaceuticals in aquatic environments in spite of taking a large varietyof pharmaceuticals.
Most of investigators have performed the photo-degradation studies by sunlight and/or UV radiation using an appropriately
devices equipped with a mercury lamp（Hg-vapor lamp）or a xenon lamp（Xe lamp）. The radiation intensity is ca. 300 W/m2（daily
average）in the case of solar light1) and 400 800 W/m2 in the cases of Hg-vapor lamp2) or Xe lamp3) which consume a relatively longradiation time（about 10 hours）to obtain the significant fate of pharmaceuticals. In addition, these radiation conditions require
some cooling system to prevent heat affection. If the photo-degradation study can be performed faster and easier, it will become
to assess the photo-degradation products readily. Therefore, we developed the very simple and fast method using a UV-LED
lighting device and a DART-TOF mass spectrometer4).
A small size UV-LED lighting device has been available recently for UV cure processing on pinpoint. At present time, there areseveral devices giving ultra violet light（ λmax： 365 nm）and high power radiation intensity（to 9500 mW/cm2 ）with no heataffection. We used the cheap model with radiation intensity of ca. 4000 mW/cm 2（40000 W/m 2 ）and beam diameter of 4 mm
（ULEDN-101, NS-Lighting）. After preparation of sample solution using methanol and/or acetonitrile（ca. 5 mg/ml）, a portion ofseveral micro-L was applied on the reverse-phase type of TLC plate（RP- 2 F254, Merck）using a glass capillary tube and UV light
was irradiated for appropriate time（ within 120 sec）on the sample spot（diameter： ca. 3 mm）. DART-Mass spectrometry wasutilized to detect the photo-degradation products on the TLC plate, because the DART ionization provides direct analysis underatmospheric pressure and few fragmentation of molecular ion. In this study, we used DART-SVP100（IonSence）for ionization ofsamples and Accu-TOF mass spectrometer（JMS-T100LC, JEOL）for acquisition of mass spectral data.
In this conference, we will present our high advantage method for exploring photo-degradation products of pharmaceuticals byshowing the results in the cases of Ibuprofen, Naproxen, Ketoprofen, Indomethacin and Diclofenac which are generally used as anon-steroidal anti-inflammatory drugs（NSAID）.
Reference
1 ）V. Matamoros, et.al., Water Air Soil Pollut., 196, 161-168（2009）2 ）J. L. Packer, et.al., Aquat. Sci., 65, 342-351（2003）
3 ）M. W. Lam, S. A. Mabury, Aquat. Sci., 67, 177-188（2005）4 ）T. Tashima, et.al., The Abstract of 59th Annual Conference on Mass Spectrom. JPN., 1 P-43, 75（2011）

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 40: Environment II

PTh-111

11:10 － 12:20

Hydrophilic-interaction liquid chromatography (HILIC)-tandem mass
spectrometry for quantification of oseltamivir and zanamivir in surface
water and sediment samples

Ryohei Takanami, Hiroaki Ozaki, Rabindra Raj Giri, Shogo Taniguchi, Shintaro Hayashi
Osaka Sangyo University, Osaka, Japan

Keywords:
Influenza, oseltamivir, zanamivir, river-water, sedimentNovel aspects:
Novel SPE and HILIC-MS/MS methods are developed for extraction and quantitation of oseltamivir and zanamivir simultaneously inenvironmental waters. Oseltamivir is detected in sediment samples for the first time.
Abstract:
Oseltamivir and zanamivir are the most prescribed antiviral drugs against influenza in Japan. Oseltamivir and its metabolite havebeen frequently detected in surface water bodies in Japan during the last few years particularly in seasonal influenza period
posing the threat of emergence of drug-resistant genes in human pathogens. Although zanamivir is placed in the third position interms of its use as an antiviral drug in Japan, its occurrence and fate in environmental waters are not known until now. Thissituation may be partially attributed to the lack of a suitable sample treatment and analysis methods for the drug. Therefore,
suitable and sensitive sample pretreatment and analysis methods are highly desirable for quantitation of the drug in environmental
waters. Furthermore, optimized pretreatment and analysis methods for both oseltamivir and zanamivir would be the best choicefor their precise and rapid quantitation in water samples.
This article discusses the occurrence of oseltamivir phosphate（OP）, oseltamivir carboxylate（OC）and zanamivir hydrate（ZH）inwater and sediment samples from Neya River in Osaka using a recently developed novel solid-phase extraction（SPE）method for
simultaneous recovery of the compounds and hydrophilic-interaction liquid chromatography（HILIC）-tandem mass spectrometry（MS/MS）method for quantitation. Water and top sediment samples at three representative points（ST-1, ST- 2 and ST- 3 ）from
the top towards the mouth along Neya River were taken during the 2011/2012 seasonal influenza period. The ST- 1 is characterized
by very small river flow and relatively bigger size sand particles in sediment. The ST- 2 is characterized by drastically large river
flow due to wastewater treatment plant discharge in the upstream side and medium sand particles in sediment. The ST- 3 ischaracterized by very large river flow and silt particles in sediment.
The OP, OC and ZH were not found in both water and sediment at ST-1. They were present in water, but not in sediment at ST-2.
The scenario was completely different for ST-3. The three compounds were found in water samples, while OP only was present in
the sediment at ST-3. Thus, the compounds were found in abundance in water phase, while OP alone was detected in thesediment from Neya River. Batch adsorption test results for the compounds in ultrapure water with river sediment showed thehighest adsorbed fraction（24%）for OP, while the values for OC and ZH were 3 % and 1 % respectively. Moreover, the fractions inwater phase for OC and ZH were about 97% and 93% respectively. The value for OP was only 6 %. These results clearly indicatedthe highest adsorption affinity of OP to sediment, while the affinities for OC and ZH were negligibly small. It may be evident fromthese results that OP is more likely to be adsorbed to sediments than OC and ZH.
A newly developed SPE method for simultaneous recovery of oseltamivir and zanamivir together with a hydrophilic-interaction
liquid chromatography（HILIC）-tandem mass spectrometry method were employed for accurate quantitation of the drugs in riverwater and sediment samples. The results demonstrated abundance of OP and OC in river water during 2011/2012 seasonal
influenza period, while OP only exhibited high potential for adsorption to sediment. It became evident that monitoring the drugs,
particularly OP, in water phase alone is not enough in assessing the threat of emergence of drug-resistant genes in humanpathogens.

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 40: Environment II

PTh-112

13:30 － 14:40

Determination of chlorinated/brominated polycyclic aromatichydrocarbons (Cl/BrPAHs) in flue gas and ash from waste incinerator

Liang Tang1, Yuichi Miyake1, Yuichi Horii2, Kiyoshi Nojiri2, Nobutoshi Ohtsuka2, Takashi Amagai1
1

University of Shizuoka, Shizuoka, Japan, 2Center for Environmental Science in Saitama, Kazo, Japan

Keywords:
halogenated PAHs, organohalogen compound, by-products
Novel aspects:
Gas chromatography coupled to high-resolution time-of-flight mass spectrometry （GC-HRTOF-MS） was applied for thecomprehensive analysis of ClPAHs and BrPAHs congeners including unidentified congeners.
Abstract:
Chlorinated polycyclic aromatic hydrocarbons（ClPAHs）such as chlorobenz［a］anthracene（ClBaA）and chlorobenzo［a］pyrene（ClBaP）have received worldwide attention because of their environmental persistence and widespread distribution. Horii et al.
have showed that several ClPAHs and BrPAHs elicit dioxin-like activity with potencies comparable to those of several mono-ortho
polychlorinated biphenyl（PCB）congeners. Recent reports have showed the occurrence of ClPAHs and BrPAHs in flue gas and fly
ash from municipal and industrial waste incinerators. However, little is known about congener profiles and distributions of ClPAHs
and BrPAHs in flue gas, fly ash, and bottom ash from waste incinerators, due to the lack of individual analytical standards. Several
tens of individual ClPAHs and BrPAHs were therefore synthesized in our laboratory. In this study, we measured individual
concentrations of 26 ClPAHs and 15 BrPAHs in flue gas, fly ash, and bottom ash from 8 waste incinerators. In addition, gas
chromatography coupled to high-resolution time-of-flight mass spectrometry（GC-HRTOF-MS）was applied for the comprehensive
analysis of ClPAHs and BrPAHs congeners including unidentified congeners. Results showed that monochloropyrene（ 1 -ClPyr）and
6 -ClBaP were the dominant compounds in flue gas samples. The profiles of halogenated PAHs were similar to the profiles
reported previously for urban air. Concentrations of chlorinated phenanthren and pyrene in flue gas were significantly correlatedwith the corresponding parent PAH concentrations. Significant correlation between ΣClPAH and ΣPAH concentrations suggeststhat direct chlorination of parent PAHs is the mechanism of formation of ClPAHs during incineration of wastes. Furthermore, highly
chlorinated PAHs and brominated/chlorinated PAHs were found in samples by the comprehensive analysis using GC-HRTOF-MS.

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 40: Environment II

PTh-113

11:10 － 12:20

SELECTIVE EXTRACTION OF ORGANOHALOGENS FROM GCxGC-HRTofMS
DATA FOR GLOBAL ANALYSIS OF ENVIRONMENTAL AND BIOLOGICALSAMPLESShunji Hashimoto, Yasuyuki Zushi, Akihiro Fushimi, Yoshikatsu Takazawa, Kiyoshi Tanabe,
Yasuyuki Shibata
National Institute for Environmental Studies, Tsukuba, Japan

Keywords:
direct-sample-introduction, thermal-desorption, exact-mass, POPs, unknown-chemicals
Novel aspects:
It is new to extract compounds on demand from a complehensive GCxGC-HRTofMS data that includes huge number of chemicals
whiout pre-setting targets.
Abstract:
There are various chemicals around us. Some of them cause problems such as environmental pollution or have adverse effects onorganisms. As the number of undesired chemicals grows, they become increasingly expensive in terms of time and cost to monitor
and count. This can be partly attributed to problems with analytical methods, which require a lot of time and resources, andwhere a skilled user is needed to measure trace levels of compounds precisely in the presence of a large amount of interference,and because the methods are individually optimized for each target substance. We are developing and improving methods andtools to overcome the problems related to the techniques used for analyzing organic environmental pollutants. We are currently
developing a new apparatus consisting of a comprehensive two-dimensional gas chromatograph（GC×GC）, which is a highperformance technique for the separation of chemical compounds, directly coupled with a quadrapole type tandem mass
spectrometer（MS/MS）and a high resolution time-of-flight mass spectrometer（HRTofMS）. We have shown that halogenatedcompounds can be detected comprehensively and selectively in environmental samples by employing a neutral loss scan（NLS）
on a GC×GC-MS/MS. However, the sensitivity of the NLS on this instrument was insufficient to detect trace levels oforganohalogens and incapable of identifying compounds due to their unit-mass records.
Here, we report our attempts at the selective extraction of a subset from the GC×GC-HRTofMS data in order to detect andidentify trace levels of organohalogens. The data was obtained by measuring several environmental and biological samples,
namely flyash, soil, sediment, the atmosphere and human urine. For global analysis, some samples were measured without any
purification using a 6890GC（Agilent Technologies）with a KT2004 GC×GC system（Zoex）coupled with a JMS-T100GC（JEOL）
HRTofMS. A column pair consisting of 5 % phenyl/phenyl-methyl silicone and 50% phenyl/phenyl-methyl silicone was employed as
the liquid phase of the GC capillary columns for measurements with the GC×GC system.
Prior to data analysis, 5 -15 gigabytes（GB）of raw data from the HRTofMS were converted into 0.5-2.5 GB of data in the netCDF（AIA）format. We attempted to extract only the mass spectra of organochlorines or organobromines using the several methods
that we developed.
As a result, after data analysis under various mass resolution conditions, we achieved the selective extraction of the mass spectraof chlorinated or brominated compounds under a high mass resolution condition that exceeded approximately 10,000 for somesamples. In contrast, low mass resolution below 1,000 was unable to extract the organohalogen data effectively. The results of thisstudy show that data obtained with a high-resolution time-of-flight mass spectrometer are valuable for the global analysis oforganohalogens, and probably of other compounds if specific data extraction methods can be devised. However, to improve thedata extraction performance the deconvolution of peaks and mass spectra is required because many compounds are co-eluted
even when using a GC×GC depending on the sample matrix.

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 40: Environment II

PTh-114

13:30 － 14:40

Electrospray mass spectrometric observation of the interaction between
environmental pollutants and biologic compounds

Hiroshi Moriwaki, Takahiro Nakagawa
Shinshu University

Keywords:
electrospray ionization； interaction； zinc pyrithione
Novel aspects:
Solutions containing amino acids and environmental pollutants were measured by ESI-MS for the purpose of gaining informationon the behavior of the pollutants with amino acids and peptides.
Abstract:
Abstract
Interactions between biologic compounds and environment pollutants often cause various toxicities, such as defective
development. Therefore, it is very important to understand the interactions.
Electrospray ionization mass spectrometry（ESI-MS）is a very powerful method for the characterization and identification ofinteractions between polar species, because the technique is a very soft ionization method. The technique has been frequently
used for the analysis of the interactions between biological materials and chemicals（H. Moriwaki, 2002）or metal ions（H.
Moriwaki, 2003）.
However, it is very difficult to research the interaction between proteins and substances by ESI-MS, because there are many
complex binding sites and forms between proteins and substances. There are several examples where an investigation of asolution of a target substance with amino acids by ESI-MS was studied in order to initially serve as a simple model for complex
interactions of the substance in proteins（C. L. Gatlin and F. Tureek, 2000）.
In this presentation, solutions containing amino acids and environmental pollutants were measured by ESI-MS for the purpose ofgaining information on the behavior of the pollutants with amino acids and peptides. Zinc pyrithione（ZnPT）and toxic metal ionswere selected as targets of this study（H. Moriwaki et al., 2009）.
Zinc pyrithione（ZnPT）is the zinc chelate of 2 -pyridinethiol- 1 -oxide. This compound is widely used as a bactericide, fungicideand algicide in various products, such as antidandruff shampoos. A few studies have examined the toxicity of ZnPT on aquaticorganisms, and it has been clarified that ZnPT can be potentially highly toxic（K. Goka, 1999）. Therefore, it is important tounderstand the influence of the compound on wild life and the mechanisms of the toxicity of ZnPT in order to determine theappropriate use of ZnPT. The ZnPT complex（［ZnPT-ligand + Amino acid］+）, in which the ligand of ZnPT was exchanged by theamino acid, was detected in ZnPT solutions mixed with one of 20 amino acids by ESI-MS. Histidine and cysteine, in particular,
showed a high reactivity with ZnPT, while serine and glycine showed a low reactivity.
In addition, electrospray mass spectrometric observation of the interaction of toxic metal ions with amino acids has been alsostudied in the same manner as ZnPT.
The procedure described in this study is very simple and suggested only a simple model for the reaction of biological materials
with the pollutants. However, it provides significant information for estimating the behavior of the pollutants within the living body.

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 40: Environment II

PTh-115

11:10 － 12:20

Characterization of organic pollutants in River Water by GC/MS ion
profilesZaharie Moldovan1, Olivian Marincas1, Veronica Avram1, Florina Tusa1, Alfredo C Alder2

National Institute for Research and Development of Isotopic and Molecular Technology, Cluj-Napoca, Romania, 2Swiss
Federal Institute of Aquatic Science and Technology, EAWAG, Environmental Chemistry Department, Dbendorf,
Switzerland

1

Keywords:
River pollutants, ion profiles, GC/MS
Novel aspects:
The families of compounds are shown by ion chromatograms obtained on characteristic ions. Their origin is discussed incorrelation with the compounds known as molecular markers.
Abstract:
Background. The specific human activities lead to contamination of the aquatic environment with a wide variety of natural andsynthetic compounds not found prior to modern times. Due to an incomplete elimination in wastewater treatment plant（WWTP）
residue of contaminants are found both in waste and surface waters.    Many of these compounds provide a means of identification
sources of inputs and pathways of movement of chemicals through ecosystem.
In respect to this, one of priority tasks is to determine the environmental distribution of organic pollutants in the surface waters.
Both anthropogenic and naturally occurring compounds are found mixed together in recent environmental samples and several ofthese compounds may be used as tracers to study natural processes affecting the fate and effects of chemical contaminants inwater.
Objective. In the south-eastern of Europe there is little information on river water status1. The existent data refers at some metal
trace in very critical sites, general quality studies by analyzing the benthic macrofauna but information on anthropogenic organic
pollutants as individual compounds is limited to only few emerging contaminants2. Therefore a systematic structural investigationof individual molecular pollutant present in environmental water at regional level is crucial.
The present paper purpose is the Mass Spectrometric（MS）characterization of organic pollutants to evaluate the degree ofcontamination of the river waters in high populated catchments from South-Eastern of Europe. The samples were collected fromfew places along of the Prut River, Border River between Romania and Republic of Moldova. In the Prut Basin live more than 1000
000 inhabitants.
Methods. The samples were collected by three techniques： a）grab sample, b）using composite sample devices and c）bypassive samples devices.The GC/MS analyses were performed using a Thermo Electron Polaris Q mass spectrometer operated in
EI mode to 70 eV. The gas chromatograph was equipped with a capillary column HP- 5 MS（30x0.25mm）with 0.25film thickness. The
temperature was programmed from 900C to 3150C.
Results. The families of pollutants was visualised by chromatograms on diagnostic ions（base ion or ion of high intensity andmolecular ion）3. A systematic study of individual compound as structure and quantity in region of border Romania-Moldavia wasdone based on experimental data on a big number of samples. Also was established the environmental molecular markers as
identification of source pollution. The molecular marker compounds will be used in the subsequent monitoring activities to obtainthe complete image of the specific pollutantsin the Prut Basin in different seasons. An number of 67 of compounds were identified.
The quantity of pollutants was calculated using isotopic labeled compounds. The structure and quantity of detected compounds
are discussed in relation to sampling methods and collection places. The obtained profiles of pollutants are a function of theirsources and reflect the industrial and domestic activities at regional level.
References：［ 1 ］Loos R. et al. EU-wide survey of polar organic persistent pollutants in European river waters. Environmental Pollution. 157, 561568（2009）［ 2 ］Moldovan Z. et al. Environmental exposure of pharmaceuticals and musk fragrances in Somes River before and after
upgrading the municipal wastewater treatment plant Cluj-Napoca, Environ. Sci. Pollut. Res. 16, S46-S54,（2009）［ 3 ］McLafferty F. W., Turecek F., Interpretation of Mass Spectra, Universities Science Books, Mill Valley, CA, 1993

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 40: Environment II

PTh-116

13:30 － 14:40

A Sensitive measurement of sucralose and acesulfame in inland andoffshore waters around Japan by LC/MS and LC/MS/MS.

Shigeru Suzuki1, Atsuko Hasegawa2, Rina HIguchi1, Takahiro Yamaguchi1, Hisato Nishikawa1,
Takuhei Miyaki1, Chiaki Oshima1, Ryosuke Otsubo1
1

Chubu University,Aichi,Japan, 2Kanagawa Environmental Research Center, Kanagawa Japan

Keywords:
sucralose, acesulfame K, environmental water
Novel aspects:
comprehensive research of sucralose and acesulfame K in environmental waters in and around Japan island
Abstract:
Introduction： Sucralose and acesulfame K are widely used artificial sweeteners, which are not likely to undergo a chemicaltransformation through not only natural processes but also biological reactors. Because of increasing consumption of sucralose
and acesulfame K, the environmental behavior and the fate of the persistent sweeteners have been a concern. But the data aremostly limited to Europe, in particular northern Europe, and few other areas. Here we report the distribution of the artificial
sweeteners in water in and around Japan islands.
Method： River water samples were collected in areas around Lake Biwa and Lake Suwa. Seawater samples between northern
Japan and Sakhalin were collected by auto sampling device equipped in a cursing ship. Sample water was prepared around pH 3
for collecting sucralose（log Kow 0.78）by solid phase extraction（SPE）. For collecting acesulfame（log Kow -2.3）by SPE,
dihexylammoniumacetate（DHAA）was added to the water sample neutralized with ammonium hydroxide. The river water
samples were measured by LC/MS using dihexylammoniumacetate as an ion pair reagent in HPLC, and seawater were measured
by LC/MS/MS with triethylamine in mobile phase.
Results： Recovery efficiencies of sucralose and acesulfame from river water fortified with the sweeteners（every 5 ng/mL）were
108% on average（RSD 2.4%）and 109%（RSD 6.8%）, respectively. Those of seawater fortified with the sweeteners（every 0.1 ng/mL）were 103% on average（RSD 11%）for sucralose and 91%（RSD 5.1%）for acesulfame.
River waters were sampled around Lake Biwa and Lake Suwa. The concentrations of sucralose and acesulfame in the 25 rivers
around Lake Biwa were 1 - 960 ng/L（120 ng/L on average）and 18 - 1500 ng/L（220 ng/L on average）, respectively. The river ofwhich the sweeteners’ concentrations were higher were tended to be higher in the biochemical pollution indexes such as TOC,
BOD, COD, total-N, total-P. The concentrations of sucralose and acesulfame in the 5 rivers around Lake Suwa were 0.1 0.59 ng/L
（2.4 ng/L on average）and 2.0 3.1 ng/L（0.29 ng/L on average）.
Seawater was sampled at 30 sites with GPS data, time and general water qualities. The concentrations of sucralose andacesulfame were 0.2 to 42 ng/L（3.0 ng/L on average）and 0.6 to 69 ng/L（9.0 ng/L on average）, respectively. The concentrations inseawater were almost one hundredth of those in river waters. Concentration ratios of sucralose to acesulfame in seawater werevaried from 0.01： 1 to 11：1, which were almost similar to those in river waters（from 0.01： 1 to13： 1 ）. It is suggested that theconcentrations between the two sweeteners had almost no relation to each other both in sea water and river water, and the
behaviors of the two sweeteners might be different in environmental waters.
Acknowledgements： This research was kindly supported by the grant from the Sumitomo Foundation. The seawater samples
were collected by Dr. Kunugi of Tokyo University of Science, highly sensitive LC-MS-MSLC/MS/MS measurements were taken by
Mr. Takeda of AB Sciex Japan. The authors sincerely appreciate their contributions.

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 40: Environment II

PTh-117

11:10 － 12:20

Real-time ambient air monitoring using selected ion flow tube-mass
spectrometry (SIFT-MS)

Vaughan S Langford1, Murray J McEwan1,2
1

Syft Technologies Limited, Christchurch, New Zealand, 2University of Canterbury, Christchurch, New Zealand

Keywords:
SIFT-MS Air-Analysis Pollutants VOC Real-time
Novel aspects:
Real-time monitoring（without sample preparation）for volatile organic compounds in the vicinity of an elementary school over an
extended period.
Abstract:
Selected Ion Flow Tube Mass Spectrometry（SIFT-MS）is a real-time analytical technique that detects volatile organic compounds
and certain inorganic gases down to part-per-trillion levels with no sample preparation. These characteristics mean that SIFT-MS
can easily be applied to real-time detection of volatile organic air pollutants. This paper presents results from a air monitoring
undertaken at Shu-Lin Elementary School in Taoyuan County, near Taipei, Taiwan R.O.C. from 19 to 21 July 2011.
SIFT-MS uses chemical ionization reactions coupled with mass spectrometric detection to rapidly quantify targeted VOCs. VOCs
are identified and quantified in real time from whole-gas samples based on the known rate coefficients for reaction of thechemically ionizing species（so-called reagent ions）with the target analytes. The soft chemical ionization used in SIFT-MS yields asmaller range of product ions than is common in electron impact mass spectrometry（as used by gas chromatography mass
spectrometry（GC-MS）, for example）. Hence the need for gas chromatographic separation of the sample is circumvented,
speeding sample throughput and providing instantaneous quantitation of VOCs. Use of several rapidly switchable reagent ions to
independently quantify target analytes also greatly reduces interferences, markedly increasing the specificity of SIFT-MS versus
other whole-gas analysis technologies.
The high-speed analysis provided by SIFT-MS allows the instrument to provide continuous monitoring. Concentration data wereextracted from full scan data for 48 compounds, many of which had been previously identified at the site using GC-MS. A numberof these compounds exhibited interesting trends over the sampling period, including toluene, C3-alkylbenzenes（e.g. mesitylene）,
methanol, isopropyl alcohol, acetone and N,N-dimethylmethanamide. Measured concentrations range from sub-ppb to peak levels
for methanol of nearly 250 ppb.

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 40: Environment II

PTh-118

13:30 － 14:40

Development of UPLC-MS/MS method with large volume injection forsimultaneous determination of regulated pesticides in drinking water.

Jun Yonekubo, Nobutake Sato
Nihon Waters K.K.,Japan

Keywords:
LC/MS/MS,pesticides,drinking water
Novel aspects:
Direct injection of large volume samples onto this system eliminated the need for sample preparation prior to analysis.This
methods achieved good sensitivity,linearity,reproducibility for all targeted pesticides in regulation purpose.
Abstract:
Introduction
Rapid and highly sensitive analysis of drinking water is essential for protecting human health and well-being. The assurance ofclean safe drinking water has become more critical given the potential of accidental or international contamination, which has
increased in recent years.
Highly efficient water treatment processes allow for the effective removal of the majority pesticides that have entered water
sources, however, drinking water regulations still require testing to ultra-trace concentrations.
This requirement has led to multiple approaches for enriching samples before instrumental analysis, with solid phase extraction（
SPE ）prior to LC/MS/MS a popular choice. In addition to this online pre-concentration and large volume injection, using
specialized injection systems have been employed to introduce samples to LC/MS/MS systems. These techniques have been bevery successful, but can add time, resources, and complexity to analysis.
Cleaner aqueous samples, such as drinking water are highly compatible for direct injection onto a LC/MS/MS system, but large
multi-analyte determinations require extremely fast systems with high-sensitive detection.
In this study, 30 pesticides which was regulated as Complementary Items by Ministry of Health, Labour and Welfare（ MHLW ）in
Japan, is targeted. The use of large volume direct injection for the rapid, precise, and high-sensitive analysis of these 30 pesticides
in drinking water is developed.
Experimental
UPLC conditions
LC System： ACQUITY UPLC H-class, Run time： 10 min.
Column： ACQUITY UPLC HSS C18, 2.1 x 100 mm, 1.8 um
Mobile phase： 0.05 % HCOOH aq. / CH3OH gradient
Flow rate： 0.4 mL/min. Injection volume： 50 uL
MS conditions
MS System： Xevo TQD
Ionization mode： positive/negative switching
Capillary voltage： 0.5 kV, Source temp.： 150 &#8451；
Desolvation gas/temp.： 1000 L/Hr（ 400&#8451； ）, Acquisition mode： SRM
Results
Sample throughput
Rapid this system separations allowed a high-throughput analysis with all pesticides of interest eluting before 6.5 min and total
runtime of 10 min for each sample.
This system was operated with load-ahead enabled. This allows for the next sample to be ready to inject immediately after theprevious sample has completed, which helps optimize instrument efficiency.
High-sensitive detection
Detection of pesticides to lower concentrations was achieved using large volume injection into Xevo TQD. Up to 50 uL injection,
there are not observed any deterioration of peak shape and separation. Amount of on-column pesticides with large volume（50 uL）injection was 10 times higher than normal（ 5 uL ）injection. Improvement of sensitivity which was performed to increase of
injection volume is able to a few or several ppt level detection.

This level of sensitivity allows detection of pesticides to 1 /100 of a desired value and is match for requirement of the regulation.
Linearity and Reproducibility
External calibration（ point ）of target pesticides was performed at concentrations around the regulatory level for each pesticides.
Good linearity was achieved for all compounds analyzed with typical coefficient of determinations（ r2 ）of > 0.99.
According for the procedure of testing pesticides in drinking water, Reproducibility of this method was verified at 4 or 20 ppt
concentrations. MHLW testing procedure mentioned in the method accuracy, Coefficient of variation（ CV ）at 1 /100 of eachpesticides desired value, It should be 20% or less in principle. % RSD of all compounds were within permissible range.
Conclusion
Direct injection of large volume samples onto this system eliminated the need for sample preparation prior to analysis.
This methods achieved good sensitivity and linearity, reproducibility for all targeted pesticides in regulation purpose.

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 40: Environment II

PTh-119

11:10 － 12:20

Development of Automated Identification and Quantification System witha Database

Kiwao Kadokami1, Terumi Miyazaki5, Chiaki Karaki1, Daisuke Jinya2, Tomomi Iwamura2, Kaori Ohkubo4,
Takashi Miyawaki3, Yoko Nakazono4, Kouji Takahashi3

The University of Kitakyushu, Kitakyushu, Japan, 2Kitakyushu City Institute of Environmental Sciences, 3Fukuoka
Institute of Health and Environmental Sciences, 4Saga Prefectural Institute of Public Health and Pharmaceutical Research,
5
Nippon Steel Kankyo Engineering
1

Keywords:
GC-MS, LC-TOF-MS, TIM, Comprehensive analysis
Novel aspects:
Novel GC-MS and LC-TOF-MS databases that can identify and quantify micropollutants in environmental and food samples without
the use of standards are reported.
Abstract:
To thoroughly examine environmental pollution by chemicals, a large number of chemicals should be analyzed. However, because
it is known that thousands of chemicals exist in the environment, it is very difficult to analyze all of toxic chemicals simultaneously
because of the huge cost and the large amount of labor needed using existing methods. In order to solve this challenge, we havedeveloped two Automated Identification and Quantification System with a Database（AIQS-DB）for GC-MS and LC-TOF-MS, which
can measure a large number of chemicals without the use of standards.
In the AIQS-DB for GC-MS, GC retention times, calibration curves, and mass spectra of nearly 1000 semi-volatile organic
compounds were registered, and the GC retention times of registered chemicals in actual samples were predicted from theretention times of n-alkanes measured before sample analysis. Differences between predicted and actual retention times wereless than 3 s, an accuracy that is nearly identical to that obtained by analysis of standard substances. The reproducibility ofquantification was examined on four GC-MS systems in four laboratories using 114 substances with a wide range ofphysicochemical properties and which are usually difficult to measure with a GC.
When the performance of a GC-MS passed the designated criteria, the reproducibility of quantification results of 47 substances
was almost the same as that obtained by a conventional method that uses calibration curves prepared before sample analysis.
Although the reproducibility of 42 substances was slightly lower than that by the conventional method, the reproducibility isreliable enough except for in cases that require a high reliability. However, the dispersion of results of 25 substances was large
under the GC conditions used. The causes that affect the dispersion of quantification results are functional groups and thestructure of molecules. The effects of the functional groups are hydroxyl amino > nitro group. The effects of the number of them
are multiple > single. The position of them can also have different effects： farther > nearer. In the case of the structure ofmolecules, the effects are as follows： side chain > straight chain > single benzene > multiple benzenes. In terms of sensitivity,
more than 90% of the chemicals in the AIQS-DB could be detected at a sensitivity sufficient for all practical purposes（100 pg orless）.
The AIQS-DB for LC-TOF-MS has been constructed according to the same concept as the GC-MS method. In order to measure asa large number of chemicals as possible, the combination of an ODS column and acetate buffer-methanol eluent（pH 6.8）and
positive ionization were used. Retention times of chemicals in the AIQS-DB were predicted from relative retention times of internal
standards. Differences between predicted and actual retention times were within +/- 30s as long as LC conditions were constant.
From correct prediction of retention times and high-resolution mass spectra obtained by TOF-MS, identification of chemicals canbe consistently performed. Quantification values are also constant as long as LC conditions are constant； reproducibility（RSD）
was less than 20%, which is slightly worse than the conventional method that calibration curves are prepared just before sample
analysis. However, the value of simultaneous measurement of a large number of chemicals compensates for lower accuracy.
Because each chemical in both AIQS-DB systems can be determined in 1 h, hydrophilic and hydrophobic micropollutants insamples can be measured efficiently and inexpensively by using both the AIQS-DB systems, and to which new substances caneasily be added.

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 40: Environment II

PTh-120

13:30 － 14:40

Metal speciations in environmental and clinical applications

Yeuk-Ki Tsoi, Kelvin Sze-Yin Leung

Hong Kong Baptist University, Hong Kong

Keywords:
elemental-speciations, LC-ICP-MS, IIP, SPME, DRC
Novel aspects:
high-throughput hyphenated instrumentation； SPME-LC for speciation, modified material for sample preconcentration andsimultaneous speciation； selectivity modulation using ion-imprinting technique
Abstract:
In the vast subject of trace analysis, evolutionary instrumentation and material developments are the current trends to re-inventthe definitions of sensitivity and selectivity in the field of elemental speciations, which in fact has become an indispensible tool forenvironmental management and clinical monitoring. In the world’s raising knowledge and demands for the quality of humanhealth, the strategies of method development is conforming to the stringent requirements of analytical standards. A diversity of
research work in this presentation aims to facilitate convenient execution of speciation protocols in routine monitoring processes.
The novelty is exhibited at four levels, featuring high-throughput instrumental methods, simplified extraction with commercial
tools, modified material for specialized applications and modulation of selective material with ion-imprinting technique.
In the first of our presented work, seven species of these elements, namely Cr（III）, Cr（VI）, As（III）, As（V）, monomethylated
As, Se（IV） and Se（VI）, were simultaneously determined by liquid chromatography-inductively coupled plasma-mass
spectrometry（LC-ICP-MS）with dynamic reaction cell（DRC）that offers optimized sensitivity at sub-ppb levels to the selected
elements. The high through-put of this method has fulfilled the hustle routine of environmental water analysis［ 1 ］.
Aiming at clinical monitoring application, the second part demonstrates a method of solid-phase microextraction-liquid
chromatography（SPME-LC）developed for MeHg and EtHg speciation in complex biological matrix［ 2 ］. Organomercury
speciation in patients’ urine has revealed source and pattern of Hg exposure which should allow rapid diagnostic whereby
appropriate clinical treatment can be applied.
The third part presents a novel laboratory-made SPE column packed with tetrabutylammonium hydroxide-immobilized activatedcarbon（AC-TBAH）for Se（IV）and Se（VI）enrichment. The assigned anion-exchange functionality on the porous AC surface
offered outstanding enrichment factors for ppt-level speciation with LC-ICP-DRC-MS［ 3 ］. Speciation analysis in natural water
samples further validated the robustness of the material which hence represents a low-cost substitute for anion exchange resins
for routine applications.
Material design has been proven very useful for modulating selectivity for trace element analysis via the synthesis of ion-imprinting
polymer（IIP）, such as one demonstrated in final part of the presentation. After a series of selectivity investigations, imidazolebased arsenic imprinted polymer was selected for selective extraction and analysis of trace As ions in environmental application［ 4 ］. Tunable selectivity through monomer selection revealed a promising advantage of imprinting technique for futurespeciation method development. With growing maturity of speciation research, routine implementation in environmental andbiological areas should make progress towards a sustainable human well-being.
［ 1 ］Y.-K. Tsoi, K. S.-Y. Leung, J. Anal. Atom. Spectrom. 25（2010）880.
［ 2 ］Y.-K. Tsoi, S. Tam, K. S.-Y. Leung, J. Anal. Atom. Spectrom. 25（2010）1785.
［ 3 ］Y.-K. Tsoi, K. S.-Y. Leung, J. Chromatogr. A 1218（2011）2160.
［ 4 ］Y.-K. Tsoi, Y.-M. Ho, K. S.-Y. Leung, Talanta 89（2012）162.

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 40: Environment II

PTh-121

11:10 － 12:20

Occurrence of 92 pharmaceuticals in river water in agricultural or urban
areas

Koya Komori, Mizuhiko Minamiyama, Yutaka Suzuki
Public Works Research Institute, Tsukuba, Japan

Keywords:
Pharmaceuticals, Sewered Area, Unsewered Area
Novel aspects:
The objective of this study is to determine the occurrence of pharmaceuticals in river waters characterized by land use of theirriver basins, such as agricultural or urban areas.
Abstract:
In recent years, physiologically active substances（e.g., pharmaceuticals）detected in the water environment have become an
emerging public concern. Human-use pharmaceuticals enter raw sewage via urine and feces or by improper disposal. These
pharmaceuticals are discharged from private households and hospitals, and eventually reach wastewater treatment plants. Ifpharmaceuticals are only partially eliminated, residual quantities enter the water environment. However, little information isavailable about the occurrence of pharmaceuticals in river water. The objective of this study is to determine the occurrence ofpharmaceuticals in river waters characterized by land use of their river basins, such as agricultural or urban areas.
We conducted sampling on six fine days at two stations of a river in an agricultural area（St.1 and St.2）and two stations at a riverin an urban area（St.3 and St.4）. The catchment areas of each sampling site are 4.6km2（St.1）, 4.1km2（St.2）, 3.9km2（St.3）and 5.4km2（St.4）. About 90 % of land utilization of the catchment area of St.1 and St.2 consists of rice field, dry field, artificial forest, andbroadleaf forest. Land utilization of the catchment area of St.3 and St.4 consists of a residential area（38% and 36%）, school zone,
open space, dry field, native grassland and artificial forest（54% and 56%）, respectively. The catchment area of St.1 and St.2 is an
unsewered area, and St.3 and St.4 are partly sewered areas. The percent of sewered population of the catchment area of St.3 andSt.4 is 74% and 31%, respectively. The populations of each sampling site are 1,062 persons, 407 persons, 25,71 persons and 28,171
persons for St.1, St.2, St.3 and St.4, respectively. The population densities of the river basins are 231 persons/km2 of St.1, 99 persons/
km2 of St.2, 6,594 persons/km2 of St.3 and 4,661 persons/km2 of St.4.
The analytical method of pharmaceuticals for river water samples is as follows： first, a 200ml sample was filtered through a 0.7-μm pore size glass fiber filter. After filtration of the sample, solid phase extraction was performed using an Oasis HLB cartridge.
Subsequently, the cartridge was eluted with methanol. After solvent removal, the residue was dissolved in acetonitrile/water
solution, which was then analyzed by LC/MS/MS. The procedure demonstrated in this study is innovative in terms of simultaneous
analysis of 92 substances.
We identified a total of 48 substances（e.g., anti-inflammatory drugs, anti-epileptic drugs, antihypertensives, antibiotics, etc.）inriver waters in agricultural and urban areas. The observed average concentration of pharmaceuticals ranged from 0.6ng/L to 14ng/
L, 0.2ng/L to 52ng/L, 0.2ng/L to 670ng/L and 0.4ng/L to 1,500ng/L for St.1, St.2, St.3 and St.4, respectively. The numbers ofpharmaceuticals detected in surveyed rivers were different. We found 15, 25, 37 and 45 for St.1, St.2, St.3 and St.4, respectively. In
agricultural areas（St.1 and St.2）, the numbers are much less than those in urban areas（St.3 and St.4）. And also, theconcentration of detected pharmaceuticals in agricultural areas（St.1 and St.2）is lower than urban areas（St.3 and St.4）and more
pharmaceuticals were detected in St.4 than in St.3, which might be caused by the insufficient and discharge of wastewater in thecatchment area of St.4. The number of detected pharmaceuticals is large when the population density is large, which is assumed
to be caused by the pharmaceuticals used in society. In agricultural areas, detected numbers of pharmaceuticals were much lessthan those in urban areas and the concentration of detected pharmaceuticals in agricultural areas was also lower than in urban
areas.

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 40: Environment II

PTh-122

13:30 － 14:40

Application of tandem mass spectrometry for the structure confirmationof a wide range of peptides synthesized by cyanobacteria Woronichinia
naegeliana

Beata Bober1,2, Zbigniew Lechowski2, Jan Bialczyk2, Piotr Suder3, Ken-ichi Harada1

1
Meijo University, Nagoya, Japan, 2Jagiellonian University, Krakow, Poland, 3University of Science and Technology,
Krakow, Poland

Keywords:
Cyanobacteria, peptides, MS/MS, structure confirmation
Novel aspects:
The structure confirmation of a wide range of bioactive peptides synthesized by cyanobacteria Woronichinia naegeliana can be
successfully performed using MS/MS.
Abstract:
Introduction
The eutrophication of water caused by human activity has influenced the increasing of mass appearance of cyanobacteria. The
cyanobacterial blooms are undesirable because of ecological as well as health reasons. Cyanobacteria have a remarkable ability
to synthesize and release into the water a wide range of bioactive secondary metabolites. Most of them are oligopeptides, whichin terms of molecular structure are divided into linear（aeruginosins, microginins）, cyclic（anabaenopeptins, cyanopeptolins,
microcystins, cyclamides）and multicyclic（microviridins）. Some of them have been recognised to cause accute or chronic toxicity
and might be reasons for serious health problems, others could be an attractive resources for new drug discoveries. The
determination of these compounds is of great significance because the cyanobacteria that produce them often occur in reservoirsof water designated for consumption. Woronichinia naegeliana（Unger）Elenkin is a species of cyanobacteria belonging toChroococcales and is appearing increasingly frequently in fresh water areas worldwide. Outbreaks, particularly in late summer
and autumn, have been confirmed in Europe, North America and Australia. However, to date its secondary metabolites have notyet been determined adequately. As a result of the potentially significant consequences that can result from a bloom of W.
naegeliana, research has been undertaken to identify its secondary metabolites. Taking into consideration limitations of applicatedanalytical methods such a high performance liquid chromatography（lack of standards）and nuclear magnetic resonance（a highamount of sample is required）, only the using tandem mass spectrometry（MS/MS）with electrospray ionization allows to confirm
structures of compounds synthesized by W. naegeliana with a high degree of specifity.
MethodField samples of Woronichinia naegeliana were collected from blooms in Dobczyce Reservoir（southern Poland）. The obtained
extract from the lyophilized cyanobacteria cells was concentrated by solid phase extraction（SPE）. Separation and preliminary
analysis of the samples were carried out using a high performance liquid chromatography system with photodiode array detector（HPLC-PDA）. The isolated fractions were analyzed with a mass spectrometer with an electrospray ion source（ESI-MS）. The
positive-ion mode was applied. The scan range was m/z 200-1400 in the MS and MS/MS modes. Because purified standards of the
majority of metabolites produced by cyanobacteria are not available for identification, confirmation of the identity of a given
compound was done by interpretation of the MS/MS spectra.
Results and Discussion
The obtained product ions were quite effective for the amino acid sequencing of linear as well as cyclic cyanopeptides. In thecase of cyclic peptides, the characteristic fragment or series of fragment indicating the cleavages at definite peptide bonds were
helpful for the interpretation of fragmentation pattern. On this base the peptides isolated from field samples of W. naegeliana fell
into four classes： microginins（microginin 757, microginin 91E, microginin FR3, microginin FR 4 ）, cyanopeptolins（cyanopeptolin B,
cyanopeptolin C, cyanopeptolin D, cyanopeptolin 880, micropeptin 478-B, micropeptin 88D）, anabaenopeptins（oscillamide B）and
microcystins（trace amounts of microcystin-LR）. The molecular masses of the determined metabolites range from 700 to 1100 Da.
The particular groups of structurally related compounds showed similarity of the fragmentations patterns. These results confirm
the usefulness of MS/MS for the determination of linear and cyclic cyanopeptides with a high structural diversity.

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 40: Environment II

PTh-123

11:10 － 12:20

A search for active ingredients in cigarette smoke that modify significant
biomolecules

Shizuyo Horiyama1, Chie Honda1, Kiyoko Suwa1, Kiyoharu Nishide1, Yuta Takahashi1, Kazuki Nakamura1,
Masaru Kunitomo1, Hirofumi Sato2, Motohiro Shizuma2, Mitsuo Takayama3
1
3

Mukogawa Women’s University, Nishinomiya, Japan, 2Osaka Municipal Technical Research Institute, Osaka, Japan,
International Graduate School of Arts and Sciences, Yokohama, Japan

Keywords:
Cigarette smoke extract, L-tyrosine, LC/MS, LC/MS/MS, GC/MS
Novel aspects:
We found out that active ingredients in CSE readily react with Tyr to form various Tyr derivatives containing a new compound,suggesting that cigarette smoke can modify biogenic amino acids.
Abstract:
Cigarette smoke contains a number of toxic chemicals, many of which contribute to the pathogenesis of smoking-related diseases
such as chronic obstructive pulmonary disease, vascular disease and cancer. Modification of biomolecules by the harmful
chemicals present in cigarette smoke is thought to mediate the adverse health effects of smoking. The chemical analysis of suchactive ingredients in cigarette smoke is one of the most challenging tasks for analysts, and extensive work has been done using
many analytical techniques. Despite such work, there have been no reports regarding the analysis of reaction products from thecompounds incigarette smoke and functional biomolecules.
The purpose of this study is to search for biomolecules modified by active ingredients in cigarette smoke. We tried to identify the
products formed by reaction of gas-phase cigarette smoke extract（CSE）, from which nicotine and tar had been removed, with
L-tyrosine（Tyr）on the assumption that Tyr is a key target amino acid in proteins for cigarette smoke toxicity. A highly sensitive
LC/MS/MS system was utilized for detection and identification of trace amounts of there action products. We also tried to identify
and quantify the active ingredients in CSE by GC/MS or LC/MS.
CSE was prepared by bubbling into phosphate-buffered saline（PBS）（ 1 mL per three cigarettes）the main stream of smoke（gasphase）from which the particulate phase including tars and nicotine had been removed 99.998% by passage through a Cambridge
filter. The pump flow rate was kept constant（ 1 L/min）and smoke was bubbled only for 1 min after lighting a cigarette. The
resulting solution was designated the 100 % CSE solution and stored at -80&#8451； until it was used. AQuattro Premier triplequadrupole LC/MS（Micromass, Manchester, UK）with an ESI source was used for the positive and negative ion mode Q 1 scanand MS/MS analysis coupled to the Alliance HT 2795 Separations Module（Waters Co., Milford, MA, U.S.A.）. Amass spectrometer（Automass SUN, JEOL Ltd., Tokyo, Japan）equipped with a GC（6890N, Agilent Technology Inc., Santa Clara, CA, U.S.A.）was used
for identify and quantify the active ingredients in CSE.
We found that CSE readily reacts with Tyr at body temperature（37&#8451；）to form various Tyr derivatives, which can bedetected using a LC/MS and LC/MS/MS. From among these derivatives, we could identify two acetylated compounds, N-acetylTyr and O-acetyl-Tyr, and a new compound, Mr251（Tyr+70）.
Next, to identify and quantify the compound of Mr 251（Tyr+70）in CSE we used GC/MS analysis. We found that two peaks
indicate Mr 70 from m/z 70 chromatogram of CSE. From the result of the library search of their mass spectra, we identified thepeak for tR 9.5 min as crotonaldehyde and the peak for tR 6.3 min as methyl vinyl ketone, as candidate ingredients. These retention
times were the same as those of their respective authentic samples. Thus, to clarify the structure of compound Tyr+70, theauthentic crotonaldehyde and methyl vinyl ketone were directly reacted with Tyr at 37&#8451；. The reaction products wereanalyzed by LC/MS in the SRM mode. The peak of compound Tyr+70 appeared on the SRM spectra of the mixed solution of Tyrand methyl vinyl ketone, but was hardly detected on that of the mixed solution of Tyr and crotonaldehyde. The analytical data ofcompound Tyr+70 synthesized by reaction of methyl vinyl ketone with Tyr completely agreed with those of compound Tyr+70
obtained by reaction of CSE with Tyr. This indicates that methyl vinyl ketone in CSE reacts with Tyr to produce compound Tyr+70.
The structure of compound Tyr+70 was identified as N-（ 3 -oxobutyl）-Tyr［ 3 -（ 4 ’-hydroxyphenyl）- 2 -（ 3 “-oxobutylamino）
propanoic acid］.

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 40: Environment II

PTh-124

13:30 － 14:40

Development of smoke diagnostic assays: When the smoke clears, will it
end up in the wine bottle?

Yoji Hayasaka, Gayle Baldock, Mango Parker, Kevin Pardon, Cory Black, Markus Herderich
The Australian Wine Research Institute

Keywords:
volatile phenols, phenolic glycosides, HPLC-MS/MS, smoke, bushfires
Novel aspects:
The newly developed methods based on the analysis of grapes and wine for phenolic glycosides induced by smoke exposure
were proven to be sensitive and reliable as smoke diagnostic assays
Abstract:
Smoke from forest/bushfires across winemaking regions in Australia as well as overseas has caused much concern among grapegrowers and winemakers. Wines made from smoke-affected grapes have been described as ’smoky’, ’dirty’, ’ashtray’ and ’burnt’.
To aid purchasing, harvesting and winemaking decisions following a smoke event, winemakers and grape-growers clearly needreliable diagnostic strategies to assess the impact of smoke exposure in grapes as early as possible, ideally before harvest.
Measurement of the smoke marker compounds guaiacol and methylguaiacol in grapes has been commonly carried out as thesmoke diagnostic assay and can identify severely smoke-affected samples. However winemakers have reported increasing
numbers of wine samples developing undesirable smoke-related characters during and after winemaking, even when grape
guaiacol concentration was as low as natural abundance. Additional challenges are that guaiacol can be found in non-smoked
grapes as a natural component, and is extracted from toasted oak, so interpretation of wine data is complicated. Theseobservations strongly suggested that the better smoke marker compounds to assess the extent of smoke exposure on grapes andwine were needed.
To find new smoke marker compounds and develop practical and reliable smoke diagnostic assays, various mass spectrometrictechniques were applied to the following experiments；· analysis of smoke generated by a forest fire to find major smoke components· uptake of the major smoke components by grapes· metabolism of the major smoke components in grapes· identification of metabolites of the major smoke components in grapes· extraction of the metabolites from grapes to wine
· evaluation of the metabolites as smoke marker compounds· development of methods for the quantification of the metabolites used as smoke diagnostic assays
As a result, various glycosidic forms of guaiacol, methylguaiacol, syringol, methylsyringol, o-, p- and m-cresol, and phenol wereselected as smoke marker compounds. Advantages of the use of these glycosides were as follows；· when a grapevine is exposed to smoke, the amount of the volatile phenols taken up by grapes can be related to the intensityand duration of smoke exposure· once taken up by grapes, the volatile phenols are rapidly metabolized into their more stable and non-volatile glycosidic forms· the smoke induced glycosides persist and accumulate in grapes until the time of harvest, therefore, the amount of the grape
glycosides can be correlated to the intensity of smoke exposure, and possibly also used to quantify extended or repeat smoke
exposure· the phenolic glycosides are not present in non-smoked grapes and are also not found（unlike free guaiacol）in oak in significant
concentrations；· the phenolic glycosides are easily extracted into wine and act as a pool of precursors to release volatile phenols during alcoholic
and/or MLF fermentation, aging, and storage. This implies that measurement of phenolic glycosides can be used to estimate thepotential of grapes to produce smoke-related taints after fermentation and at the time of consumption.

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 40: Environment II

PTh-125

11:10 － 12:20

Ions Observed in DART-MS Analysis of Pharmaceuticals Containing
Various Functional Groups on Normal and Reverse Phase TLC Plates

Kaori Asano1, Toshio Tashima2, Takeyuki Suzuki1, Tsuyoshi Matsuzaki1, Takeshi Nakano3

The Institute of Scientific and Industrial Research, Osaka University, 2GeneStem Co., Ltd, 3Research Center for
Environmental Preservation, Osaka University

1

Keywords:
TLC-MS, DART, Pharmaceuticals, PPCPs
Novel aspects:
This report is the first detailed study on DRAT mass spectrometry of pharmaceuticals containing various functional groups in TLCDART/MS.
Abstract:
Thin Layer Chromatography（TLC）is one of the most popular chromatographic methods and widely used as separation andpurification techniques in a variety of fields such as pharmaceutical science, forensic medicine, organic synthetic chemistry,
botanical science and environmental analysis. TLC is an inexpensive and easy operation method in comparison to highperformance liquid chromatography（HPLC）, gas chromatography（GC）and capillary electrophoresis（CE）. However, theidentification of compound by TLC analysis often becomes difficult because of its low specificity. To dissolve this problem, mass
spectrometry（MS）has been generally used for identification of compounds separated on a TLC plate. In the previous time, thestructural elucidation by MS analysis has been carried out after extraction of the target compound from a TLC plate. This off-line
TLC-MS method is the relatively much time-consuming technique. Thus, various ionization modes have been investigated toanalyze the compounds on TLC plate directly, so far the following ionization techniques have been utilized in a direct sampling
TLC-MS analysis； Fast atom bombardment ionization method（FAB）, Matrix associated laser desorption ionization method（MALDI）, Electrospray ionization method（ESI）, Desorption electrospray ionization method（DESI）, Direct analysis in real time
ionization method（DART）, and so on.1)
Among the direct sampling TLC-MS analyses, TLC-DART/MS seems a relatively easy method if a target compound is not nonvolatile, because the ionization of compound on a TLC plate can be performed under atmospheric pressure without solvents andmatrixes. However, it is relatively difficult and inconvenient to cut a glass type of TLC plate to a narrow width strip（below
ca.10mm）after separation of samples on a TLC plate. A new type of DART ion source was introduced in 2009, which allowed theangle of DART gas stream to be adjusted. The new model shall make it easy to access to more wide surfaces compared with theconventional DART ion source（horizontal type）.2)
On the other hand, the only protonated molecules ions have been discussed in the majority of publications on DART mass
spectrometry. In the case of TLC-DART/MS, there are few publications about the detailed investigation on DART mass spectra ofcompounds containing different functional group.3) Therefore, we started investigation about the characteristics of the DART mass
spectrometry to use the TLC-DART/MS method for analysis of pharmaceuticals and personal care products（PPCPs）.
At the first time, we selected the typical pharmaceuticals containing various functional groups, which occurred as the major
PPCPs in aquatic environments. Most of compounds were obtained by extraction with methanol from the ethical pharmaceuticalproducts, if necessary, were purified by re-dissolving with acetonitrile and/or solid-phase extraction. The silicagel60 F254 and RP- 2
F254 were used as normal-phase and reverse-phase TLC plates, respectively. Both of them were a glass type of TLC plates（20 X 20
cm, stationary-thickness： 0.25 mm）purchased from Merck. The glass strips（100 mm X ca.10 mm）were prepared by cutting TLC
plates and washed by development of methanol in a glass developing chamber. After the sample solutions were prepared in aconcentration level of 0.1 to 5 mg/mL using appropriate solvent, a portion of 1 or 2 μL was applied on a TLC plate using a glass
capillary. DART-MS system consisted of DART-SVP-100（IonSence）for ionization of samples and Accu-TOF mass spectrometer（JMS-T100LC, JEOL）for acquisition of mass spectral data.
In this conference, we will present the information on DART mass spectrometry of pharmaceuticals spotted on normal- andreverse-phase TLC plates.
Reference
1 ）Sy-Chyi Cheng, et.al., J. chromatogr. A, 1218, 2700-2711（2011）2 ）E.S. Chernestsova, et.al., Rapid Commun. Mass spectrum., 25, 2275-2282（2011）3 ）E.S. Chernestsova, et.al., J. Anal. Chem., 66（13）, 1348-1351（2011）

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 40: Environment II

PTh-126

13:30 － 14:40

Concentration profiles of PCB congeners in Steller sea lion, Hokkaido,
Japan

Keiko Kubo1, Katsuyuki Yamaguchi2, Wakana Yamada3, Tsuyoshi Ishinazaka4, Masaki Mitsuhashi6,
Kaoru Hattori5, Shunitz Tanaka1

Hokkaido University, Sapporo, Japan, 2Hokkaido Research Organization, Sapporo, Japan, 3Hokkaido University,
Hakodate, Japan, 4Shiretoko Nature Foundation, Rausu, Japan, 5Fisheries Research Agency, Kushiro, Japan, 6Hokkaido
Research Organization, Kushiro, Japan

1

Keywords:
PCBs, congeners, fetus, liver, blubber
Novel aspects:
The distributions of PCB congeners in SSL were described. The concentration profiles of PCB congeners in the liver and blubber
were different among males, females and fetuses.
Abstract:
Introduction
PCBs still have been the major contaminants in the marine mammals at the top of the marine food-web. Especially, liver andblubber were thought to be most accumulative organs of PCBs in their body. The high risks of PCBs on the immunity obstruction
and the cacochymia in marine mammals are feared. Therefore, more detail survey about PCB congeners in marine mammals isnecessary.
In this study, PCB congeners in the liver and blubber of Steller sea lion（Eumetopias jubatus： SSL）from the coastal Hokkaido,
Japan were measured by HRGC-HRMS to describe the distribution of PCB congeners in the males, females and fetus of SSL.
Materials and Method
The liver and bluber samples of SSL were obtained from commercial fishery-related control killed and bycatch animals at the
Shakotan area and Nemuro Strait, Hokkaido, Japan, during winter season in 2008 and 2010. These samples were measured by anisotope dilution method with 13C-labeled internal standard substances. The liver samples of eight males, two females and threefetuses were analyzed. The blubber samples of four males and three females and three fetuses were analyzed. These samples
include three pairs of mother and fetus.
Results and Discussion
In the males, #153（in IUPAC#）was predominated in the both organs of the liver and blubber, followed by #138, #99 and #118. The
concentration levels of #28, #31, #177 and #199 were appeared to be considerable differences between these tow organs. It
seemed that each organ had specific accumulative property of PCB congeners. The total PCB concentrations（ΣPCBs）of males in
the liver and blubber were 3,100±1,300 ng/g-fat and 3,000±830 ng/g-fat, respectively.
Between genders, the concentration profiles of major congeners in the blubber were differed, significantly. Meanwhile in the liver,there were no obvious differences in the congener profile between the male and female. In general, gender differences depend on
the experience of pregnancy and lactation. ΣPCBs of the female in the liver and blubber were 1,200±810 ng/g-fat and 910±410 ng/
g-fat, respectively. ΣPCBs in female were 61%-71% less than in males.
We analyzed PCBs in the liver and blubber from three pairs of mother and fetus to describe the detail profiles and to compare
the concentrations between them. In the blubber, #153 and #138 were dominated congeners except one fetus. The majorcongeners in the blubber and liver of fetus were #153, #138 and #118 except one fetus. The average of ΣPCBs in the liver andblubber of the fetus was 710±430 ng/g-fat and 1,600±900 ng/g-fat, respectively. It was explained that ΣPCBs in the blubber of fetus
was about 27%-85% higher than those of mother. These fetuses already have been contaminated during gestational period. It wasthought that the concentration levels of these fetuses might depend on each mother’s PCB levels.
The concentration profiles of congeners in the liver and blubber were different between the liver and blubber of males, theblubber of genders, each pair of mother and fetus. The level of ΣPCBs in male SSL from Hokkaido was as the same level as thereport in 14 years ago（Kim et al.,1996）. The PCB levels of congeners in SSL from Hokkaido showed how PCBs still remained inmarine environment of the coastal Hokkaido. More investigations of PCB congeners in SSL are needed to study the mechanism of
the accumulation and metabolism.

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 40: Environment II

PTh-127

11:10 － 12:20

Investigation of metabolites formed during activated sludge treatment ofclarithromycin

Masakazu Morita1, Koya Komori2, Mizuhiko Minamiyama2
1

NS Environmental Science Consultant Co., Tokyo, Japan, 2Public Works Research Institute, Ibaraki, Japan

Keywords:
clarithromycin, wastewater, activated sludge treatment, metabolite
Novel aspects:
During activated sludge treatment, sulfate conjugation that is one of the phase II reactions observed as mammal’s drug
metabolism may occur in CAM.
Abstract:
The occurrence of antibiotics in aquatic environments is of ecotoxicological concern because of potential ecosystem alteration.
Prolonged exposure to low doses of antibiotics leads to the selective proliferation of resistant bacteria, which could transfer the
resistance genes to other bacteria species. Since most antibiotics used in human treatment are eventually disposed of with
wastewater, wastewater treatment plants play a key role in removing antibiotics and preventing them from reaching the receiving
water bodies.
Clarithromycin（CAM）is a half synthetic macrolide antibiotic which is induced from the antibiotic erythromycin. In Japan, CAM has
been detected at low microgram / liter in some untreated wastewater samples. It is understood that CAM is one of the drugs thathas ecological risk in aquatic environments. In most of the investigations reported to date, the efficiency of CAM removal during
wastewater treatment is determined by measuring the disappearance of the parent compound. But little attention has been given
to the identification and quantification of transformation products formed during wastewater treatment.
In the present study, the activated sludge treatment that was the typical wastewater treatment method was reproduced in thelaboratory, and screening of major metabolites of CAM was conducted. To investigate the microbial degradation of CAM in theaeration tank, a batch reactor（ 5 L）loaded with freshly collected activated sludge from the municipal wastewater treatment
plant was spiked with the parent compound at a concentration of 20 μg/L. Negative control experiments（without CAM, withactivated sludge）and abiotic experiments（with CAM, without activated sludge）were also performed. For the screening of
metabolites of CAM using the liquid chromatography / tandem mass spectrometry（LC/MS/MS）system equipped with anelectrospray ionization（ESI）source, the samples were pre-concentrated by solid-phase extraction.
Analysis of samples from batch cultures with activated sludge revealed the formation of a metabolite with m/z 828（retention timeof 15.70 minutes）using LC /（+）ESI-MS［corresponding to protonated molecule（M+H）+］. This metabolite was not observed innegative control and abiotic samples, suggesting that this peak is unique to the biotransformation of CAM in the presence ofactivated sludge. The protonated molecule of this metabolite（m/z 828）is hereinafter termed as M828. Because CAM was detected
at m/z 748（retention time of 15.35 minutes, hereinafter termed as P748）as the protonated molecule（M+H）+, M828 increased 80
units more than P748. To facilitate identification of M828, fragmentation studies on P748 and M828 were performed by LC /（+）ESIMS/MS. A minimum fragment ion to which 80 units shifted in the fragment ion of M828 compared with the fragment ion of P748 was
m/z 238. This fragment ion corresponds to fragment ion m/z 158 of P748. There is a possibility of causing the shift of 80 units in M828
with desosamine because fragment ion m/z 158 of P748 is derived from desosamine, which is one of the sugars that composes
CAM. Results from the analysis of LC /（-）ESI-MS reveal that m/z 826 was detected only with the sample from the batch culture
with activated sludge［corresponding to deprotonated molecule（M-H）-］. The deprotonated molecule of CAM was not detected.
Hence m/z 826 in a negative ionization is presumed to be a deprotonated molecule of a metabolite of CAM, which corresponds toM828 in a positive ionization. A negative ionization is often caused by the elimination of protons in the acid group. One possible
biotransformation that causes the change of +80 units is sulfate conjugation. During activated sludge treatment, sulfate
conjugation that is one of the phase II reactions observed as mammal’s drug metabolism may occur in CAM.

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 40: Environment II

PTh-128

13:30 － 14:40

A NEW CHEMOMETRIC APPROACH FOR THE QUANTITATION OF
STEROIDAL COMPOUNDS IN WASTEWATER BY GCxGC-TOFMS

Matias Kopperi, Jose Ruiz-Jimenez, Kari Hartonen, Marja-Liisa Riekkola
University of Helsinki, Finland

Keywords:
GCxGC-TOFMS, chemometrics, emerging organic contaminants, steroids, wastewater
Novel aspects:
The quantitation of unknown emerging organic contaminants with a chemometric model based on partial least squares regression
equations, which were built up using six commercial steroid standards.
Abstract:
An increasing variety of chemical compounds including pharmaceuticals, personal beauty products and insect repellents areoffered daily to consumers. The final destination for most of these compounds is the environment as raw compounds and their
metabolites, some of which are potentially hazardous for the environment. In order to speed up the process of regulation andassessment of environmental threats, it is essential to develop rapid and efficient methods for the analysis of these contaminants.
In this study, exhaustive sample preparation method was first developed for the determination of steroidal species in thewastewater samples aiming at the clarification of the distribution of free and conjugated steroids in wastewater and suspended
particles. Special emphasis was put on the applicability of comprehensive two-dimensional gas chromatography coupled with
time-of-flight mass spectrometry（GCxGC-TOFMS）to screen steroidal contaminants in wastewater. Although the resolution andsensitivity of GCxGC-TOFMS is superior over those of conventional GC-MS techniques, the lack of available commercial standards
can hinder the usefulness of the technique, especially if the goal of the analysis is the quantitation of the identified compounds.
Several approaches based on the use of surrogates can be found in the literature for the quantitation of compounds identified byMS, but frequently they lack the accuracy. In this study a new approach based on partial-least-squares regression（PLSR）was
developed for the quantitation of emerging steroidal compounds in water and sludge samples taken from the largest wastewater
treatment plant of Finland.
Six commercial steroidal compounds injected to the GCxGC-TOFMS at seven concentration levels ranging from 0.1 to 10 ng, wereused as reference compounds for the development of the PLSR equation, which was successfully exploited for the determinationof more than 40 steroidal compounds present in the analyzed samples.

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 40: Environment II

PTh-129

11:10 － 12:20

High-Resolution Tandem Mass Spectrometry Analysis of the Interactionsof Oligonucleotides with Selected River Basin Specific Pollutants

Janna Anichina, Andre Schreiber, Ron Bonner, Takeo Sakuma
AB SCIEX, Concord, Canada

Keywords:
Oligonucleotides, organic micro-contaminats, high resolution tandem mass spectrometry, micro-flow liquid chromatography, noncovalent interactions
Novel aspects:
For the first time the reactivity of selected organic micro-contaminants towards oligonucleotides was assessed using highresolution electrospray ionization tandem mass spectrometry combined with the micro-flow liquid chromatography.
Abstract:
Since a great number of organic compounds are annually released into the environment, the necessity to assess in a timely
manner the potential risks associated with these chemicals along with the products of their environmental transformation is ofhigh priority. In order to reduce the number of candidates for full-scale animal studies, potential toxicity of the compounds ofinterest can be rapidly assessed in simplified model systems. In this study we employed high resolution electrospray ionization
tandem mass spectrometry（ESI-TOF/MS/MS）along with micro LC-ESI-TOF/MS to study interactions of selected river basin
pollutants with model nucleic acids. The micro-contaminants were chosen in the context of the European Union Water
Framework Directive（EU WFD）based on the assessment study for 500 organic substances observed in the four European river
basins of the Elbe, Scheldt, Danube and Llobregat.
Two AB SCIEX LC-MS systems were used in this study： a hybrid linear ion trap-triple quadrupole system and a hybrid quadrupoletime-of-flight instrument with Turbo V™ sources and Electrospray Ionization（ESI）probe in the negative ion mode. A high pressure
micro-flow LC system was utilized for chromatographic separation. Interactions of two decameric oligonucleotides（ODNs）, d（ 5 ’-GCGCATGCGC- 3 ’）and d（ 5 ’-GCGCGCGCGC- 3 ’）with diazinon, diuron, alachlor and bis（ 2 -ethylhexyl）phthalate were
investigated in the direct infusion mode and with the micro-flow LC separation. The AB SCIEX PeakView TM software with aprototype oligonucleotide fragmentation interpretation tool was used for the data analysis.
In this study we examined the interactions of two self-complementary decameric ODNs with four river basin specific pollutants.
ESI-MS/MS analysis of the incubation mixtures of diuron with two selected ODNs indicated the formation of 1 ： 1 adducts ofboth single-stranded oligonucleotides at a molar ratio of 10 or higher. Collected high resolution MS and MS/MS data were used toconfirm the elemental composition of the ions of interest as well as to gain an insight into the structure of the adducts.
Tandem mass spectrometric measurements of the 1 ： 1 adducts of diuron with both single-stranded ODNs demonstrated that
their dissociation proceeds via the loss of a neutral diuron molecule at a relatively low value of the laboratory frame collisionenergy. When highly negatively charged diuron：ODN adducts（charge states of 5 -, 6 - or 7 -）were exposed to the collisioninduced dissociation（CID）we have also observed a competing charge-separation channel to produce a deprotonated diuron
moiety and an oligonucleotide ion in（n- 1 ）- charge state with n=5,6,7 respectively. Based on this observation we hypothesize a
non-covalent mode of binding in the 1 ： 1 complexes. Under the studied experimental conditions we did not detect diuron
adducts with the ODN duplexes.
The results of the experiments performed with the studied ODNs and diazinon were similar to those observed for diuroncontaining species. However, the formation of the 1 ： 1 adducts was observed at a higher excess of diazinon（50-fold or higher）.
CID measurements of the diazinon adducts demonstrated an earlier（compared to diuron）CID onset which indicates a weakerbinding with the ODNs. No detectable adducts of alachlor with either ODNs were formed even at 1000-fold excess of thecompound.
This study has demonstrated an elegant application of high resolution tandem mass spectrometry combined with the micro-flow
liquid chromatography for rapid screening of the reactivity of high priority environmental contaminants toward DNA models.

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 40: Environment II

PTh-130

13:30 － 14:40

Formation of Hydroxy Polychlorinated Biphenyl

Shiho Fukuzawa1, Takeshi Morita1, Masahiro Shimase1, Akinobu Kunitake1, Takanori Sakiyama2,
Tameo Okumura3

1
Nippon Steel Kankyo Engineering Co., Ltd., Chiba, Japan, 2Osaka City Institute of Public Health and Environmental
Sciences, Osaka, Japan, 3Environmental Pollution Control Center, Osaka Prefectural, Government, retirement

Keywords:
Hydroxy-PCB, PCB, formation mechanism, UV-light, soil
Novel aspects:
Congener specific formation of OH-PCB with UV-light in silica gel or without light in soil
Abstract:
Hydroxy polychlorinated biphenyl（OH-PCB）is one of an intravital metabolite of polychlorinated biphenyl（PCB）. Many cases
about measurement OH-PCB in biological samples were reported and they pointed out that some of the isomer has an affect on
thyroid hormone action or female hormone action. In the case of environmental samples Sakiyama et al.1) indicated the presenceof OH-PCB in aqueous environment and bottom sediments. The result suggests that PCB is also hydroxylated in environment byphysicochemical reaction or metabolic reaction of living organisms.
At our previous research 2) on the actual situation of OH-PCB in the soil there are correlations between the OH-PCB concentrations
and the PCB concentrations regardless of whether the soil is contaminated. We also reported that OH-PCBs are formed frommixture of PCBs（KCmix）under various conditions. No report is available that actually researched about the formation mechanismof OH-PCBs from dominant PCB congeners. In order to confirm the mechanism of OH-PCB formation, we selected 10 dominantcongeners of PCB（#3, #12, #26, #31, #52, #101, #118, #153, #180, #209）as parent to hydroxylate under two conditions as follows. The
first was irradiating UV-light to the silica gels, and the second was keeping the soils under light-shielding with addition individual
PCB congeners. The OH-PCBs formed in the test samples were pretreatment through several steps and then methylated foranalysis. The methoxy-PCBs were detected based on mass spectra characteristics after gas chromatography separation. In
addition, they were identified by comparing their retention characteristics with methoxy-PCBs those were methylated from theavailable OH-PCB standards provided by reagent manufacturer（47 congeners）and Okumura synthesized（80 congeners）. We
present the new findings about congener specific formation of OH-PCB that will be the help for the following studies on OH-PCB inenvironmental samples.
Reference literature
1 ）16th Symposium on Environmental Chemistry, Sakiyama et al., 408-409（2007）
2 ）17th Symposium on Environmental Chemistry, Shimase et al., 480-481（2008）

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 40: Environment II

PTh-131

11:10 － 12:20

High Resolution LC-MS for Screening and Quantitative Analysis of
Antibiotics in Drinking Water using an Orbitrap and Online Sample
Preparation

Jonathan R Beck, Dipankar Ghosh, Charles T Yang
Thermo Fisher Scientific, San Jose, USA

Keywords:
Antibiotics, Orbitrap, Online SPE, PPCPs
Novel aspects:
High resolution mass spectrometry plus online pre-concentration to screen, quantify and confirm 27 antibiotics in water samples at
low ppb levels
Abstract:
Most current methodologies for the quantitation of antibiotics in drinking water revolve around analysis using triple quadrupole
platforms with offline sample preparation. The method described here utilizes LC-MS（/MS）with a Orbitrap instrument using highresolution accurate mass and online sample preparation. This work will describe a method to do screening and quantitation of 27
antibiotics at ppb and sub ppb levels in drinking water using online pre-concentration together with high resolution accurate massconfirmations of the compounds.
A standard curve containing 27 compounds was spiked in neat solution ranging from 100 pg/mL levels to 250 ng/mL levels was
injected in triplicate and screening of different water samples（municipal tap water and bottled water）was analyzed for atargeted list of the 27 compounds. The sample was also screened for other possible unknown pesticides compounds. The
spectrometer was set to a resolving power of 70,000（FWHM）at m/z 200 in full MS mode to minimize matrix interferences, anddata dependant all-ion-fragmentation（AIF）was collected to quantify and qualify. The data was then compared to a current MS/
MS library for confirmation, and calibration curves were generated for the individual target compounds.

Calibration lines were generated for the compounds analyzed, and the limits of detection（LOD）varied from 100pg/mL to 500pg/
ml based on the individual compounds based on a 1 mL injection. For confirmation of each compound, the exact mass of thecompound, its isotope pattern and as well as the MS/MS spectrum produced were collected and compared against afragmentation library which prevented“false positives“ in the results. One of the main challenges using a high resolution accuratemass system is data mining. In this work we will show the data to screen, quantify and confirm in a single run therefore, spending
less time on the instrument for repeat runs. Furthermore, the online pre-concentration step allows for low detection limits of thesecompounds without the time consuming steps of offline solid phase extraction of 1 L sample volumes.

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 40: Environment II

PTh-132

13:30 － 14:40

Rapid screening and confirmation of emerging contaminants in UK riverwaters by UHPLC-IT-TOF

David R Baker1, John Bagnall2, Alan J Barnes1, Barbara Kasprzyk-Hordern2, Neil J Loftus1
1

Shimadzu Co., Manchester, UK, 2University of Bath, Bath, UK

Keywords:
river water screening environment I.T-TOF
Novel aspects:
Presented is a fast screening and confirmation method, applied to UK river waters, using UHPLC-IT-TOF with software（MetID）toaid in rapid and relatively straightforward interpretation of large data files
Abstract:
The extensive environmental distribution of emerging contaminants and their potential ecotoxicological effects at very low
concentrations has attracted increasing interest amongst researchers, regulatory authorities and the public worldwide. Emerging
organic contaminants constitute a diverse set of compounds including pharmaceuticals and personal-care products（PPCPs）,
drugs of abuse and their metabolites, polar pesticides and their degradation/transformation products, perfluorinated compounds（PFCs）and organophosphorous flame retardants. The attention given to these contaminants is warranted as, amongst a numberof reasons, many of these contaminants are relatively small molecules which may not be effectively removed during drinking water
treatment.
Typically multi-residue methods by tandem mass spectrometry provide routine analysis in identifying emerging contaminants dueto their sensitivity and selectivity. These are however limited to unit resolution and have low sensitivity in full scan. For thesereasons the employment of high resolution mass spectrometry has become increasing popular in environmental analysis using fullscan. Time of flight technology offers the possibly of accurate mass and isotope distribution analysis of targets compounds,
however without specialist software, typically data analysis can be labour intensive.
In this study we address the discussed challenges by presenting a fast screening and confirmation method using UHPLC-IT-TOF（Shimadzu Corporation）with software（MetID, Shimadzu Corporation）to aid in rapid and relatively straightforward interpretationof large data files.
River water was collected from a major river in the UK and screened for the presence of over 100 emerging organic contaminants.
Samples were extracted using Oasis HLB cartridges. UHPLC separation was achieved using a Kinetex XB-C18 100 x 2.1mm column,
with 0.2 % formic acid in both the aqueous and organic（methanol）phases, and separated by binary gradient over 25 minutes.
LCMS-IT-TOF data was acquired in the mass range 70-900 Da in both positive and negative ionisation modes using fast（100ms）
polarity switching.
Preliminary data indicated several organophosphorous flame retardants were detected in addition to pharmaceutical compounds
and an insect repellent compound. With these preliminary identifications, comparison to authentic standards will enable final
confirmation without the expensive task of purchasing authentic standards for all possible screened compounds.

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 40: Environment II

PTh-133

11:10 － 12:20

Degradation of Pentachlorobenzene by Fungi Screened from Nature

Ajeng A Sari1, Sanro Tachibana2, Kazutaka Itoh2

The United Graduate School of Agriculture Sciences, Ehime University, Matsuyama, Japan, 2Department of Applied
Bioscience, Faculty of Agriculture, Ehime University

1

Keywords:
degradation, pentachlorobenzene, white rot fungi, metabolite
Novel aspects:
Limited information about degradation of pentachlorobenzene, a newly POPs, by white rot fungi and its metabolite products
Abstract:
Introduction: Pentachlorobenzene is used as an intermediate in particularly the fungicide pentachloronitrobenzene. It has also
been used as fire retardant. This compound was added in 2009 to the list of POPs compounds covered by the Stockholm
Convention. Considering pentachlorobenzene potential negative effects, it is necessary to address the environmental persistenceof this pesticide and to develop effective methods for remediation by miroorganisms. White rot fungi are well known for theiroutstanding ability to produce extracellular oxidative enzymes e.g. lignin peroxidase（LiP）, manganese peroxidase（MnP）andlaccase, which are involved in the degradation of either lignin than a wide range of pollutants include pentachlorobenzene. Other
non-ligninolytic enzymes may participate in the transformation of pentachlorobenzene. The objective of these studies is to screen
the fungi from nature having ability to degrade pentachlorobenzene on solid medium and in liquid medium, to investigate the
enzyme activities secreted by selected fungi and to investigate the major metabolic products.
Experiment: Fungi screened from nature were inoculated for 7 d on a malt extract agar medium containing 1 mg
pentachlorobenzene. Liquid culture experiment was conducted using 20 ml of malt extract medium in a 100 ml Erlenmeyer flask.
After inoculation fungus for 7 d, 0.1 mM pentachlorobenzene solution was added and then incubated for 15 and 30 d. After aperiod of culture, the sample was extracted using ethyl acetate and purified using silica gel column chromatography eluent withhexane：dichloromethane（ 3 ： 1 ）. The concentrate dissolved in toluene was analyzed by GC-MS Shimadzu QP-2010 equippedwith a TC- 1 column（30 m, id： 0.25 mm）. The carrier gas was helium delivered at a constant flow rate of 1.5 ml/min with columnpressure of 100 kPa and interface temperature of 120 °C. The temperature program was started at 120 °C for 1 min, raised 20 °C/
min to 180 °C, then 2 °C/min to 210 °C, then 5 °C/min to 310 °C, and maintained at 310°C for 3 min to allow the eluting peak toexit the column. For enzyme activity analysis, after period culture, the extracellular fluid was collected, filtered through a 0.2 µm
membrane filter and measured by using Spectrophotometer. Enzymes that checked were laccase, MnP, LiP, 1,2 dioxygenase and 2,3
dioxygenase. For investigation of metabolic products, the samples were extracted with ethyl acetate and then the identification of
metabolic products was performed in comparison with authentic standards.
Results and discussion: Cultivation of 4 fungi screened from nature on solid medium containing pentachlorobenzene indicatedthat growth of the fungi was obtained maximum of 100 % growth by 10th to 15th d. On solid medium, U80 showed the mostdegrading pentachlorobenzene fungus. In liquid medium, U80 degraded pentachlorobenzene at 63% on 30 d. All enzymes tested
were produced by U80 during pentachlorobenzene degradation which the high enzyme activity were obtained in dioxygenase and
LiP. By comparing retention times and mass spectra with standard compounds by GC/MS, U80 produced 5 metabolite products.
Pentachlorobenzene was initially dechlorinated to form tetrachlorobenzene, which was converted to trichlorobenzene,
dichlorobenzene and chlorobenzene. Dechlorination was occured by presence of LiP. Moreover, dichlorobenzene could form
4 -chlorophenol or 2,4-dichlorophenol. Dichlorophenol could be identified as a metabolite from dichlorobenzene or a product ofdichlorobenzene dihydrodiol decomposition. It was caused by monooxygenase or dioxygenase because the initial attack ondichlorobenzene at the 1 or 2 position could form 4 -chlorophenol or 2,4-dichlorophenol.

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 40: Environment II

PTh-134

13:30 － 14:40

Analysis of thermal products of chlorpyrifos using LC/FTMS

Yoshinari Yamoto1, Takanori Sakiyama2, Hideya Kawasaki1, Ryuichi Arakawa1, Takeshi Nakano3

Kansai University, Osaka, Japan, 2Osaka City Institute of Public Health and Env. Sciences, Osaka, Japan, 3Osaka
University, Osaka, Japan

1

Keywords:
chlorpyrifos, thermal products, LC/FTMS, isomers containing chrlorine
Novel aspects:
The thermal products of chlorpyrifos were identified to be its polymeric species containing a wide variety of structural isomers
with different binding sites of chlorine atoms.
Abstract:
Introduction
Chlorpyrifos（O,O-diethylO-3,5,6-trichloropyridin- 2 -yl phosphorothioate）is an organophosphate insecticide that inhibits
acetylcholinesterase and is used to control insect pests. Chlorpyrifos have been used in the lawn on a golf course. It was used fortermite control as chlordane alternatives. Sakiyama et al. have reported that 2,3,7,8-TCDD- N-analogue is formed in pyrolysis
experiments of chlorpyrifos by GC/HRMS analysis.1 We should consider carefully burning scrap woods of houses contaminated bychlorpyrifos. It is suggested that the influence appears. In this study, the further thermal products are examined by LC/FTMS andthe thermal degradation pathways are proposed.
Methods
All pyrolysis experiments were carried out in sealed brown glass ampoules（10 ml）with about 2 mg of chlorpyrifos and
3,5,6-trichloro-pyridinol（3,5,6-TCP）at temperature between 300 °C and 380 °C. After cooling to room temperature, the ampoules
were opened carefully and the reaction products were extracted with toluene. The toluene was concentrated to 1 ml undergentle nitrogen stream. The thermal products of chlorpyrifos and 3,5,6-TCP are analyzed with an Exactive orbitrap mass
spectrometer（ThermoFisher Scientific Inc.）equipped with positive electrospray ionization（ESI）probe and Accela LC system（ThermoFisher Scientific Inc.）with a column of Inertsil ODS- 3 5 um 2.1×250mm（GL Science）. The best LC separation for thethermal products was achieved using（a）ammonium acetate aqueous solution（ 5 mM）and（b）acetonitrile for the gradientconditions.
Result and discussion
Analysis of chlorpyrifos after thermal treatment at 300 ~ 380 °C was performed by LC/FTMS. The peak intensity of 3,5,6-TCP（m/z=
195.91）was found to increase with decrease in that of chlorpyrifos. The results indicated that the formation of 3,5,6-TCP wasprobably due to the heating of chlorpyrifos. In addition, the thermal treatment of 3,5,6-TCP showed that the intensity of dimer,
trimer and tetramer ions increased as the peak intensity of 3,5,6-TCP decreased in the mass spectra. The analysis of separation
behavior for the LC-MS chromatogram indicated that these polymeric species contained some structural isomers. The new finding
obtained from the present LC/FTMS measurements is that the heating of chlorpyrifos generates its oligomers by polymerization.
The oligomers are shown to have a wide variety of isomers with different binding sites of chlorine atoms.

1 ）Sakiyama T, Weber R, Behnisch P, Nakano T, PreliminaryAssessment of Dioxin-Like Compounds In/From Chlorpyrifos - A
PotentialPrecursor of the Pyridine Analogue of 2,3,7,8-TCDD, Organohalogen compounds： Vol. 73, 146-149 (2011)

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 40: Environment II

PTh-135

11:10 － 12:20

Behavior of Hexabromocyclododecane (HBCD) stereoisomers inwater,sediment, and biological samples.

Hitomi Hasegawa1,2, Masatoshi Watanabe1, Shigeru Suzuki2
1

Nagoya City Env.Sci.Res.Inst., Nagoya, Japan, 2Chubu University, Kasugai, Japan

Keywords:
brominated flame retardants, 1,2,5,6,9,10-hexabromocyclododecane, LC/MS/MS, environment
Novel aspects:
HBCDs regional contaminations in Nagoya city were revealed by measuring river waters, sea waters, sediments, fishes and breast
milks.
Abstract:
1,2,5,6,9,10-hexabromocyclododecane stereoisomers（HBCDs）are used as flame retardants in thermal insulation building materials,
upholstery textiles, and electronics. Since 2004, the production and use of pentamix- and octamix- polybrominated diphenyl ethers（PBDEs）have been restricted in Europe. HBCDs might be used as an alternative for PBDEs, and are the second highest-volume
brominated flame retardants（BFRs）used world wide, after tetrabromobisphenol A. Because of the widespread use and thephysical and chemical stability, HBCDs are now ubiquitous contaminants in the environment. In Nagoya city, HBCDs were
frequently measured in environment. It is required to reveal their environmental distribution, behavior and time trends.
Here we report the concentrations of HBCDs in water, sediment, and biological samples, and analyze the biomagnification
potential, the stereoisomer profiles, and the time trends.
Analysis was undertaken by liquid chromatography/ tandem mass spectrometry（LC/MS/MS）on a diastereoisomer basis. Ninetythree samples（22 of river and sea water samples, 27 of sediment samples, 24 of fish and shellfish samples, and 20 of breast milk
samples）in Nagoya city were measured.
HBCDs were detected in 18 water samples, of which concentrations of total isomers ranged from 0.4 to 280 ng/L. γ-HBCD
dominated over the other isomers in water samples. Similarly, sediment samples were dominated by γ-HBCD, which was
detected in all samples, ranged from 0.2 to 27 ng/g-dry. Time trend analysis by boring sediment core samples in Nagoya port
showed a remarkable increase in HBCD concentrations since about 2000. This might be reflected by changing use of HBCDs from
PBDEs according to regulation of EU on the production and use of two PBDEs.
In fish and shellfish samples, the maximum HBCDs concentration was about 5 -10 times that reported in earlier Japanese studies.
α-HBCD was the predominant isomer of HBCDs and was found in all the samples in levels from 24 to 6300 ng/g-lipid. On the other
hand, γ-HBCD was predominant in some samples, and the isomer patterns of HBCDs were similar to those in industrial products.
Samples in which γ-HBCD concentrations were higher might be collected near the source.
In breast milk samples, α-HBCD was also predominant in HBCDs and was found in all samples in concentrations from 1.5 to 13 ng/
g-lipid. The concentrations of HBCDs in the samples of the same person decreased from 7 to 1.5 ng/g-lipid during 9 days to 300
days of post parturition.

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 40: Environment II

PTh-136

13:30 － 14:40

LC/MS/MS determination of hair-dye ingredients in products, water andurine:estimation of human exposure and environmental release.

Mari Takazawa, Shigeru Suzuki
CHUBU University,Aichi,Japan

Keywords:
exposure, environment, release, hair-dye
Novel aspects:
Human exposure and environmental release of hair-dye ingredients were examined by developing measurement method by LC/
MS/MS.
Abstract:
Introduction：Over 5,000 different chemicals are used in hair-dye products, some of which are reported to be carcinogenic and/or
allergic. Aminophenol, diaminotoluene, aminocresol and resorcinol are well used and hazardous ingredients of hair-dye. The
authors developed an LC/MS/MS method for simultaneously determining the hair-dye ingredients and applied it to the estimationof human exposure and environmental release of the ingredients.
Methods：（ 1 ）measurement of the chemicals in shampoo and rinse water
After coloring, the hair was shampooed with one push（16g）of shampoo bottle. Then the hair was rinsed with water（ 5 L）, which
was collected in a plastic bucket. A hundred milliliter of the rinsed water was loaded to Solid Phase Extraction Cartridges（C18and Oasis MCX plus were combined in series）. The elute with methanol（ 5 ml）from C18 and the elutes with methanol（ 5 ml）,
ammonia/methanol solution（ 1 /19, 5 ml）from Oasis MCX plus were separately concentrated to near 0.5ml under nitrogen gas
stream. The concentrates were diluted with acetonitrile, filtrated and prepared in 2 ml for LC/MS/MS analysis.
（ 2 ）measurement of the chemicals in urine.
Within 24 hours after coloring the hair, all the urine were separately collected in glass bottles and stored in cool box. Every ten
milliliter of urine was diluted with 40ml of water, and applied to the SPE. Samples were prepared with similar way to above.
Predominant result：（ 1 ）determination of hair-dye ingredients in shampoo and rinse water
For the SPE method development, five kinds of SPE cartridges were examined, i.e. Slim-J PRS, AC-2, NEXUS, HLB and MCX.
Although good recovery was not obtained with any of them, MCX had a potential to collect hydrophilic hair-dye ingredients byconnecting it with other SPE cartridge. It was the best in the recoveries to connect C18 cartridge upstream of MCX. With the seriesconnection of C18 and MCX, the recoveries of 5 -amino-o-cresol, 2,5-diamino-toluene, p-amino-phenol and resorcinol spiked inshampoo and rinse water had improved from 15.5% to 96%, from 39% to 76%, from 21% to 56%, and 0 % to 28%, respectively. An
effect of pH on recovery was examined, because the MCX performs the best under pH 3 &#65374；4. But it was concluded thathigher recovery efficiency was obtained under pH value of 9.5 than that of 3.
A shampoo and rinse water sample was analyzed at 0, 2, 4, 8 and 24 hours afterward. The examined hair-dyes were two kinds, i.e.
black and golden. The ingredients of the dyes differ to each other； 2,5-diamino-toluene and p-amino-phenol are contained in theblack dye, and 5 -amino-o-cresol, p-amino-phenol and resorcinol are in the golden. With progress of time, the concentration of
every substance decreased. In particular p-amino-phenol which was contained in golden hair-dye showed remarkable decrease inconcentration from 4 hours after the shampoo. And as for resorcinol, the rate of the decrease was much slower than that ofothers. Further study is required to pursue the persistence of resorcinol.

（ 2 ）determination of hair-dye ingredients in urine
It is still under examination. Because of many impurities in urine, more efficient sample preparation method is required. .

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 40: Environment II

PTh-137

11:10 － 12:20

Qualitative analysis of waste leachate by using exact mass of LC/
Q-ToFMS/MS

Akira Murakami1, Shigeru Suzuki1, Masahiko Takino2
1

CHUBU University,Aiti,Japan, 2Agilent Technologies,Tokyo,Japan

Keywords:
LC/Q-ToFMS/MS, MetFrag, irregular dumping
Novel aspects:
The simultaneously fractionation method followed by LC/ToFMS/MS was developed. The exact masses of product ions andprecursor ions were analyzed by using MetFrag.
Abstract:
Introduction：
Environmental pollution by chemical substances has been a serious problem. In our daily life the various types of chemical
substances are used and wasted. The authors aim to develop a method comprehensively separating chemicals according to theirproperties and qualifying them with LC/Q-ToFMS/MS. Here we report a qualitative analysis of a leachate from irregular waste
dumping area by a provisional qualification method with LC/Q-ToFMS/MS.
Methods ：To separate a variety of chemicals, a solid phase extraction（SPE）method using C18 cartridge followed with ion exchange
cartridges（Oasis MAX, WAX, MCX, WCX）was developed. Chemicals collected in the SPE cartridges were fractionated with theway as follows； Two liter of water sample was loaded into C-18. Substances collected in C18 were eluted with 10mL of methanol.
The water passed through C18 was divided into four equal parts of 2 L. The every 500mL of C18 passed fraction was separately
loaded into Oasis MCX, MAX, WCX and WAX. From the MCX and the WAX, collected substances were eluted with 10mL of
methanol/ammonium solution. From the MAX and the WCX, collected chemicals were eluted with 10mL of methanol/formic acidsolution. All the fractions were concentrated to 2 mL in volume and served for accurate mass measurement using LC/Q-ToFMS/MS.
The exact mass spectra were analyzed with ’MetFrag’（Leibniz-Institut für Pflanzenbiochemie）by ways as follows； the elemental
compositions of intact molecular ions were analyzed with the mass accuracy of better than 3 ppm. Then molecular structuresthat agree with the elemental composition were extracted from ’ChemSpider’（chemical structure database by UK in the 26 million
structures）and the structures of which predicted fragments match the sample spectrum（with mass accuracy of better than 6ppm）were chosen.. Chemicals in a leachate from irregularly dumped waste were qualified by the method written above.
Predominant result：
Hundreds or more candidates of chemicals were extracted by simply eliminating chemicals of which intact molecular ions didn’t fit
the mass spectra with the mass accuracy of better than 3 ppm. It could be possible to identify some chemicals from the sample
mass spectra with the combination of the product ion analysis with mass accuracy of better than 6 ppm. In a leachate from
irregular waste disposal site, pharmaceuticals, flame retardants, surfactants and other chemicals were found； VALGANCICLOVIR（0.28ppm Antiviral agents）, Etodroxizin dimaleate（1.91ppm Sedative ）. Phosphoricacid-tris-（ 2 -butoxyethyl）（0.75ppm）,
Dimethylaminophenol（2.19ppm Resin hardener）and a polyethoxylate［-OCH2CH2-］surfactant were found. The LogP values of thesubstances above were ranged from-1.27 to 3.50.
The phosphorus-based flame retardant would be from industrial waste. The pharmaceuticals might be from a different sourcefrom industrial wastes. The presented method is provisional until it has been validated with various environmental samples.
With the presented SPE method, hydrophilic and hydrophobic substances could be simultaneously collected, fractionated andqualified by LC/Q-ToFMS/MS.

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 40: Environment II

PTh-138

13:30 － 14:40

Investigation of Perfluorinated compounds in Osaka-bay over past threeyears

Shusuke Takemine1,2, Chisato Matsumura1, Katsuya Yamamoto1, Masahiro Tsurukawa1,2,
Takeshi Nakano2, Akira Kondo2

1
Hyogo Prefectural Institute of Environmental Sciences, Kobe, Japan, 2Graduate School of Engineering Osaka University,
Osaka, Japan

Keywords:
Perfluorinated compounds, Osaka-bay, Environmental investigation, LC-MS/MS
Novel aspects:
Contamination of PFCs in Osaka-bay has been insufficient information.This research presented the characteristic distribution andthe change of concentrations of PFCs in Osaka-bay.
Abstract:
Perfluorinated compounds（PFCs）have been widely used by various consumers and industrial products for over 50 years. PFCs
are used for various products commencing with surface protectors in carpets, paper, packaging, and fire fighting foams. However,
PFCs, such as perfluorooctane sulfonate（PFOS）and perfluorooctanoic acid（PFOA）have been found to be persistent,
bioaccumulative, and toxicant Furthermore, many studies have been reported on global distribution of PFOS, PFOA and thehomologues, such as perfluorinated carboxylic acids（PFCAs）and perfluorinated alkyl sulfonates（PFASs）. PFCs have been
detected in human bloods, biota, sea water, and remote areas such as the Arctic.
In Japan, PFCs contamination, especially PFOA, in Keihanshin area（the area over Osaka prefecture, Kyoto prefecture, and Hyogo
prefecture）were recognized by many studies. On the other hand, fluoro resin manufacturers and fluoro resin suppliers are
reducing emissions and product content of PFOA, precursors, and higher homologues based on the USEPA’s“2010/15 PFOA
Stewardship Program“. Some companies are using PFCs of C 6 such as Perfluorohexanoic acid（PFHxA）as alternative materials.
Composition of PFCs in the environment may change reflecting these activities. In addition, serum half-life in rats and monkey andsome toxic properties of PFHxA was lower than PFOA. Therefore, it is considered that risk of PFHxA is smaller than PFOA.
In this study, to confirm the concentration and composition of PFCs in the coastal region of Osaka-bay, Japan, total of 12 PFCs,
which were PFOA, PFOS and the homologues, were investigated. The coastal region of Osaka-bay is one of the main denselypopulated and industrial areas in Japan. It seems that the concentrations of PFCs in the sea reflect emission from points and/or
non-point source of the coastal land area. The sea water samples were collected in 2008, 2009, and 2010. The samples were stored
in 4 °C until analysis. The sample was extracted by a solid phase extraction cartridge. The loaded cartridge was eluted by
Methanol. The eluted solution was concentrated to 1 mL. The final solution was carried out by using liquid chromatography（LC）-tandem mass spectrometer（MS/MS）. The analysis was conducted using negative electrospray ionization with multiple reaction
monitoring.
PFHxA and PFOA were detected in all samples. Concentration of PFHxA ranged from 11 to 760 ng/L. Concentration of PFOA
ranged from <MQL to 62 ng/L. Composition ratio of PFHxA was dominant in all samples. The concentrations in the coast had beenreported few ng/L or few hundred ng/L by other studies. The concentrations of PFHxA and PFOA in this study were of similarvalue to other literatures. However, the concentration of PFCs in pacific, which had been considered that there is no influence
from defined source, had been reported few pg/L or few ten pg/L. It seems that there is discharge of PFCs from the coast to thesea. Additionally, as for the characteristics of spatial distribution of PFCs, concentrations at closed-off section were high. PFCs
might be discharged from closed-off section. Concentrations of PFHxA were increased by one order of magnitude from 2008 to
2009. It seems that amount of the discharge had mounted in around 2008.

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 40: Environment II

PTh-139

11:10 － 12:20

Computational Chemistry Study on Negative Ion Chemical Ionization
Mechanism of Peroxyacetyl Nitrate

Yasuyuki ITANO1, Mamoru SAKAI1, Yasuhiro SADANAGA2, Hiroshi BANDOW2Osaka City Institute of Public Health and Environmental Siences, Osaka, Japan, 2Osaka Prefecture University, Sakai,
Japan

1

Keywords:
Electron capture chemical ionization, DFT, MOPAC, Photochemical oxidant
Novel aspects:
The fragmentation and ionization mechanisms of PAN was theoretically elucidated. This helps us to guess the major NICI/MS
fragment ions of unidentified PANs.
Abstract:
Atmospheric photochemical reactions of non-methane hydrocarbons（NMHCs）and nitrogen oxides（NOx）produce wide varietyof peroxyacyl nitrates（PANs, RC（O）OONO2）. They transport NOx far downwind to affect the photochemistry in remote areas.
PANs affect adversely on human, and their mutagenic activities are species-dependent. Recently, negative ion chemical ionization/
mass spectrometer（NICI/MS）was reported as a sensitive and selective detector for PANs. On the other hand, their exlosive andthermally unstable nature makes it difficult to synthesize their authentic standards. In this study, the behaviors of peroxyacetyl
nitrate（PAN, CH 3 C（O）OONO 2 ）, the most common class of PANs, in the NICI/MS source was investigated by means ofcomputational chemistry study to fully understand the production mechanism of the dominant ions by means of density functional
theory and semi-empirical molecular orbital calculations.
The NICI process was initiated by the electron attachment on PAN. The resulting PNA- seemed highly unstable with respect to
the O-O bond dissociation. Besides, the O-N bond seemed also dissociable for PAN- at the Frank-Condon state. The possible
neutral products from the two dissociation channels were electrophilic, and therefore could capture additional electrons to ionizein the NICI/MS source. Accordingly, NO3 -, NO2 -, CH3C（O）O-, and CH3C（O）OO- were possible product ions during the NICI/MS
analysis of PAN.

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 40: Environment II

PTh-140

13:30 － 14:40

Analysis of inadvertent PCBs contained in consumer goods

Masanobu Yokota1, Akiko Sawada1, Yoshikuni Deguchi1, Takeshi Nakano2
1

Kaneka Techno Research Corporation, Hyogo, Japan, 2Osaka University, Osaka, Japan

Keywords:
inadvertent PCB congener yellow pigment
Novel aspects:
Some PCB congeners showing irregular composition profile were found in some pigments, and becoming social concern. Analyses
of inadvertent PCBs contained in consumer goods were carried out.
Abstract:
Although production and use of polychlorinated biphenyls（PCBs）were banned in early 70s in Japan, safe storage and disposal of
PCBs are still social concern. In addition to that,“irregular“ PCB congeners, showing different composition from PCB products,
were found unexpectedly in wastewater（ 1 ）followed by in air and river water（2, 3 ）, giving rise to another social and technicalconcern. The origin of such“irregular“ congeners has been studied and elucidated（ 2 ）； they are from polychlorobenzidine dyes
and production process thereof and released to the environment finally.
Among 209 PCB congeners, 3,3’-dichlorobiphenyl（PCB 11）and 2,5,2’,5’-tetrachlorobiphenyl（PCB 52）are the most typicalinadvertent ones. These congeners in some organic pigments showed different composition profile from the one seen in ordinary
PCB products. This means that some portion of PCBs found in the environment may be the byproducts of pigment. Little attention
had been paid for them until recently, though US Code of Federal Regulations pointed out the unintentional formation of suchbyproducts in the yellow pigments in 1979（ 4 ）.
Based on such a background, Japanese Government placed an official request for emergency survey of PCB content as for several
certain pigments against manufacturers and importers thereof in February 2012（ 5 ）. Several consecutive actions have been
undertaken so far.
From a point of risk management, it is more important to know the exact congener composition than to know total PCB content,
as every congener has its own toxicity varying from the top to the bottom. For such a precise purpose, powerful high resolution
GC-MS is preferably used instead of conventional GC-ECD, because GC-ECD, often used for PCB determination in electricalcondenser oil, cannot differentiate congener peaks.
Organic pigments have replaced traditional Cadmium Yellow aiming to avoid cadmium, and they are now used for wide variety ofindustrial products as well as daily consumer goods. It is quite rational to think“Do yellow retail products carry such congeners?“
Some analytical results are available in the literature（ 6 ）, however ordinary consumers may know very little about the material
composition of consumer goods. That is why upstream compound maker may have it closed with secrecy reason. Therefore weare somehow anxious about yellow stuff, whether it contains unintentional PCB congeners or not.
Analyses of inadvertent PCBs contained in yellow consumer goods such as crayon, ink, paint, lacquer, adhesive tape and so on
were carried out by utilizing capillary GC equipped with high resolution MS in this study. In comparison to the environmental
samples such as air and water, sampling itself was easy as for consumer goods. However, it seemed to be difficult to constructtypical one standard method so as to cover all samples we dealt. From an experimental viewpoint, clean-up protocol shall be
modified case by case according to the property of test sample.
Analytical results obtained are to be presented in this paper.
References
1 ）Litten, S.； Fowler, B.I.； Luszniak, D. Chemosphere, 2002, 46, 1457-1459.
2 ）Hu, D.； Martinez, A.； Hornbuckle, K. C. Environ. Sci. Technol. 2008, 42［21］, 7873-7877.
3 ）Du, S.； Belton, T. J.； Rodenburg, L. A. Environ. Sci. Technol. 2008, 42［11］, 4044-4051.
4 ）40 Code of Federal Regulations； Environmental Protection Agency： Washington DC, 1979, Part 761.31.（g）.
5 ）METI Information； http：//www.meti.go.jp/english/press/2012/0210_04.html
6 ）Rodenburg, L. A.； Guo, J.； Du, S.； Cavallo, G.J. Environ. Sci. Technol. 2010, 44［ 8 ］, 2816-2821.

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 40: Environment II

PTh-141

11:10 － 12:20

Method development for simultaneous analysis of hydroxylated
polychlorinated biphenyl by GC-ECNI/MS in biota sample

Eguchi Akifumi1, Nomiyama Kei1, Katsuhiko Nakagawa2, Tanaka Kouki2, Miyagawa Haruhiko2,
Tanabe Shunsuke1
1

Center for Marine Environmental Studies (CMES), Ehime University, Matsuyama, Japan, 2Shimadzu Corporation, Japan

Keywords:
hydroxylated polychlorinated biphenyls, biota samples, GC-LRMS（ECNI）, isotope dilution
Novel aspects:
The present study is to develop the fast and high sensitive methods for the analysis of OH-PCBs encompassing a wide range ofhomologues in blood sample using GC-LRMS（ECNI）.
Abstract:
Halogenated phenolic compounds are known to be strongly retained in human and wildlife blood. Among those compounds, alarge number of hydroxylated polychlorinated biphenyls（OH-PCBs）have earlier been reported in blood from humans, terrestrial
and marine mammals.
Some analytical methods, which employed mainly gas chromatography coupled to high-resolution mass spectrometry（GC-HRMS）
using electron ionization mode（EI）have already been applied for OH-PCBs analysis. Although GC-HRMS（EI）can provide highsensitive and selective data, the instruments are extremely expensive for routine and high through-put analysis and require
advanced technical skills for operation. A gas chromatography coupled to low-resolution mass spectrometry（GC-LRMS）with
electron capture negative ionization mode（ECNI）is widely used analytical instruments in environmental chemistry.
The objective of the present study is to develop the fast and high sensitive methods for the analysis of OH-PCBs encompassing awide range of homologues in blood sample by isotope dilution method using GC-LRMS（ECNI）analysis.
In biota sample, extracts of human serum（ 2 - 3 g）and whole blood（10g）samples from human and wildlife were analyzed.
Briefly, blood samples were denatured with 6 M HCl and 2 -propanol, and extracted with 50 % methyl t-butyl ether（MTBE）/
hexane. 13C12-labeled PCBs as well as OH-PCBs were spiked as internal standards. The organic phase was partitioned into neutral（containing PCBs）and phenolic（containing OH-PCBs）fractions with KOH（ 1 M ethanol/H 2 O, 1 ：1, v/v）. The alkaline phase（containing OH-PCBs）was acidified to pH 2 with sulfuric acid and re-extracted with MTBE/hexane. OH-PCBs fraction was passed
through non-activated silica-gel column chromatography. OH-PCBs were derivatized with trimethylsilyldiazomethane. The
derivatized solution was further cleaned up by using activated silica-gel column. Identification and quantification of MeO-PCBs
were analyzed by GC-LRMS（ECNI）and compared to GC-HRMS（EI）.
Firstly, we compared the instrumental detection limit（IDL）of OH-PCBs in GC-LRMS（ECNI）and GC-HRMS（EI）. The IDL wasdefined as 3 times the standard deviation（SD）of 5 replicate injections of a low concentration standard solution of OH-PCBs（ 1pg ml-1）. The IDLs were 4 ´OH-triCB29（22 pg）, 4 ´OH-tetraCB79（14 pg）, 4 OH-pentaCB107（0.65 pg）, 4 OH-hexaCB146（1.6pg）,
4 OH-hexaCB159（0.90 pg）, 4 ´OH-hepta-CB172（0.81 pg）,4OH-heptaCB187（0.57 pg）and 4 OH-octaCB201（0.63 pg）by GC-ECNIMS analysis, and compared to 4 ´OH-triCB29（0.82 pg）, 4 ´OH-tetraCB79（0.56 pg）, 4 OH-pentaCB107（0.34 pg）, 4 OHhexaCB146（0.24pg）, 4 OH-hexaCB159（0.23 pg）, 4 ´OH-hepta-CB172（0.40 pg）,4OH-heptaCB187（0.20 pg）and 4 OH-octaCB201（0.52pg）by GC-HRMS（EI）. The sensitivity of penta- to octa- OH-PCBs determined using the GC-LRMS（ECNI）was in agreement with
the GC-HRMS（EI）method. However, sensitivities of GC-LRMS（ECNI）to tri- to tetra- OH-PCBs of GC-LRMS（ECNI）were lower thanof GC-HRMS（EI）.
The recoveries of 13C12 labeled OH-PCB inblood samples were 61-113 %. The method repeatability was in the range 6.7-10.4% and0.6-2.5% relative standard deviation（RSD）for the GC-LRMS（ECNI）and GC-HRMS（EI）systems, respectively.
In conclusion, the GC-LRMS（ECNI）and GC-HRMS（EI）systems were found to be equally well suited for determination for OHPCBs in biological samples at the pg-levels order of penta- to octa-OH-PCBs.

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 40: Environment II

PTh-142

13:30 － 14:40

Suspended solid as a disturbance of PFOS analysis in case of wastewater

Hitomi Oka1, Akiko Sawada1, Yoshikuni Deguchi1, Takeshi Nakano2
1

Kaneka Techno Research Corporation, Hyogo, Japan, 2Osaka University, Osaka, Japan

Keywords:
PFOS SS surrogate recovery COP 4
Novel aspects:
Recovery of surrogate substance in PFOS analysis was investigated to improve current method, and the effect of chain length ofperfluorinatedalkane on the recovery is to be discussed.
Abstract:
Perfluorooctane Sulfonate（PFOS）is one of the Perfluoroalkane Sulfonates（PFASs）and is also the terminal degradation productof many perfluoroalkyl compounds（PFCs）. Specific properties of PFCs such as water repellency, thermal stability, and surface
tension lowering, made these compounds important both for commercial and technical aspects.
As a consequence, PFOS having several emission paths to the environment had been broadly spread out over 50 years. Recently
PFOS was designated and restricted as persistent organic compounds（POPs）by the Stockholm Convention on POPs（ 1 ）.
In Japan, PFOS was specified to the first class chemical under the Chemical Substance Control Law followed by filing technicalguideline for disposal of PFOS containing garbage in March 2011（ 2 ）. Several kinds of standard analytical method using LC-MS/
MS（ 2 - 4 ）had been proposed for surface water prior to that.
Many papers pointed out the technical difficulties arising from the contamination owing to fluorine resin used for connectors or
tubing material in the apparatus, even when clean water was analyzed（ 5 ）.
Other than contamination, we encountered different difficulties when we applied current analytical methods to the wastewater inthe beginning of our study. We experienced low recovery of surrogate substance that was added to the sample to ensure theprecision. Low surrogate recovery did not meet the requirements prescribed in the current analytical standards designed forsurface water.
We found the reason for the difficulties. On one hand, it is known among the lab analysts that concentration of PFOS is prone to
be decreased probably owing to the adsorption toward labware. But on the other hand, little attention was paid for theadsorption onto suspended solid（SS）in case of surface water, in spite of numerous efforts to develop sampling and analyticalmethods for PFOS, since SS content in surface water is generally enough low（ 6 ）.
Development of harmless disposal method undergoes, analysis of wastewater we concern shall be required. Proper care should
be taken for adsorption on SS for the analysis of water containing high SS.
Among elution conditions of PFOS, eluent composition was intensively investigated, and in addition we found both quality andquantity of SS affected on surrogate recovery, leading to an improvement toward current method. We would suggest theimportance of SS remained on the filter upon analytical results.
The last portion of this study relates to the effect of perfluoroalkane chain length on the recovery, and results are to be discussed.
Acknowledgements
The authors thank Mr. Shusuke Takemine of Hyogo Prefectural Institute of Environmental Sciences for helpful discussions about
PFOS nature.

References
1 ）UNEP, The new POPs under the Stockholm Convention, http：//chm.pops.int/pogrammes/newpops/the 9 newpops/tabid/672/
language/en-us/default.aspx
2 ）Ministry of the Environment technical guideline, http：//www.env.go.jp/recycle/misc/pfos/tptc.pdf
3 ）ISO 25101：2009, http：//www.iso.org/iso/catalogue_detail.htm?csnumber=42742
4 ）Ministry of the Environment Survey Manual, http：//www.env.go.jp/water/chosa/h20-03.pdf
5 ）Yamashita, N.； Kannan, K.； Taniyasu, S.； Horii, Y.； Okazawa T.； Petrick, G.； Gamo, T. Environ. Sci. Technol.2004, 38［21］,5522-5528.
6 ）Nakayama, S.F.； Strynar, M. J.； Reiner, J. L.； Delinsky, A. D.； Lindstrom, A.B. Environ. Sci. Technol.2010, 44［11］, 4103-4109.

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 40: Environment II

PTh-143

11:10 － 12:20

Analysis of Metabolites emitted by Soil-Derived Fungi using Ion Mobility
Spectrometry based on GC/MS Data Analysis

Shoko Ichii1, Tomoko Kimura2, Haruna Tanaka1, Sachiyo Kaneko3, Yoko Kiuchi3, Takahito Suzuki4,
Toshiki Sugai5, Takae Takeuchi1,6Department of Chemistry, Graduate School of Humanities and Sciences, Nara Women’s University, Nara, Japan,
Department of Chemistry, Faculty of Science, Nara Women’s University, Nara, Japan, 3Department of Biological
Science, Graduate School of Humanities and Sciences, Nara Women’s University, Nara, Japan, 4Department of
Biological Science, Faculty of Science, Nara Women’s University, Nara, Japan, 5Department of Chemistry, Faculty of
Science, Toho University, Funabashi, Chiba, Japan, 6National Institute of Advanced Industrial Science and Technology(AIST), Osaka Japan
1
2

Keywords:
MVOC； Metabolite； Ion Mobility Spectrometrty； Mass Spectrometry； Cultural Properties
Novel aspects:
The microbial volatile organic compounds emitted from fungi may be applicable to a notification of fungal growth in theenvironment in order to preserve cultural properties using MS or IMS.
Abstract:
Contaminations of fungi were found on the mural paintings in the famous Takamatsuzuka tumulus in Japan［ 1 ］. Fungi oftencause serious damage to cultural properties. The purpose of our work is to detect fungal growth by monitoring Microbial Volatile
Organic Compounds（MVOCs）emitted from fungi at cultural sites.
Ion Mobility Spectrometer（IMS）is suitable for on location measurements because it is portable. Furthermore, it is a powerful tool
to simultaneously detect multiple compounds. However IMS alone cannot be used to identify these MVOCs. In order to identify
these compounds, we also must use GC/MS. In this study, metabolites emitted by soil-derived fungi were analyzed using both Ion
Mobility Spectrometry and Mass Spectrometry.
Alcohols, aldehydes and ketones were found as MVOCs in most of Aspergillusfumigatus, Aspergillus nidulans, Fusarium solani and
Penicillium paneum. On the other hand, sesquiterpenes were found in only specific fungi. Because each fungal strain had acharacteristic compound（sesquiterpene）, these compounds are useful in identifying their respective fungi. As the number ofspores increased with the fungal cultivation period, it was found that the amount of these MVOCs, ketones, aldehydes andalcohols also increased. Therefore, 3 -octanone is suited to be an indicator of the size of fungi（fungal amounts）because of thepositive correlation found between the number of spores and the amount of the compound. On the other hand, sesquiterpenes
showed a peak of MVOCs at a particular period right before spore reproduction and therefore are useful to identify both fungal
species and their reproduction periods［ 2 ］.
Ion Mobility Spectra of volatile metabolites emitted from A. nidulans were measured using the IMS-MINI Ion Mobility Spectrometer（I.U.T. GmbH, Germany）. In the IMS driftgram of MVOCs from A. nidulans, a large peak appeared at 6.36ms which correspond to“Reaction Ion Peak“（RIP）. From the assignment based on the comparison with GC/MS data analysis of MVOCs from A.
nidulans cultivated under the same condition as samples measured by IMS； it was found that relatively smaller peaks appeared
at 7.69, 8.31 and 9.68ms were assigned to phenyl acetaldehyde, 3 -octanone and bisabolene, respectively. A peak at 7.50 ms
corresponds to 2 -octen- 1 -ol and 1 -octen- 3 -ol. The IMSdriftgram of volatile metabolites emitted P. paneum showed the peakcorresponding to beta-Caryophyllene. This compound is one of sesquiterpenes and an unique MVOC for P. paneum. Therefore, it
was concluded that P. paneum can be monitored using only a driftgram of MVOC from P. paneum.
The IMS drift time for these compounds was also calculated using Monte Carlo simulations.
The calculated drift time was consist with the experiments.
References［ 1 ］T. Kiyuna, K.-D. An, R. Kigawa, C. Sano, S. Miura, J. Sugiyama, Mycoscience2008, 49, 298.
［ 2 ］T. Takeuchi, T. Kimura, H. Tanaka,a S. Kaneko, S. Ichii, M, Kiuchi and T. Suzuki, Surf. Interface Anal. 2012, 44(6), in press.

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 40: Environment II

PTh-144

13:30 － 14:40

Measurement of brominated flame retardants in the environment nearfactories by LC/MS/MS.

Itaru Oue, Shigeru Suzuki

CHUBU University,Aichi,Japan

Keywords:
BFRs, road-dusts, LC/MS/MS, factories
Novel aspects:
BFRs in river waters, sediments, effluents and road-dusts near factories were determined by LC/MS/MS.
Abstract:
Introduction： Brominated flame retardants（BFRs）are widely used in plastics, rubbers, textiles and other products for fire
protection, and are discharged into the environment through air emissions, water discharges, wastes and end products. In theprevious studies, the authors found unexpected contaminations with BFRs in not only plastic products such as baby toys, chew
toys and household products, but in road dust around the non BFR plastic moulding factories. Here we present a researchpredominantly taken place around factories by measuring BFRs in effluents, river waters, sediments and road dusts.
Method： Waters, sediments and road dusts were collected in rivers, effluent water ways or road sides near the factories. Water
samples were added with formic acid（500μL to every 500mL of water）and stored in cool and dark place. Sediment samples werecollected in glass bottles and stored in freezer. Road dusts were sampled in glass bottles and stored in cool and dark place.
Water sample was filtered and the filtrate was extracted by solid phase extraction（SPE）with C18 sorbent（Inert Sep C18, 500 mg）
which was conditioned with methanol（ 5 mL）and distilled water（ 5 mL）in the order. The sample loaded cartridge was cleaned
by flushing with 50%methanol（ 1 mL）, and then the target substances ware eluted from the cartridge with methanol（ 5 ml）andacetone（ 5 mL）. The residual suspended solids on the filter and the glassware used for sample preparations were rinsed in theorder with acetone（ 5 mL）and dichloromethane（ 5 mL）, which were added to the C18 extract. The extract was concentrated
with nitrogen, and the solvents were exchanged to acetonitrile followed by LC/MS/MS（with API3000）. Sediments and road dusts
were extracted in the order with acetone（ 5 mL, two times）and dichloromethane（ 5 mL, two times）. The extract wereconcentrated and cleaned up by SPE with C18, and measured by LC/MS/MS.
Predominant result： ： The method recoveries were studied by analyzing five replicates of samples spiked with
Hexabromocyclododecane（HBCD）, tetrabromobisphenol A（TBBPA）, tribromophenol（TBP）and bisphenol A（BPA）. Recoveries
of HBCD, TBBPA, TBP and BPA were 63% with relative standard deviation（RSD）of 8 %, 102% with RSD of 17%, 98% with RSD of 12%
and 106% with RSD of 4 %, respectively. It would be the reason for the lower recovery of HBCD（around 60%）that the HBCD isextremely hydrophobic substance, which might be adsorbed on the surface of glassware, SPE adsorbents and other hydrophobic
materials.
In research of a dyeing processing factory, water concentrations of TBP and BPA were 1.3-6.3ppt and 9.0-950ppt, respectively. On
the other hand, those of HBCD were lower than limit of detection in most samples excepting in the samples of the effluent andnear the effluent discharge point.in a river. All other results will be presented in the poster.

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 40: Environment II

PTh-145

11:10 － 12:20

Determination of sulfonamides and tetracyclines in livestock wastewaterusing hybrid ion trap - time of flight mass spectrometer

Youngmin Hong1, Hyunook Kim2

1
Dong-il Shimadzu Corp., Seoul, Korea, 2Department of Energy & Environmental System Engineering, The University ofSeoul, Seoul, Korea

Keywords:
Sulfonamide, Tetracycline, Ion Trap, Time of Flight, Wastewater
Novel aspects:
Analytical method of sulfonamides and tetracyclines using hybrid ion trap - time of flight MS was described. Sample extraction was
carried out with a HLB and a MCX cartridges.
Abstract:
Veterinary drugs are widely used in large quantities for treatment of disease or to promote animal growth. Nowadays, veterinary
drugs are recognized as not only medicines but also as ’Newly Emerging Contaminants’ in the environment. The aim of this study
is to monitor organic compounds in aqueous samples using Liquid chromatograph coupled to hybrid ion trap - time of flight mass
spectrometer. The analyzed compounds were 3 sulfonamides（i.e., sulfathiazole, sulfamethazine, and sulfamethoxazole）, and 2
tetracyclines（i.e., oxytetracycline and chlortetracycline）in samples from a livestock wastewater treatment plant（WWTP）.
Sample extraction was carried out with a hydrophilic-lipophilic balance and a mixed-mode cation exchange solid phase extraction
cartridges. Average recoveries of sulfonamides at fortification levels from 1.0 and 4.0 μg L-1 in effluent of a local domestic WWTP
were 73-95 % and 89-104 % , respectively, while tetracyclines at 0.4 and 4.0 μg L-1 were 76-104 % and 101-107 % , respectively. The
method detection limits were 22.8, 23.0, 25.8, 23.6, and 9.8 ng L-1 for sulfathiazole, sulfamethazine, sulfamethoxazole, oxytetracycline,
and chlortetracycline, respectively. The maximum concentration of sulfonamide and tetracycline residues detected in samples
were 49.5 and 4.1 μg L-1, respectively. The developed method could be applied successfully to quantitate residual sulfonamides
and tetracyclines in animal wastewater. The analysis of the samples showed that more than 90% of target pharmaceuticals were
removed in the animal WWTP consisting of a biological process, a UF membrane, and a coagulation process.

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 40: Environment II

PTh-146

13:30 － 14:40

Dechlorane Plus, a highly chlorinated flame retardant in Japaneseenvironment samples.

Takanori Sakiyama1, Takeshi Nakano2
1

Osaka City Institute of Public Health and Environmental Sciences, Osaka, Japan, 2Osaka University, Osaka, Japan

Keywords:
Dechlorane Plus, Japan, Environment
Novel aspects:
Dechlorane Plus was determined in environment samples for the first time in Japan.
Abstract:
Dechlorane Plus（DP）is a highly chlorinated flame retardant, was developed to replace Mirex（Dechlorane）that was banned in
the 1970s. It is manufactured about 450 ton per year by Occidental Chemical Corporation（OxyChem）in the United States. In 2006,
the first of sightings of this chlorinated flame retardant were in the Great Lakes region, and the investigation reports are increasing
rapidly recently in the world, mainly in North America. More recently, a newly DP production facility was discovered in China. It isestimated that Chinese capacity of DP production has reached 2,000 ton in 2006.
However, to our knowledge, no data for levels of DP in environmental samples were available in Japan. Therefore, we measured
DP by using the GC / high resolution MS in EI mode, and identified this compound in the environmental samples collected fromJapanese urban area. DP was detected in house dust, deposit of the window frame, road sediment, garden soil and sediment
samples at concentrations ranging from 2.9-42ng/g-dry,240-270ng/g-dry, 74-150ng/g-dry, 1.7ng/g-dry, 17-140ng/g-dry, respectively.
Further, we showed the mean of anti-DP fractional abundance（fanti）value in these Japanese samples was 0.65, 0.83, 0.80, 0.81, 0.81,
respectively. This compound had not been identified in the environment of Japan before our report. In this conference, we will
indicate the detailed additional data of DP in surface sediment samples of the urban river in Japan.

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 40: Environment II

PTh-147

11:10 － 12:20

Multi-residue method for rapid screening of veterinary drugs in muscle
matrices by UHPLC-MS/MS

Youngmin Hong1, Sujeong Park2, Insun Lee1, Minhye Lee1
1

Dong-il Shimadzu Corp. , Seoul, Korea, 2Animal, Plant and Fisheries Quarantine and Inspection Agency, Seoul, Korea

Keywords:
Multi-residue screening, Veterinary drugs, UHPLC-MS/MS
Novel aspects:
A rapid multi-residue screening method was developed for the 180 veterinary drugs and their metabolites using a UHPLC-MS/MS.
Abstract:
A rapid multi-residue screening method was developed for the simultaneous analysis of about 180 veterinary drugs and their
metabolites using a ultra-high-performance liquid chromatograph coupled with tandem mass spectrometer（UHPLC-MS/MS）. The
screened veterinary drugs belonged to amphenicols, anthelmintics, benzimidazoles,
β-lactams, coccidiostats, ionophores,
macrolides, non-steroidal anti-inflammatory agents, quinolones, sulfonamides, tetracyclines and tranquilizers. The drugs wereextracted from bovine, porcine, and chicken muscle samples with acetonitrile water（ 4 ：1, v/v）containing 2 mM ammonium
formate, and the extracts were applied to the dispersive solid phase extraction and n-hexane clean-up procedure. Reverse-phase
LC separation was accomplished on a C18 column and gradient elution with a mobile phase consisting of（A）water containing
0.1% formic acid and（B）acetonitrile containing 0.1% formic acid. The method had a chromatographic total run time of 5 min. The
separated compounds were detected with a Shimadzu LCMS-8030 tandem quadrupole mass spectrometer operating with anelectrospray ion source（ESI）in positive and negative switching mode by applying a time scheduled multiple reaction monitoringof 2 or 3 transitions. The developed method was validated according to the EU Commission Decision 2002/657/EEC for aquantitative screening method. All the validation data, such as the mean accuracy, the repeatability, the within-laboratory
reproducibility and the detection capability, CCβ accuracy were within the required limits.

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 40: Environment II

PTh-148

13:30 － 14:40

Development of LC-MS/MS method to monitor pharmaceuticals inenvironmental wastewater

Youngmin Hong, Insun Lee, Minhye Lee
Dongil Shimadzu, Seoul, Korea

Keywords:
Pharmaceutical, Wastewater, UHPLC-MS/MS
Novel aspects:
An analytical method for screening and confirming of 30 pharmaceuticals in water and wastewater samples was developed usinga UHPLC-MS/MS.
Abstract:
A rapid and reliable LC-MS/MS method was developed for the simultaneous identification, confirmation and quantification of 30
pharmaceuticals in water and wastewater samples. Two distinct chromatographic conditions were used according to the polarity.
The method used sub two micron size C18 and hydrophilic interaction chromatography columns. The separated compounds weredetected with a Shimadzu LCMS-8030 tandem quadrupole mass spectrometer operating with an electrospray ion source（ESI）in
positive and negative switching mode by applying a time scheduled multiple reaction monitoring of 2 or 3 transitions. The
analytical performance of the method was evaluated for sample collected from a local domestic wastewater treatment plant.
Sample extraction was carried out with a hydrophilic-lipophilic balance and a mixed-mode cation exchange solid phase extraction
cartridges. To minimize the matrix effect, matrix-matched standards were analyzed in a wastewater effluent. Under the study, allcompounds gave good sensitivity over a level of ng L-1 with r2 values of 0.99 or greater. Average recoveries of all compounds at 10
and 1,000 ng L-1 were about 54-105%.

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 40: Environment II

PTh-149

11:10 － 12:20

Analysis of Ultraviolet Absorbers in Urine Samples by Functionalized
Nanomaterial-assisted Electrospray Mass Spectrometry

Tzung-Jie Yang, Maw-Rong Lee

National Chung Hsing university, Taichung, Taiwan

Keywords:
Ultraviolet Absorbers； Functionalized Nanomaterial-assisted Electrospray Mass Spectrometry
Novel aspects:
The nanomaterial-assisted electrospray ionization offers a high specific and high throughput screening for trace analysis.
Abstract:
Functionalized nanomaterials assisted electrospray ionization for ananlysis of ultraviolet absorbers in urine was developed in thisstudy. The electrospray was performed with high voltage on a laboratory-made screen-printed plate with coating a layer of silver
or graphite. The ultraviolet absorbers studies in this study are including 3 -（ 4 -methylbenzylidene）-camphor（ 4 -MBC）,
2 -ethyl- 4 -（dimethylamino）benzoate（OD-PABA）and 2 -hydroxy- 4 -（octyloxy）-benzophenone（BP-12）. The analytes wereextracted with nanomaterials that included iron oxide-coated silicon dioxide nanoparticles, multiwall carbon nanotubes（MWCNTs）and graphene oxide（GOx）was dipped on the screen-printed substrate to evaluate the effect on the ionization
efficiency. After extraction, the extract or nanomaterial without doing any desorption. From the results, the MWCNTs can enhance
the intensity of the signal detected and short the analytical time. This technique demonstrates that the nanoparticles assisted
electrospray ionization offers a high specific and fast screening in trace analysis.

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 40: Environment II

PTh-150

13:30 － 14:40

Determination of diuretics in urine using immobilized-multiwalled carbon
nanotubes hollow fiber liquid-phase microextraction combined with liquid
chromatography-mass spectrometry

Tse-Tsung Ho, Maw-Rong Lee

National Chung Hsing university, Taichung, Taiwan

Keywords:
Diuretics； multiwalled carbon nanotubes； hollow fiber liquid-phase microextraction； liquid chromatography-mass spectrometry
Novel aspects:
The laboratory-made immobilized-multiwalled carbon nanotubes（I-MWCNTs）in hollow fiber liquid-phase microextraction（HFLPME）combined with LC-MS for analyzing diuretics in urine.
Abstract:
Diuretics, which increase urine flow from the kidneys, are used for treatment of heart conditions, liver, kidney and lung disease,generally to reduce salt or water retention. The Medical Commission of the International Olympic Committee（IOC）included
diuretics as a banned substance since 1986. This study was to evaluate a sample treatment technique using immobilizedmultiwalled carbon nanotubes（I-MWCNTs）in hollow fiber liquid-phase microextraction（HF-LPME）combined with liquid
chromatography-mass spectrometry for diuretics analysis in urine samples. The optimal conditions of sample extraction and mass
spectrometry have been studied. The detection limits for the diuretics studied were found to be in the range of 0.19 to 0.96 ng/mL
with the relative standard deviation（RSD）below 11.1%. No carryover effect was found, and every laboratory-made I-MWCNTs HFLPME could be reused for extraction up to 50 times with recovery efficiency above 85%. The I-MWCNTs HF-LPME has been proveneffectively for increasing extraction efficiency and reducing matrix interference from urine. The method developed offers not only
very high sensitivity for determination of trace diuretics in urine, but also reduced extraction costs for having a long use times of
the I-MWCNTs HF. The method is recommended for determination of trace diuretics in urine for its elegance, simplicity and highsensitivity even in the presence of high levels of interference.

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 40: Environment II

PTh-151

11:10 － 12:20

In-line preconcentration capillary electrophoresis-electrospray ionizationmass spectrometry for the analysis of haloacetic acids in tap water

Sih-Hua Hung, Guor-Rong Her

National Taiwan University, Taipei, Taiwan(R.O.C)

Keywords:
haloacetic acids, in-line preconcentration, CE-MS, tap water
Novel aspects:
A simple and sensitive FASI-CE-ESI-MS/MS method was developed for in-line monitoring haloacetic acids in tap water.
Abstract:
Haloacetic acids（HAAs）belong to an important class of disinfection byproducts that are being regulated. A strategy based on inline preconcentration CE-ESI-MS/MS was developed for determination of haloacetic acids（HAAs）in tap water. The field amplified
sample injection（FASI）technique was used for in-line preconcentration of five HAAs including monoacetic acid（MCAA）,
dichloroacetic acid（DCAA）, trichloroacetic acid（TCAA）, dibromoacetic acid（DBAA）and monobromoacetic acid（MBAA）. In
FASI-CE-MS/MS analysis, 50% MeOH containing 2.5%ammonium acetate（pH 3.5）was used as background electrolyte and sheathliquid. Low sheath flow interface was used to preserve separation condition and to stabilize electrospray ionization process. With
30 second hydrodynamic injection of a water plug followed by 20 second electrokinetic injection of the sample, FASI enrichment
factors in a range of 1000-3000 were obtained. Detection limits of HAAs were in a range of 0.01-0.1 μgL-1. In tap water analysis, three
HAAs including TCAA, DCAA and MBAA were detected at a concentration about 0.1- 1 ppb, 0.1- 1 ppb and 0 -0.1ppb, respectively.
The feasibility of FASI-CE-ESI-MS/MS in real time analysis is demonstrated by continuous monitoring HAAs in tap water.

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 40: Environment II

PTh-152

13:30 － 14:40

Effects of silver on Chlamydomonas reinhardtii; insights from proteome
analysis and physiological endpoints

Marc J Suter, Smitha Pillai, Holger Nestler, Rene Schoenenberger, Renata Behra, Kristin Schirmer
Eawag, Dubendorf, Switzerland

Keywords:
proteomics, silver, Chlamydomonas reinhardtii, ecotoxicology
Novel aspects:
systems biology view of silver-induced stress response in green algae. Linking of molecular responses to physiological effects
Abstract:
Classic ecotoxicology has focused upon a bottom-up approach to understand stressor effects in which a few genes, proteins, orbiochemical reactions are studied at a time. The invention of new technologies in the last decade has enabled the analysis ofwhole transcriptome（gene transcripts）, proteome（proteins）and small cellular molecules（metabolite profiling）resulting inwhole system approaches. By complementing the traditional approach with the system biology it is possible to define the genetic,
protein, and biochemical reactions as integrated and interacting networks of anorganism. We use such a systems biology
approach to understand the biological responses of Chlamydomonas reinhardtii to ionic silver, which is toxic to a variety ofaquatic organisms in the nanomolar range. Specifically, the proteome profiling by Multidimensional Protein Identification
Technology（MudPIT）allows determination of differential expression of proteins.
MudPIT analysis of C.reinhardtii exposed to silver allowed discrete identification of roughly 2500 proteins in each sample with false
discovery rate set to 2 % , representing major cellular processes. Enrichment analysis showed significant regulation of several
biological pathways. Key among them were photosynthesis, ATP synthesis and tetrapyrolle synthesis with all being severely downregulated. Differently, some pathways that were up-regulated were the lipid synthesis, oxidative stress response, proteolysis andcell wall synthesis. Our results provide the first insights into the mechanisms of toxicity of ionic silver. Silver is taken up into thecells via active metal transporters and inhibits key proteins involved in photosynthesis and ATP synthesis. Silver also induces
oxidative stress as deduced from the induction of oxidative stress response proteins such as GPXH. The up-regulation of lipid
synthesis also indicates an autophagy response. Importantly, we could link the changes at the proteome to the physiological stateof the algae on exposure to silver.

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 40: Environment II

PTh-154

13:30 － 14:40

on-site analysis of gases emitted from soils using MULTUM-S II

Takahiro Anan1, Shuichi Shimma2, Yo Toma3, Yasuyuki Hashidoko4, Ryusuke Hatano4, Morio Ishihara1,
Jun Aoki1, Michisato Toyoda1
1Osaka University,Osaka,Japan, 2National Cancer Center Research Institute, Tokyo, Japan, 3Ehime University, Ehime,
Japan, 4Hokkaido University, Hokkaido, Japan

Keywords:
on-site analysis, N 2 O
Novel aspects:
We detected N 2 O in the atmospheric concentration level（314ppb）by using the MULTUM-S II within one minute.
Abstract:
Recently, miniature mass spectrometers which can be used for on-site analysis have been designed and developed. These
instruments are expected to have widespread applications. In our laboratory, a miniaturized multi-turn time-of-flight mass
spectrometer“MULTUM-S II“ was designed and constructed. Although the size of newly developed“MULTUM-S II“ was 45 cm x
25 cm x 64 cm, it has capability of high mass resolution of more than 30, 000.
We attempted to carry out real-time monitoring of the microbial production in the soil and consumption of gaseous compounds（N2O, CO2, CO and CH4）by using this MULTUM-S II. In this study, we attempted to monitor of N2O concentration in real-time.
N2O is known as one of the important greenhouse gases, and its warming effect is 298 times higher than that of CO2. Atmospheric
N 2O concentration level is 314ppb and increases to several hundred ppm by bacterial activities. N2O is mainly produced by N
fertilizer in agricultural soils. Therefore, on-site high performance N 2 O analyzer has been required to elucidate generating
mechanism and estimate reduction procedure of generated N2O from agricultural soils. However, N2O is measured by using a gas
chromatography（GC）with an electron capture detector（ECD）so that it is difficult to apply GC/ECD on the field due to theradioisotope（63Ni）in the detector. On the other hand, we can measure N2O in the field by GC/MS. In this method, it takes about
ten minutes to separate CO2 and N2O in a separation column. If we could separate CO2 and N2O by using a high-mass resolution
mass spectrometer, the measurement time would be reduced significantly. However, required mass resolution which is toseparate CO2 and N2O doublet is larger than 8, 000 due to the mass difference is 0.0113 u. Therefore, conventional miniaturized
instrument can’t be separate the doublet. MULTUM-S II can provide high mass resolution in the compact size. Therefore, we
attempted to separate CO 2 and N 2 O doublet. In this experiment, we used 5 m PLOT column to separate N 2 O, CO 2 and main
components of air（N2 and O2）. When large amounts of N2 and O2 are injected into the ion source at the same time, low amountN2O signal is suppressed. Using this procedure, we detected N2O in the atmospheric concentration level（314ppb）. This result
was significant increase compared to direct sample injection. Furthermore, the measurement time was within one minute. We
also developed an automatic sampler to inject these gases into MULTUM-S II in one minute intervals for automatic real-timemonitoring. As a result, real-time monitoring of atmospheric N2O concentration and its variation of emission from soils can beachieved by the combination of the automatic sampler and the MULTUM-S II .

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 40: Environment II

PTh-155

11:10 － 12:20

Level of Dechlorane Plus in ambient air and development of monitoring
methodKoichiro MATSUMOTO1, Yuko ODASHIMA1, Yuji KASHIMA1, Takanori SAKIYAMA2, Takeshi NAKANO3

1Japan Environmental Sanitation Center, Kanagawa, Japan, 2Osaka City Institute of Public Health and Environmental
Sciences, Osaka, Japan, 3Graduate School of Engineering, Osaka University, Osaka, Japan

Keywords:
Dechlorane Plus, ambient air, NCI
Novel aspects:
Dechlorane plus were determined in air samples for the first time in Japan.
Abstract:
A chlorinated flame retardant, Dechlorane Plus（DP）（syn-, anti-）, was detected and identified in ambient air from Japanese
urban area. This study shows that DP was detected in air samples in Japan for the first time. In a moment of this research, weinvestigated the mode of ionization using GC/MS availability. GC/MS is commonly applying to determine DP with negative
chemical ionization（NCI）mode at low resolution（LR）. DP was also determined magnetic sector-type MS with electron impact
ionization（EI）mode at high resolution（HR）. R 2 for calibration curves on DP（syn-, anti-）were over 0.9999 of LR-NCI and HR-EI
method. Instrument Detection Limits（IDL）of both methods for DP（syn-, anti-）were 0.20 pg/m3, 0.23 pg/m3 and 0.34 pg/m3, 0.36
pg/m3 respectively. LR-NCI method was S/N = 50 at 0.1 ng/mL standard solution that approximately four times greater than HR-EI
method. Result of compared both methods, LR-NCI method was applying in this study.
Air samples were collected on the rooftop of Japan Environmental Sanitation Center. Samples were collected 24 hours andrepeated 4 times. All samplings were implemented duplicate. Result of measurement by GC/MS（LR-NCI）, DP air concentration
ranged from 1.9 to 21 pg/m3. Recoveries of 13C labeled DP were more than 90%. The syn- and anti- ratio were read between 0.24
and 0.48. All duplicates were considerably fit in each sample. DP concentrations were higher than Mirex to compare withEnvironmental Survey and Monitoring of Chemicals was implemented by Ministry of the Environment Japan.

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 40: Environment II

PTh-156

13:30 － 14:40

Determination of hydroxylated polycyclic aromatic hydrocarbons inmariner’s urine by high performance liquid chromatography-tandem mass
spectrometry

Tetsuya Hirai1,2, Hiroaki Kinoshita1, Hideo Okamura2, Yoshiji Yano2, Takeshi Nakano2
1

Otsuka Pharmaceutical Co., Tokushima, Japan, 2Graduate School of Maritime Sciences Kobe University, Kobe, Japan

Keywords:
Polycyclic aromatic hydrocarbons, Urine, Human exposure, LC/MS/MS, metabolite
Novel aspects:
We assess the potential health risks posed by exhaust gas from ship and obtain a better understanding of the occupational
hazards connected with PAHs exposure.
Abstract:
Polycyclic aromatic hydrocarbons（PAHs）are a group of chemicals released into the air during the incomplete burning of fossil
fuels such as gasoline and other organic substances
Some PAHs are reasonable anticipated to be human carcinogens. The carcinogenic risk to humans among 16 PAHs is classified asprobable human carcinogens by the International Agency of Research on Cancer in 2010. PAHs are also known to have endocrine
disrupting activity. PAHs are absorbed into the human body through the skin, lungs and gastrointestinal tract and are thenmetabolized to their hydroxylated PAHs（OHPAHs）and finally excreted in urine. In this study, a high performance liquid
chromatography-tandem mass spectrometry method has been developed for the simultaneous quantification of six urinary
OHPAHs, including 1 -hydroxynaphthalene（ 1 -OHNap）, 2 -hydroxynaphthalene（ 2 -OHNap）, 1 -hydroxyphenanthrene（ 1 -OHPhe）,
3 -hydroxyphenanthrene（ 3 -OHPhe）, 4 -hydroxyphenanthrene（ 4 -OHPhe）and 1 -hydroxypyrene（ 1 -OHPyr）in human urine.
Deuterated 3 -OHPhe-d 9 and 1 -OHPyr-d 9 were used for the quantification of the analyte as internal standards. Considerable
amounts of PAHs are present in the workplace. PAHs exposure is reported high in coke plants, aluminium work and paving work.
In order to assess the potential health risks posed by exhaust gas from ship and obtain a better understanding of the occupational
hazards connected with PAHs exposure, the concentration of OHPAHs in urine collected from mariners has been analyzed.
The urine sample treatment involved enzymatic hydrolysis of glucuronide and sulfate conjugates followed by solid-phaseextraction using Sep-Pak C 18 cartridge for LC/MS/MS analysis. The LC/MS/MS system consisted of an Agilent 1260 infinity series（Agilent Technologies, Santa Clara, CA, USA）and QTRP 5500 mass spectrometer（AB SCIEX, Framingham, MA, USA）. The analyte
and ISTD were separated from interference peaks on an Ascentis Express C18 column（2.1mmID × 100mm, 2.7μm particle size；
SIGMA-ALDRICH Co., St. Louis, MO, USA）. The mass spectrometer was operated under multiple reaction monitoring（MRM）
negative mode for the ion transitions m/z 143 →115（OHNap）, m/z 193 →193（OHPhe）, m/z 202 →202（OHPhe-d 9 ）, m/z 217 →189（OHPyr）and m/z 226 → 198（OHPyr-d 9 ）.
This method was applied to the analysis of OH-PAHs in 29 urine specimens（11 engineers and 18 other crews）collected from thecrews on a ship. The average concentrations of urinary 1 -OHNap, 2 -OHNap, 1 -OHPhe, 3 -OHPhe, 4 -OHPhe and 1 -OHPyr were
1.6, 2.2, 0.2, 0.2,0.1 and 0.2 μg/g creatinine in engineers and 0.5, 1.8, 0.2, 0.1, 0.1 and 0.1μg/g creatinine in other crews, respectively.
The method can be used to evaluate occupational exposure to PAHs.

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 40: Environment II

PTh-157

11:10 － 12:20

Thermal solid phase extraction for GC-MS analysis of complex samples

Vivi Kofoed-Soerensen, Asger W Noergaard, Peder Wolkoff, Per Axel Clausen
National Research Centre for the Working Environment (NRCWE)

Keywords:
GC-MS, thermal desorption, solid phase extraction, sample preparation
Novel aspects:
Thermal solid phase extraction（TSPE）is a fast and simple sample preparation for elimination of unwanted matrix compounds in
GC-MS analysis of VOC and SVOC in complex matrices.
Abstract:
Introduction
Pristine samples or sample extracts often contain matrix compounds and/or solvents, which are incompatible with gas
chromatographic analysis. Thus, an often complicated and time-consuming sample preparation with potential loss of analytes isneeded before analysis. Here we present thermal solid phase extraction（TSPE）as a simple and fast approach based on thermal
desorption（TD）for analysis of samples in complex matrixes. Samples are simply injected onto Tenax TA adsorbent tube andsolvents（e.g. methanol, ethanol or water）are subsequently removed by purging with helium at ambient temperature. VOCs andSVOCs with boiling points up to ca. 400 °C are released during the following thermal desorption whereas larger and non-volatilecompounds（e.g. sugars, proteins, fat, salts etc.）are retained by the adsorbent. Since TSPE may also be used for concentration of
trace level samples by large volume injection onto the adsorbent, it can replace the following sample preparation steps：
1 ）Removal of unwanted matrix compounds that deteriorate the analysis
2 ）Solvent change to a suitable GC solvent3 ）Up-concentration（e.g. by evaporation）of analytes before GC-MS analysis
Here TSPE has successfully been applied to the analysis of whisky and extracts of floor dust and tattoo inks.
Experimental
Single malt whiskies or methanol extracts of floor dust and tattoo inks were injected into stainless steel tubes containing Tenax
TA adsorbent. The injections were followed by purging with He（60 ml/min for 3 - 5 min）. Injection volumes were 1 -50 µl. A
thermal desorber（Perkin-Elmer ATD 400）was connected to a GC-MS system（Perkin-Elmer Autosystem XL GC/TurboMass MS or
Varian CP 3800 GC/1200 MS）. The Tenax TA tubes were desorbed for 20 min at 250 °C, using a He flow of 50 mL/min, and a cold
trap temperature of -30 °C. The cold trap was narrow bore and packed with Tenax TA for VOCs and empty for SVOCs. Flashheating of the cold trap to 300 °C transferred the analytes through the transfer line（225 °C）to the GC equipped with a 30 m VF5 ms column. The mass spectrometers were operated in electron ionization mode using full-scan（m/z 50-400）.
Preliminary results
TSPE showed good chromatographic performance and minimization of co-eluting peaks. So far the method has showed to bequantitative within the range of VOC/SVOC from toluene to di- 2 -ethylhexyl phthalate（DEHP）. The limit of detection of DEHP
was estimated to 31 ng determined as three times the standard deviation of the mean of a low-concentration standard.
Whisky： For comparison several different whiskies brands（e.g. Ardbeg, Macallan and Glenlivet）were analyzed. The resulting
chromatograms showed good chromatographic resolution and clear differences, thus facilitating（easy）distinction of the whiskiesfrom one another. Phenols, cresols, acids and esters were identified as the main eluting compounds.
Floor dust： Comparison of floor dust samples from different locations in Denmark showed that the main constituents were
phthalates（mainly DEHP）, fatty acids and hydrocarbons. The main problem for direct on-column injection of floor dust extracts
are the high content of fat. This problem was eliminated by TSPE.
Tattoo inks： A series of black tattoo inks（high content of carbon black pigment）were screened and compared for their content
of organic compounds. They contained a large number of compounds including butanediol, phenol, trichlorobenzen, Texanol, fatty
acid methyl esters, phthalates and PAHs.
The range of useful solvents is limited by the properties of Tenax TA to solvents like methanol, pentane and water. However, thismay be extended to other solvents by use of other adsorbents.

TSPE has a large potential as a fast analytical method with minimal sample preparation for volatile and semi-volatile organic
compounds in a wide range of sample matrices.

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 40: Environment II

PTh-158

13:30 － 14:40

Organic coatings of engineered nanomaterials characterized by mass
spectrometry

Per Axel Clausen, Renie Birkedal, Vivi Kofoed-Sorensen, Asger W Norgaard, Keld A Jensen
National Research Centre for the Working Environment, Denmark (pac@nrcwe.dk)

Keywords:
Engineered nanomaterials, surface coating, GC-MS, LC-MS, NALDI
Novel aspects:
This is the first general approach using mass spectrometry for quantitative characterization of organic surface modifications ofengineered nanomaterials.
Abstract:
Introduction
Engineered nanomaterials（ENM）are often chemically surface modified in order to tailor their physical-chemical properties for
specific applications. For example, lipophilic surfaces are needed for incorporation of ENM in polymers whereas hydrophilic
surfaces are used in water-based paints. These surface modifications may have influence on the toxicological and environmental
properties of the ENM, but they are often trade secrets. Therefore, analytical procedures are needed to reliably enable detection
and quantification of unknown ENM surface modifications. Only a limited number of publications describe quantitative methods
for this type of application and despite of the obvious advantage of using mass spectrometry（MS）only a few studies describe theuse of MS. Here we present an approach, based on several MS techniques, to characterize high temperature extractable andpredominantly non-covalent bounded organic coatings of ENM.
Experimental
Thermogravimetric analysis（TGA）was used to identify ENM with organic coating. The following ENM with more than 1 wt %apparent coating were investigated： Organoclays, graphite, synthetic amorphous silica, titanium dioxide, silver, calcium carbonate,iron oxide, and nickel-zink-iron oxide. The ENM were extracted with pressurized liquid extraction（PLE）using methanol at 200 °C
and 140 bar. The extract was centrifuged and the supernatant used for the succeeding analysis.
MS combined with on-column gas chromatography（GC-MS）or thermal desorption was used to analyze the extracts for volatileorganic compounds. A 30 m 5 % phenylmethylsilicone column was used for GC and the MS was run in electron ionization（EI）
mode and the mass range was m/z 50 to 500. The identification of organic compounds was based on searches in the NIST 2011 MS
library and authentic standards.
Quadrupole time-of-flight MS combined with liquid chromatography（LC-MS）or direct infusion was used for analysis of nonvolatile organic compounds in the extracts using both electrospray ionization and atmospheric pressure chemical ionization. The
MS was operated in positive mode and the mass range was usually m/z 50 to 3000. Collision induced dissociation was used forstructure determination.
Nanostructured surface-assisted laser desorption ionization time-of-flight MS（NALDI-TOF）was used for characterization ofpolymeric compounds. 1 µl of either a suspension of the ENM in methanol or the PLE extract was deposited on the NALDI target.
Sodium trifluoro acetate was used as cationizing agent. Spectra were acquired in the reflector-positive mode and mass range wasm/z 50-5000. The identification of the organic compounds from the NALDI-TOF results was based on pattern recognition, literature,
and GC-MS data.
Results
A wide range of organic compounds was extractable from the ENM and their identity was confirmed by authentic standards inseveral cases. For example organoclays were surface functionalized with quaternary ammonium compounds containing benyzyl
and alkyl side chains to make them miscible with polymers. Silver was surface modified by 2 -pyrrolidone or coated with
polyethoxylated nonionic surfactants. Graphite, calcium carbonates, iron oxides were coated with a mixture of fatty acids andfatty acid methyl esters - probably added to prevent agglomeration. Other compounds were silanes and siloxanes from synthetic
amorphous silica and nickel-zink-iron oxide. Most of the extracted compounds are presumably intentionally added for different
purposes. Some of the surface modifications have already been shown to influence the toxic properties of the ENM.

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 40: Environment II

PTh-159

11:10 － 12:20

Simultaneous determination of cationic and anionic compounds using ahigh-speed polarity switching ESI and an online-SPE LC-MS/MS

Shoji F Nakayama1, Yayoi Suzuki1, Tairo Ogura2, Ichiro Hirano2, Mitsuha Yoshikane3

National Institute for Environmental Studies, Tsukuba, Japan, 2Shimadzu Corporation, Kyoto, Japan, 3The University ofTokyo, Kashiwanoha, Japan

1

Keywords:
Online SPE LC-MS/MS, high-speed polarity switching, simultaneous determination, phthalate monoesters, phthalate diesters
Novel aspects:
Developed was simultaneous quantitation technique of cationic and anionic compounds which requires small volume ofenvironmental samples and thus enables high through put screening of contaminants of emerging concern.
Abstract:
Mounting evidence suggesting ubiquitous presence of newly or unexpectedly detected chemicals, namely contaminants ofemerging concern（CECs, e.g. pharmaceuticals and personal care products： PPCPs, perfluorinated compounds, steroid hormonesand others）, has lead public concern and government actions. The CECs consist of hundreds of compounds, both cationic andanionic, and exist in the environment in generally trace levels. Those compounds typically enter the environment through sewage
systems since the current wastewater treatment technologies are not designed for decomposing CECs, which is considered as animportant exposure route for aquatic ecosystem. Some of the CECs are also found to resist drinking water treatment, which could
result in human exposure. To establish an effective monitoring system for wastewater influent and drinking water, a high-throughput analytical method is in acute need.
For the analysis of trace level compounds, the current sampling methodologies often involve large volume sample collection toconcentrate analytes in pretreatments, which limits sample transportation and introduces complex sample pretreatments. Recent
advances in online solid phase extraction（online SPE）technology may be one of the solutions to this limitation since it requires
relatively small volume of samples（typically 1 -10 mL）and introduces all the injected volume into an analytical system. A newly
developed high-speed polarity switching technology is capable of simultaneously determining cationic and anionic compounds
including PPCPs, steroid hormones and other CECs.
An online SPE liquid chromatography tandem mass spectrometry（online SPE LC-MS/MS； Shimadzu Corporation, Kyoto, Japan）
was used to develop a simultaneous quantitation method for the analysis of phthalate di- and mono-esters（PEs）in finished water.
One mL of raw samples was injected after receiving stable isotope labelled PEs. Two binary pumps were used for sample cleaning
with an SPE column（MASK-ENV, Chemco Scientific Co., Osaka, Japan）and another two pumps were used for separation by ananalytical column（Shim-pack XR-ODS）. For both sets of pumps, scrubber columns（Shim-pack XR-ODS）were inserted just after
mixing chambers to reduce contaminations from the analytical system.
Method detection limits（MDLs in ng/ml）were as follows： monometyl-（MMP, 0.203）, monoetyl（MEP, 0.162）, mono-n-butyl（MnBP,
0.214）, monobenzyl（MBzP, 0.201）, mono- 2 -ethylhexyl-（MEHP, 0.213）, mono- 2 -ethyl- 5 -hydroxyhexyl-（MEHHP, 0.223）phthalates；
dimethyl-（DMP, 0.144）, dietyl-（DEP, 0.179）, di-n-butyl-（DnBP, 1.43）, butylbenzyl-（BBzP, 0.117）, di-（ 2 -etylhexyl）-（DEHP, notcalculated due to blank contamination）phthalates. Recoveries ranged 56 % -134 % . DnBP and DEHP were detected even inprocedure blanks suggesting that contaminations occurred during sample preparations or analytical procedures. Phthalatemonoesters were not detected or below MDLs in the most of samples although MEHP was detected in one sample（0.471 ppb）.
Since this method is capable to detect very low levels of PEs, it is essential to minimize contaminations from sampling to analysis
in order to achieve lower MDLs for phthalate diesters. In the presentation, preliminary result of the application of online-SPE-LCMS/MS system for the measurement of phthalate esters will be discussed.

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 40: Environment II

PTh-160

13:30 － 14:40

The analysis of olefine and aromatic hydrocarbon in hydrocarbon mixture
using Multi-Dimensional GC/GCMS system

LiBin Liu, GuiXiang Yang, TaoHong Huang, Lei Cao
Shimadzu Co.,Beijing, China

Keywords:
hydrocarbon mixture,Multi-Dimensional GC/GCMS system
Novel aspects:
MDGC/GCMS system can show superiority on the analysis of complicated matrix sample such as hydrocarbon mixture,etc.
Abstract:
The component of hydrocarbon mixture is very complicated, including even and odd number carbon hydrocarbon. Its analysis
with conventional gas chromatographic approaches is a big challenge and can give us inaccurate or even false results because
overlapping is always happened. Many measures were taken to avoid interferences, such as improving sample preparation or
using high selectivity detectors. Multi-dimensional GC/GCMS system with multiple heart-cutting is one of the powerful tools. Multidimensional GC/GCMS can improve resolution beyond that of the regular GC analysis as it re-introduces the dissolved componentof interest into another column. In other words, only part of the peak of the component that was insufficiently separated on thecolumn where the sample initially passed through（called the“ 1 st column“）is introduced（heart-cut）to a column of anothertype（called the“ 2 nd column“）, so that insufficiently separated components can be separated. A device called a“switching
device“ is used for heart-cut introduction of peaks eluted from the 1 st column to the 2 nd column. As a switching device, therecently developed Multi-Deans switching unit can be used in combination with a GC-FID as the first analytical dimension and a
GCMS as the second analytical dimension. The analytes pass the first column and are detected in the FID（“stand-by mode“）orare transferred to the second column and analyzed with mass spectrometer or GC detector such as FID（“cut mode“）. By using
this system, complicated matrix analysis such as hydrocarbon mixture was done to demonstrate MDGC/GCMS system
performance.
This MDGC/GCMS system can analyze the olefine and aromatic hydrocarbon in hydrocarbon mixture. It has superiority on thedetermination of the complicated matrix sample in order to obtain more reliable analytical results.

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 40: Environment II

PTh-161

11:10 － 12:20

A Novel Sensing Material for Iron(III) Ions based on Poly (gamma-Glutamicacid)-grafted-3,4-dihydro-3-2’-ethylhydroxyl-6-methyl-1,3,2H-benzoxazine

Nuorn Choothong1, Attaphon Kaewvilai1, Apirat Laobuthee1,2, Amornrat Lertworasirikul1,21
Kasetsart University, Bangkok, Thailand, 2Center of Advanced Studies in Industrial Technology, Faculty of Engineering,
Kasetsart University

Keywords:
Poly（gamma-glutamic acid）, benzoxazine, Fe（III）ion, colorimetric sensing material, photometric titration method
Novel aspects:
The novel polymeric sensing materials was synthesized based on a simple reaction. The responsiveness of the polymer toward Fe（III）is very rapid, and clearly observable with the naked eye.
Abstract:
A novel sensing material for Fe（III）ion was prepared from poly（gamma-glutamic acid）（gamma-PGA）and 3,4-dihydro- 3 -（ 2 ’-ethylhydroxyl）- 6 -methyl-1,3,2H-benzoxazine（Mt-Bx）. Mt-Bx was used as an ionophore segment and grafted onto the gammaPGA backbone via an esterification reaction. The optimum reaction time determined by FT-IR was 2 h. The calculation basedon 1H-NMR spectrum revealed that the most attainable grafting degree was 30%. This copolymer showed a highly selective andsensitive recognition toward Fe（III）ions. A simultaneous transition of color and solubility was observed when the copolymer
formed complex with the Fe（III）ions. These responses were clearly observable with the naked eye. A quantitative analysis basedon a photometric titration method indicated that the copolymer exhibited and excellent interaction with Fe（III）ions at a
stoichiometric ratio of 1 ：390.

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 40: Environment II

PTh-162

13:30 － 14:40

Analysis of perfluorinated compounds in sediment samples fromwastewater canal of Pancevo industrial area, Serbia

Vladimir P Beskoski1, Shuusuke Takemine2, Takeshi Nakano3, Latinka Slavkovic-Beskoski4,
Gordana Gojgic-Cvijovic1, Mila Ilic1, Srdjan Miletic1, Miroslav M Vrvic1,5

Institute of Chemistry, Technology and Metallurgy, University of Belgrade, Serbia, 2Hyogo Prefectural Institute ofEnvironmental Science, Kobe, Japan, 3Center for Advanced Science and Innovation, Osaka University, Japan, 4Instituteof Nuclear Sciences, 5Faculty of Chemistry, University of Belgrade, Serbia

1

Keywords:
PFOA, PFOS, Industrial wastewater canal, Sediment, Danube River
Novel aspects:
This is the first report of presence of PFCs compounds in the sediments from Serbia. Compared to other reports, high levels of
PFOA and PFOS were found.
Abstract:
Perfluorinated compounds（PFCs）are chemicals that do not occur naturally, but have been widely used in chemical production
for some time. They are globally distributed, environmentally persistent, bioaccumulative, and potentially harmful.
Perfluorooctansulfonate（PFOS）and perfluorooctanoate（PFOA）are the two PFCs most commonly used and found in theenvironment. Together with perfluorohexane sulfonate（PFHxS）these compounds are widely employed in different industrial
processes such as in protective coatings.
The wastewater canal（WWC）Vojlovica was built in 1962 to collect the wastewater discharges from the industrial complex of thecity of Pancevo in Serbia. Industrial complex consist of a petrochemical factory（HIP Petrohemija）, an oil refinery（NIS Rafinerija,
Pancevo）and chemical fertilizers factory（HIP Azotara）. The canal is artificial with no natural flows, about 2 km long, around 70
m wide and directly connected to the Danube River. The water depth is around 12 m. The environment surrounding the canal has
been strongly affected for a long time by the presence of the industrial complex. Additionally heavy destruction during NATObombing events in 1999 resulted in contamination of air, soil, groundwater and the WWC itself.
In total, 4 sediment samples from WWC were collected. Surface sediments layer of 15 cm were taken by a Van Veen Grab
sampler, transported in glass jars and stored in the laboratory at 4 °C. For comparative purposes, the same type of sample werealso taken from the navigation canal flowing parallel to WWC but not receiving any direct discharge of industrial wastewaters.
Sampling sites are listed below：
No 1 - navigation canal；
No 2 - at the confluence of WWC with the Danube River, downstream from the industrial area and effluents；
No 3 - downstream from the fertilizer factory outlet（first effluent）；
No 4 - downstream from the petrochemical plant（second effluent）；
No 5 - downstream from the oil refinery outlet（third effluent）.
Sediment sample was extracted with methanol. MPFAC-MXA as mass-labeled surrogates was spiked into the sample. The sample
was extracted with SPE. The elution was concentrated and labeled 13C8PFOA was added as syringe spike. The each final solution
was analyzed by liquid chromatography（LC）-tandem mass spectrometer（MS/MS）using Xevo TQ（Waters）coupled withACQUITY UPLC（Waters）.
Concentrations of PFCs were determined as follows：
No 1 ： 68, 230 and 230 ng/kg-dry of PFOA, PFHxS and PFOS, respectively.
No 2 ： 80 and 2100 ng/kg-dry of PFOA and PFOS, respectively.
No 3 ： 170 and 5300 ng/kg-dry of PFHxS and PFOS, respectively.
No 4 ： 130, 170, and 5700 ng/kg-dry of PFOA, PFHxA, and PFOS, respectively.
No 5 ： 76, 66 and 420 ng/kg-dry of PFOA, PFHxA, and PFOS, respectively.
Concentrations of PFOS in the samples No 3 and No 4 are 3 -3.2 times higher compared with sea sediment in Tokyo bay 1). PFOA
and PFOS concentrations from WWC were from two to twenty fold higher comparing to sediment samples taken from Roter Mainriver（Germany）which receives treated waste waters of industrial, commercial and domestic origin from municipal wastewater
treatment plant2). Comparing to upstream Danube River bank sediment samples3) PFOS from the WWC samples were from two to
six fold higher.
This is the first study and report of presence of PFCs compounds in the samples from Serbia. Most of the PFCs are released fromfertilizer factory and petrochemical plant outlets, while oil refinery outlet mostly contribute to petroleum pollution. The exact
origin of PFCs cannot be established from one study but one of the reasons for presence of these compounds might be theirusage as components in pipes, fittings and wiring insulations.

1 ）Zushi Y. et al Environmental pollution 158, 756-763（2010）
2 ）Becker, A.M et al Environmental Pollution 156, 818-820（2008）
3 ）Clara, M.et al Water Research 43, 4760-4768（2010）

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 40: Environment II

PTh-163

11:10 － 12:20

Study of On-site sampling method for Dioxin in water with highconcentrations of suspended solids.

Takeshi Enomoto1, Miho Okimoto1, Chuanpit Boonyoy2, Areerat Jaksakul2, Ruchaya Boonyatumanond2,
Genta Takahashi5, Kenji Tawara5, Tohru Matsumura3, Takeshi Nakano4
1
JEOL Ltd.,Tokyo,Japan, 2ERTC,Pathumthani,Thailand, 3IDEA Consultant Inc.,Shizuoka,Japan, 4Osaka University, Osaka,
Japan, 5Hyogo Environmental Advancement Association,Kobe,Japan

Keywords:
Dioxin, PCB, river water, coagulant, Suspended Solids
Novel aspects:
We have been developed the water sampling method by applyingcoagulant to analysis of dioxin in water which has a large
volume of suspended solids.
Abstract:
Dioxins in water are generally collected by disk type SPE with ODS. However, a large amount of water causes clogging of the disk
and delays time of the sampling. In order to resolve this problem, adsorbents utilizing coagulation have been developed. We havebeen developed the water sampling method by applying coagulant to analysis of dioxin in water which has a large volume ofsuspended solids. SS can result from erosion from urban runoff and agricultural land, industrial wastes. Suspended solids（SS）aresolid materials, including organic and inorganic, that are suspended in the water. SS consists of silt, plankton and industrial wastes.
Samples are stably collected in spiking experiments using river water. Because this method requires minimum instruments andtakes shorter time to analysis, it has more advantage than the conventional water sampling method.
In this study, congener-specific analysis of PCDD/DF including PCBs were performed in the environmental samples using BPXDioxin, HT 8 -PCB as separation column by GC/HRMS（JEOL JMS-800D）. HT 8 -PCB capillary column（60m, 0.25mm i.d.）was used
for determination of PCB all congeners. Contribution of PCB-11 in the environmental samples such as river water and ambient air
were evaluated.
PCB congener profiles in environmental samples, such as air, water, and sediment, are similar to the pattern of a particular PCB
product used around the area. On the other hand, among biological samples, such as fish, clams, birds and humans, the congener
profiles differ drastically reflecting different half-lives of congeners in different biological systems. We would propose the source
estimation and monitoring method using PCB congener-specific analysis.

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 40: Environment II

PTh-164

13:30 － 14:40

Multi-residue analysis of pesticides in animal and fishery products, andtheir processed foods by dual-column GC-MS/MS

Eiji Ueno, Haruka Ohno, Minae Watanabe, Harumi Oshima, Eiichi Mikami
Aichi Prefectural Institute of Public Health, Nagoya, Japan

Keywords:
pesticide, animal and fishery product, processed food, dual-column, GC-MS/MS
Novel aspects:
Newly multi-residue method to enable the analysis of water-soluble pesticides such as methamidophos in processed foods suchas frozen gyoza dumplings, and highly quantification at ppb levels by dual-column GC-MS/MS.
Abstract:
In Japan, food poisoning caused by methamidophos-tainted frozen gyoza dumplings imported from China came to light in January
2008. The news has set off alarms about the safety of not only perishable foods but also processed foods. Methamidophos is awater-soluble pesticide（solubility in water >200 g/L and Kow logP =-0.8 at 20&#8451；）. The official multi-residue method foranimal and fishery products is only applied to fat-soluble compounds（Kow logP > 2 - 3 ）such as DDT’s in perishable foods.
Therefore, we developed a multi-residue method for determining many pesticides including water-soluble compounds（Kow logP>- 1 ）in animal and fishery products, and their processed foods by dual-column gas chromatography with tandem quadrupole
mass spectrometry（dual-column GC-MS/MS）.
First, 21 stable isotopically labeled pesticides, dichlorvos-d 6 , acephate-d 6 , α-HCH-d 6 , diazinon-d 10 , iprobenfos-d 7 , carbaryl-d 7 ,fenitrothion-d6, linuron-d6, metolachlor-d6, chlorpyrifos-d10, diethofencarb-d7, fosthiazate-d5, pendimethalin-d5, thiabendazole-13C6,
imazalil-d5, isoprothiolane-d4, isoxathion-d10, pp’-DDT-d8, EPN-d5, etofenprox-d5, es-fenvalerate-d7 and triphenylphosphate as theinternal standards（surrogates）for 202 representative target pesticides were selected. The chopped sample spiked with surrogates
was homogenized with 2 % acetic acid aqueous solution, and extracted with acetone-n-hexane（ 2 ： 3 ）while melting adipose
tissue under mildly acidic conditions, and then centrifuged. The organic layer and aqueous layer were filtered together. The
remaining solid contents were re-extracted with acetone-n-hexane（ 2 ： 3 ）, and then filtered. After concentration of the
combined filtrate, the aqueous residues saturated with sodium chloride was loaded onto a macroporous diatomaceous earth
column（EXtrelut NT20）, and eluted with ethyl acetate. Co-extractives were removed by a combination of gel permeation
chromatography（GPC, CLNpak EV-2000AC-12F column： 300 x 12 mm id, CLNpak EV-G-12F guard column： 100 x 12 mm id, mobile
phase： acetone-cyclohexane（ 3 ：17））and graphitized carbon solid-phase extraction（SPE, Carbo C-Avicel（ 1 ： 4 ）： 500 mg,
eluant： acetone-cyclohexane（ 3 ：17））automatically, and then by a tandem SAX/PSA cartridge SPE（Bond Elut Jr-SAX： 500 mg,
Bond Elut Jr-PSA： 500 mg, eluant： acetone-n-hexane（ 1 ： 1 ））. The cleaned sample extract was subjected to dual-column GCMS/MS（Rxi- 5 Sil MS first column： 30 m x 0.25 mm id, Rtx-200MS secondary column： 30 m x 0.25 mm id, ionization mode： EI, MS/
MS mode： MRM）.
Mean recoveries（n = 5 ）of most pesticides from ten kinds of fortified samples, beef, salmon, hen’s egg, cow’s milk, honey, kimchi,
corned beef, eel kabayaki, frozen gyoza dumpling and retort curry at 0.01 mg/kg were within a range from 70 to 120 %（relative
standard deviation values <15%）.
The method was applied to many commercial foods to demonstrate its use in routine analysis. Not a few interfering peaks
appeared on the MRM chromatograms of some food such as retort curry at ppb level detection. In these cases, the most ofinterfering peaks did not disappear by using alternative MRM transitions, so that using a single-column GC-MS/MS may result infalse positive data. To overcome this problem, GC-MS/MS was equipped with a secondary column together. As a result, nointerfering peaks on the either MRM chromatograms of almost all foods were detected. The capability of the best column
combination for GC-MS/MS allows easy identification and precise quantification of many pesticides in animal and fishery products,
and their processed foods at the ppb level such as uniform limit（0.01 mg/kg）under the Japanese positive list system.
References
1 ）E. Ueno, H. Oshima, I. Saito, H. Matsumoto, J. AOAC Int.,87, 1003-1015（2004）.
2 ）E. Ueno, J. Pestic. Sci., 35, 74-78（2010）（in Japanese）.
3 ）E. Ueno, J. Pestic. Sci., 36, 554-558（2011）（in Japanese）.

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Session 40: Environment II

PTh-165

11:10 － 12:20

Simultaneous analysis of cationic, anionic and neutral surfactants fromdifferent matrices using LCMS/MS.

Rashi Kochhar1, Shruti Raju1, Deepti Bhandarkar1, Bhairavi Saraf1, Shailendra Rane1, Jitendra Kelkar1,
Ajit Datar1, Zhaoqi Zhan2
1

Shimadzu Analytical (India) Pvt.Ltd., Mumbai, India, 2Shimadzu Asia Pacific Pte.Ltd.

Keywords:
Surfactants, Ultra fast MS, Polarity switiching, Triple Quadrupole, Industrial Waste ,Novel aspects:
LCMS/MS with fast scanning and polarity switching is a good screening technique for analysis of surfactants in areas likepharmaceuticals, forensics, effluent treatments, cosmetics etc.
Abstract:
Surfactants are compounds that lower the surface tension of a liquid, the interfacial tension between two liquids, or that between
a liquid and a solid. Surfactants may act as detergents, wetting agents, emulsifiers, foaming agents, and dispersants. They areroutinely deposited in numerous ways on land and into water systems, whether as part of an intended process or as industrial
and household waste. Some surfactants are known to be toxic to animals, ecosystems, and humans, and can increase the diffusionof other environmental contaminants. Some detergents also cause lysis of cells and tissues by disorganizing the cell membrane’s
lipidic bilayer and solubilising proteins.
It, therefore, becomes important to detect these surfactants and here we have attempted to simultaneously analyse them
The surfactant standards were dissolved in water ： methanol（ 1 ： 1 ）to ensure all surfactants can be solubilized, and subjected
to liquid chromatography using UHPLC Nexera coupled with triple quadrupole（TQ）mass spectrometer LCMS 8030, Shimadzu
Corporation. The samples were subjected to Solid Phase Extraction（SPE）and analysed using the method developed for the
standards. The chromatographic condition ensured analysis to be completed in short time, because of system tolerance to highpressure. Subsequent fast analysis was supported by Fast TQ MS.
The surfactant standard mixture was separated chromatographically and subjected to mass spectrometry. Surfactants of different
ionising tendencies were simultaneously analysed for their parent and product ions. For eg, Cetrimide, Octylphenol ethoxylate
were analysed in ESI positive mode, whereas Sodium lauryl sulphate（SLS）was analysed in ESI negative mode. Molecular ion of
cetrimide was observed with loss of bromine whereas molecular ion of SLS was observed with loss of sodium. Octylphenol
ethoxylate showed a characteristic polymeric pattern with mass difference of 44. Fast analysis was possible due to short runtimes
that could be achieved without compromising resolution in Nexera system. The ultrafast polarity switching and ultrafast scanning
capacity of the mass spectrometric system enabled analysis of different types of molecules in the same run.

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Food safety

PTh-166

13:30 － 14:40

Detection of Melamine in Human Renal Uric Acid Stone by Matrix-Assisted
Laser Desorption / Ionization Time-Of-Flight Mass Spectrometry (MALDITOF MS)

Chia-Fang Wu1, Chia-Chu Liu2,3,4,5, Jentaie Shiea6, Yi-Tzu Cho7, Yii-Her Chou2,3, Bai-Hsiun Chen8,
Chao-Yi Chien1, Shu-Pin Huang2,3, Wen-Jeng Wu2,3,9, Jung-Tsung Shen9, Mei-Yu Chang9,
Chun-Hsiung Huang2,3, Ai-Wen Chang2, Ming-Tsang Wu*1,4,10,11

Department of Public Health, Kaohsiung Medical University, Kaohsiung, Taiwan, 2Department of Urology, Kaohsiung
Medical University Hospital, Kaohsiung Medical University, Kaohsiung, Taiwan, 3Department of Urology, Faculty of
Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan, 4Graduate Institute of Medicine,
Kaohsiung Medical University, Kaohsiung, Taiwan, 5Pingtung Hospital, Department of Health, Executive Yuan, Pingtung,Taiwan, 6Department of Chemistry, National Sun Yat-Sen University, Kaohsiung, Taiwan, 7Department of Cosmetic
Applications and Management, Yuh-Ing Junior College of Health Care & Management, Kaohsiung, Taiwan, 8Departmentof Laboratory Medicine, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan, 9Department of Urology, Kaohsiung
Municipal Hsiao-Kang Hospital, Kaohsiung, Taiwan, 10Department of Family Medicine, Kaohsiung Medical University
Hospital, Kaohsiung, Taiwan, 11Center of Environmental and Occupational Medicine, Kaohsiung Municipal Hsiao-Kang
Hospital, Kaohsiung, Taiwan
1

Keywords:
melamine, urolithiasis, MALDI-TOF MS
Novel aspects:
MALDI-TOF MS could detect melamine in uric acid stone specimens, even when urinary melamine level was undetectable, which
can be an additional analytical method to evaluate for external melamine exposure.
Abstract:
Background： The link between melamine-contaminated daily foodstuffs and urolithiasis formation has drawn international
concern. However, detection of melamine levels in urine may not completely represent external melamine exposure. Thus, finding
an additional analytical method for the study of environmental melamine exposure and its adverse effect in humans is crucial.
Methods： Eleven adult patients diagnosed with uric acid urolithiasis were retrospectively analyzed. Melamine levels in theirovernight one-spot urine samples were measured by a triple quadrupole liquid chromatography tandem mass spectrometry（LCMS/MS）. The compositions of stone samples were analyzed by the Fourier transform infrared（FTIR）spectrophotometer andmatrix-assisted laser desorption / ionization time-of-flight mass spectrometry（MALDI-TOF MS）.
Results： Seven（63.6%）out of 11 patients had detectable melamine levels in their urine specimens（method of detection limit： 0.8
ng/ml）. Three patients（27.3%）were highly suspected of having melamine-containing urolithiasis in FTIR spectra. In one of those
three cases who still had available stored stone specimens, MALDI-TOF MS further confirmed melamine components in this malepatient’s stone specimens. In contrast, his urinary melamine level was below the detection limit by LC-MS/MS.
Conclusions： Direct analysis of melamine in the composition of urolithiasis by MALDI-TOF MS can be an additional analyticalmethod to evaluate for external melamine exposure.
*Correspondence： Prof Ming-Tsang Wu

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Food safety

PTh-167

11:10 － 12:20

Urinary oxidative metabolites of di(2-ethylhexyl)phthalate can predict thedaily intake of phthalate-tainted foods in Taiwanese children

I-Chen Wu1, Chia-Fang Wu2, Jentaie Shiea3, Bai-Hsiun Chen4, Jiunn-Ren Wu5, Ming-Tsang Wu*2,6,7

Division of Gastroenterology, Department of Internal Medicine, Kaohsiung Medical University Hospital, Kaohsiung,Taiwan, 2Department of Public Health, Kaohsiung Medical University, Kaohsiung, Taiwan, 3Department of Chemistry,
National Sun Yat-Sen University, Kaohsiung, Taiwan, 4Department of Laboratory Medicine, Kaohsiung Medical University
Hospital, Kaohsiung Medical University, Kaohsiung, Taiwan, 5Department of Pediatrics, Kaohsiung Medical University
Hospital, Kaohsiung Medical University, Kaohsiung, Taiwan, 6Department of Family Medicine, Kaohsiung Medical
University Hospital, Kaohsiung, Taiwan, 7Center of Environmental and Occupational Medicine, Kaohsiung Municipal
Hsiao-Kang Hospital, Kaohsiung, Taiwan
1

Keywords:
urinary phthalate metabolites, children, LC-MS/MS
Novel aspects:
Urinary oxidative metabolites of DEHP of our study children can predit their affected foods exposure.
Abstract:
Background：A major incident of phthalate-contaminated foodstuffs happened in Taiwan between April and July, 2011. Phthalates,
mainly di（ 2 -ethylhexyl）phthalate（DEHP）, were deliberately added to foodstuffs as a substitute of emulsifier. The highest
concentration of DEHP in affected foods belonged to the category of nutrient supplements which were regularly taken by children
or infant. Thus, this study investigated the correlation between the amount of DEHP intake and its major oxidative metabolites in
Taiwanese children.
Methods：Between May 31 and June 17, 2011, we established a special Phthalates Clinic for Children（PCC）at Kaohsiung Medical
University Hospital in southern Taiwan for those children who were suspected to have the intake of affected foods. Their parents
were interviewed to collect their exposure information from two sources： the official website of Taiwan Food and Drug
Administration and Bureau of Health of Kaohsiung City to calculate daily intake dose. One-spot urine specimen in each study
children was collected and measured four oxidative metabolites of DEHP, including 5 oxo-MEHP, 5 OH-MEHP, 5 carboxy-MEPP,
and 2 carboxy-MMHP, by a triple quadrupole liquid chromatography tandem mass spectrometry（LC-MS/MS）.
Results：In total, 28 children whose age £ 10 years old（pre-puberty）were studied. The daily intake dose ranged from 0 to 0.16
mg/kg/day. The intake of affected foods in 19（67.9%）and 12（42.9%）out of 28 study children exceeded the tolerable daily intake（TDI）recommended by U.S. Environmental Protection Agency（TDI < 0.02 mg/kg/day）and European Food Safety Authority（TDI< 0.05 mg/kg/day）. For urinary DEHP metabolites, after corrected by urinary creatinine（Cr）, the median levels（interquartile
range）of 5 oxo-MEHP, 5 OH-MEHP, 5 carboxy-MEPP, and 2 carboxy-MMHP were 76.09, 80.36, 109.31, and 36.84 μg/g Cr（56.21-143.32,
29.28-137.36, 45.98-200.33, and 24.74-59.40 μg/g Cr, respectively）. We found that the significant and positive correlations between
DEHP daily intake（mg/kg/day）and urinary 5 oxo-MEHP（Spearman correlation r=0.39, p=0.04）and 5 carboxy-MEPP（r=0.45,
p=0.02）, but not for 5 OH-MEHP（r=0.28, p=0.14）and 2 carboxy-MMHP（r=0.32, p=0.10）.
Conclusions：These study children were exposed to the high amount of DEHP from affected foods and some oxidative
metabolites of DEHP was correlated to their exposure dose. A further study is necessary to link these exposure indices to thehealth outcome in children.
*Correspondence： Prof Ming-Tsang Wu

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Food safety

PTh-168

13:30 － 14:40

LC-MS analysis of neonicotinoid insecticides in the crops using a novel
selective solid-phase extractant having dipole type functional group

Toshio Miwa1, Isao Saito1, Atsushi Yamamoto1, Yoshinori Inoue2, Mitsuru Saito2
1

Chubu University, Aichi, Japan, 2Nippon Filcon Co., Tokyo, Japan

Keywords:
SPE, Adsorbent, LC-MS, Neonicotinoids
Novel aspects:
We have developed a novel adsorbent which highly recognized chemical characteristics of the functional groups.
Abstract:
Neonicotinoids, which are insecticides chemically related to nicotine, act as an agonist on an acetylcholine receptor in insects, as
nicotine does. Since neonicotinoids show much lower toxicity in mammals, 7 kinds of them represented by imidacloprid are
currently the most widely used insecticide in the world. Chromatographic methods dedicated to the determination of insecticide
residues such as GC and HPLC have been reported. However, they require a fair amount of sample manipulations such as liquidliquid extraction and column cleanup procedure, and do not fit for a distribution cycle of food.
The basic chemical structure of the neonicotinoids contains both heteroaromatic ring and dipole functional group such as
nitroimine or cyanoimine. We have developed a novel adsorbent, PNP, which highly recognized chemical characteristics of thefunctional groups. The PNP adsorbent was synthesized by introducing p-nitrophenol on the hydrophilic base gel with glycidylgroup. One hundred mg dry weight of the adsorbent was packed in a solid-phase extraction cartridge. The cartridge wasconditioned by passing 10 mL of acetone, and then neonicotinoids mix solution dissolved in various solvents were loaded on it.
The solutes concentration in various eluates from the cartridge was measured by LC-MS.
Neonicotinoids in acetone or ethyl acetate were tightly entrapped on the PNP adsorbent and were easily eluted with methanol. It
appeared that development of plural interaction such as hydrogen bonding, π-π interaction, and dipole interaction raised
selectivity of the adsorbent for neonicotinoids. Acetone or ethyl acetate is suitable extraction solvent for pesticides of widepolarity ranges. Thus, the PNP adsorbent was used to determine neonicotinoids in the actual crops such as green pepper.
Quantitative analysis was done in selected-ion monitoring mode for each compound. The use of PNP adsorbent allows for clean
extracts to be obtained which dramatically reduces sample matrices.

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Food safety

PTh-169

11:10 － 12:20

Studies on Residual Characteristics of Growth Regulator 6-BA in Bean
Sprout

Wan-Hee Seo, Young-Mo Jeong, Soon-Kil Cho, Bong-Suk Oh

Jeonnam Provincial Office, National Agricultural Products Quality Management Service, MIFAFF, Korea

Keywords:
Bean sprout, Residue analysis, LC-MS/MS
Novel aspects:
We think 6 -BA will not be remained during its shipping date.
Abstract:
Bean sprout is a common food due to its cheap price and short cultivation period（ 5 - 7 days）. It is also used as an ingredient of
in-flight meal and food for astronauts. Industrial mass production of bean sprout usually accompanied diverse chemical drugs suchas seed disinfectants and growth regulators. In this study, residual characteristics of growth regulator 6 -BA were observed. During
the cultivation of bean sprout, the seeds were washed and soaked in water for 20 hours. 6 -BA was applied（75 mL/ 6 L/75 kg）tothe seeds and 1.2 kg bean sprout seeds were cultivated in plastic container for 7 days with sprinkling of water（15 min/ 4 h）.
Temperature and humidity were maintained as 18.3±0.6&#8451； and 71.0±1.6 % during the cultivation period. After 7 days, theweight of bean sprout was increased from 1.6 g to 14 g. For sample preparation, each sample was extracted with 30 mL acetonitrile
and kept in a refrigerator（-80&#8451；）for 5 min in order to separate organic and aqueous layer. The organic supernatant was
directly injected to LC-MS/MS for residual analysis of 6 -BA. The method was validated in triplicate at two different fortification
concentrations（0.1and 0.5 mg/kg）in the blank matrix. Good recoveries were observed for the target analysis and ranged between
78.2 and 84.9% with relative standard deviations of less than 10%. The limits of detection（LOD）and limits of quantification（LOQ）
were 0.003 and 0.01 mg/kg for 6 -BA. The residual amount of 6 -BA in application day was 1.60 mg/kg. A slow degradation of 6 -BA
was observed until 5 days of application with a concentration of 0.07 mg/kg. After 6 days, 6 -BA was completely disappeared in
the bean sprout samples. Based on the dissipation pattern, we think 6 -BA will not be remained during its shipping date.

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Food safety

PTh-170

13:30 － 14:40

Improvement of Determination Method for Pesticide Residues in Bean
SproutSoon-Kil Cho, Wan-Hee Seo, Young-Mo Jeong, Ji-Mi Cho

Jeonnam Provincial Office, National Agricultural Products Quality Management Service, MIFAFF, Korea

Keywords:
Improved method , LC-MS/MS
Novel aspects:
The improved method is easy and reliable for the determination of carbendazim, thiabendazole, and 6 -BA in bean sprout.
Abstract:
Bean sprout is a common food due to its short cultivation period and cheap price. An industrial mass production accompanied
diverse chemical drugs, such as seed disinfectants and growth regulators, which led to controversies regarding its safety. The
treatment of carbendazim and thiabendazole to bean sprout was legally allowed, but their residues have to be absent according
to their maximum residue limits in bean sprout. Typical methods used for the determination of the pesticide residues in bean
sprout have tended to be labor-intensive and have used large volumes of solvent, resulting in large volumes of hazardous waste. It
is also very difficult to monitoring pesticides in bean sprout due to its short distribution period. Thus, the typical sample
preparation needs to minimize the number of steps in order to reduce both time and sources of error during theses process. For
sample preparation in this study, precisely 20 grams of samples were placed into a centrifuge tube. A 20 mL portion of acetonitrile
was added, and vigorously shaken for 5 min followed by centrifugation at 3000 rpm for 5 min. The tube was kept in a refrigerator（-80&#8451；）for 5 ~ 10 minute to separate acetonitrile and water layers. A 2 mL portion of the acetonitrile layer was injected
into a LC-MS/MS. Recoveries were between 77.1 and 98.7 for carbendazim, thiabendazole, and 6 -BA. The calculated LOQs were0.01 mg/kg for 6 -BA, and 0.001 mg/kg for carbendazim and thiabendazole. These results indicate that the improved method is easy
and reliable for the determination of carbendazim, thiabendazole, and 6 -BA in bean sprout.

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Food safety

PTh-171

11:10 － 12:20

Screening of five mycotoxins by using immunoaffinity column and HPLCorbitrapMS in processed foods

Dong Sik Jeong, Seung Lim Baek, Dae Hyun Kim, Jong Ho Lee, Cheong-Tae Kim
NONGSHIM Co., LTD., Seoul, South Korea

Keywords:
Mycotoxins, OrbitrapMS, Immunoaffinity column
Novel aspects:
Applying to processing foods, Using OrbitrapMS, Muti-determination of mycotoxins
Abstract:
Introduction
The risk assessment of mycotoxins contamination of foods was important because it can cause harmful health effects（e.g.
cancer）in the humans. But the determination of mycotoxins in processing foods（e.g. snack, instant noodle）was a difficult
experiment due to residue of extremely small amounts and adverse matrix effects（e.g. fat and sugar）. The object of this study
was to development a reliable and fast analytical method using by immunoaffinity column and HPLC-Orbitrap MS system inprocessing foods.
-Five mycotoxins： Aflatoxin, DON, Zearalenone, Ochratoxin, Fumonisin
Materials & Methods-Sample preparation： The immunoaffinity column was applied to ensure efficient cleanup. The homogenized 10g sample wasweighed. 50mL of PBS（ 1 X, pH 7.4, 1 st extraction）and 50mL of 70% MeOH in DW（ 2 nd extraction）were added for extraction.
Two aliquot of the extract were transferred into immunoaffinity column for cleanup. After drying down eluant under an nitrogenstream at 50&#8451；, reconstitute with 1 mL of 40% MeOH in DW containing 1 mM ammonium acetate and 0.1% acetic acid.
-LC/MS/MS analysis： 10uL of preparation sample was injected onto a XBridge C18 150*2.1mm, 3.5uL analytical column. A gradient
LC method used mobile phases water containing 1 mM ammonium acetate and 0.1% acetic acid and methanol containing 1 mM
ammonium acetate and 0.1% acetic acid at a flow rate of 0.2mL/min. HPLC-Orbitrap MS with ESI（+,-）probe was used and scan
type was full scan.
Data & Results- MS Condition： Aflatoxin B 1 （313.0707,+）, Aflatoxin B 2 （315.0863,+）, Aflatoxin G 1 （329.0656,+）, Aflatoxin G 2 （331.0812,+）,
Fumonisin B 1 （722.3957,+）, Fumonisin B 2 （706.4008,+）, DON（355.1387,+）, Zearalenone（317.1394,-）, Ochratoxin（404.0901,+）- Linearity： The calibration curves had R^ 2 values that were greater than 0.98.
- Recovery： Each test was performed three times and two spiked level. In snacks, the mean recovery values range from 79.0% to116.7% and in instant noodles from 73.8% to 94.7%.
- Repeatability： The RSD was calculated from three replicates. In snacks, the RSD range from 10.6% to 17.9% and in instant noodles
from 8.5% to 14.4%.
- LOQ： The level of LOQ was low than the MRL established KFDA（Korea Food and Drug Administration）for mycotoxins. LOQ
range of aflatoxin, DON, zearalenone, ochratoxin, and fumonisin were 0.1, 10.0, 5.0, 0.5, and 10.0ug/kg respectively.

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Food safety

PTh-172

13:30 － 14:40

Simultaneous determination of melamine and its analogues in various
processed foods using LTQ-orbitrap HRMS

JONG HO LEE, DONGSIK JEONG, DAE HYUN KIM, CHEONG-TAE KIM
NONGSHIM Co., Ltd., Seoul, Korea

Keywords:
Melamin, LC-MS/MS, LTQ-orbitrap HRMS, GC-MS
Novel aspects:
Simultaneous analysis of melamine, ammeline, ammelide and cyanuric acid in various processed foods by LTQ-orbitrap HRMS ispresented.
Abstract:[Introduction]
Various techniques have been used to detect melamine and cyanuric acid. But limited methods including GC-MS are suitable forthe simultaneous determination of melamine, ammeline, ammelide and cyanuric acid.
However, derivatization is necessary to GC-MS detection, and comparatively poor reproducibility of the quantitative analysis may
be unavoidable due to the incomplete derivatization.
At the same time, because of the large polarity of melamine and its related compounds, RP-HPLC method has weak retention andpoor separation efficiency.
The purpose of our study was to develop a LTQ-orbitrap HRMS method for the simultaneous determination of melamine and its
analogues in processed foods（e.g. instant noodle soups and milk products）.
[Methods]
The US FDA method for melamine and cyanuric acid was followed with some modifications made to establish a method for all
four compounds.
The sample was separated on a Biobasic AX column with acetonitrile-10 mM ammonium acetate（78：22, v/v）as the mobile phaseat flow rate 0.2 mL/min, and determined by positive and negative ESI mode. Test sample was extracted with 2.5% formic acid. The
extract was centrifuged and the supernatant was determined by LC-MS/MS and confirmed by LTQ-orbitrap HRMS. Separation wasachieved within 10 minutes using a column.
SPE cartridge was used as a clean-up filter to retain matrix interferences while all the polar analytes pass through.
（15N3,13C3）-melamine,（15N3,13C3）-cyanuric acid, 13C3-ammeline and 13C3-ammelide were used as internal standard, respectively.
[Preliminary data]
Under the optimized conditions, the retention times of melamine, ammeline, cyanuric acid and ammelide were about 2.8, 3.6, 4.0
and 7.2min, respectively.
For each analyte, a single mass spectral peak corresponding to the［M+H］+ ion or［M-H］- ion was observed. Accordingly, the［M+H］+ ions were used as precursor ions for melamine and ammeline, while the［M-H］- ions were used as precursor ions forammelide and cyanuric acid.
Accuracy mass ： melamine（+, C3H6O6, 127.07267）, cyanuric acid（-, C3H3N3O3, 128.00907）, ammeline（+, C3H5N5O, 128.05669）andammelide（+, C4H4N4O2, 129.04070）
Isotope accuracy mass ： melamine（+, 13C3, 15N3, 133.07384）, cyanuric acid（-, 13C3, 15N3, 134.01024）, ammeline（+, 13C3, 131.06675）
and ammelide（+, 13C3, 132.05076）
The limit of detection was 0.01 mg/kg and the limit of quantification was 0.5 mg/kg. The RSDs of the retention times and peakareas were 0.02~0.80 and 1.01~2.80%, respectively. The mean recoveries were varied from 85.0 to 102.1% for four targets in the various
instant noodle soups and milk products.
The method was validated and good results were obtained with respect to precision, repeatability and spiked recovery,Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Food safety

PTh-174

13:30 － 14:40

Determination of DNA adducts originating from methyleugenol using
isotope-dilution UPLC-ESI-MS/MS

Wolfram Engst1, Kristin Herrmann1, Fabian Schumacher1, Simone Florian1, Klaus E Appel2,
Hansruedi Glatt1

German Institute of Human Nutrition Potsdam- Rehbrücke, Nuthetal, Germany, 2Federal Institute for Risk Assessment,
Berlin, Germany

1

Keywords:
DNA adducts, UPLC-ESI-MS/MS, methyleugenol, MRM quantification
Novel aspects:
Development and application of UPLC-ESI-MS/MS method for quantification of DNA adducts originated from methyleugenol
Abstract:
Methyleugenol（ME）is a secondary metabolite present in many herbal spices. Former observations indicate that hydroxylation
followed by sulfation is an important bioactivation pathway of ME and other alkenylbenzenes leading to carcinogenicity in animals.
In this context, DNA adducts of activated alkenylbenzenes have been detected in vitro and in vivo using the 32P-postlabeling assay.
This method cannot provide structural information of the detected adducts. Therefore, the aim of the present study was tocharacterize the chemical structures of ME-derived DNA adducts on the basis of MS-MS fragmentation patterns. Furthermore, wedeveloped a sensitive quantification method using UPLC-ESI-MS/MS suitable for studying the bioactivation as well as adductformation potential of ME in vitro and in animal models.
The UPLC-ESI-MS/MS method developed is based on the analytical determination of adducted 2 ’-deoxynucleosides using isotopic
dilution analysis. For this purpose, stable-isotope labeled adduct standards of the ME-derived 2 ’-deoxyguanosine adduct and 2 ’-deoxyadenosine adduct were synthesized.
Extracted DNA is enzymatically hydrolyzed into 2 ’-deoxynucleosides with addition of stable-isotope labeled compounds asinternal standards. Afterwards the analytes are separated from digestion mixture ingredients by protein precipitation using
ethanol. The 2 ’-deoxynucleoside adducts are chromatographically separated within 4 minutes. MRM data are aquired in thepositive ion mode using three transitions for each adduct. All analyses are conducted with an ACQUITY UPLC（Waters）connected
to a Xevo™ TQ MS（Waters）.
By means of daughter scan experiments we were able to identify the structures of DNA adducts originated from ME, which wereconfirmed by NMR data. Consequently we generated MRM methods for these adducts. Therefore, we could identify andabsolutely quantify adducted 2 ’-deoxyguanosine and 2 ’-deoxyadenosine in samples resulting from in vitro and in vivo
experiments. The achieved sensitivity of this method is nearly one adduct per 108 nucleosides.
In summary the new developed method represents a suitable and improved alternative for detection of ME-derived DNA adducts.
Furthermore,ME-derived RNA adducts can be analyzed in the same UPLC run applying RNA adduct specific MS-MS transitions.
By means of this method we investigated the bioactivation pathway of ME via sulfation as well as the DNA adduct formation
potential in vitro（ 1 ）and in vivo. Moreover, we even could demonstrate the presence of ME-derived DNA adducts in humantissues.

（ 1 ）Herrmann, K., Engst, W., Appel, K. E., Monien, B. H., Glatt, H.（2012）Identification of human and murine sulfotransferases able
to activate hydroxylated metabolites of methyleugenol to mutagens in Salmonella typhimurium and detection of associated DNA
adducts using UPLC-MS/MS methods. Mutagenesis,（in press）doi： 10.1093/mutage/ges004.

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Food safety

PTh-175

11:10 － 12:20

Exploring the application of a universal method for pesticide screening infoods using a high data acquisition speed MS/MS

Hirano Ichiro1, Yuka Fujito2, Kiyomi Arakawa1, Yusuke Inohana1
1

Shimadzu Corporation, Kyoto, Japan, 2Shimadzu Analytical and Measuring Center Inc., Kyoto, Japan

Keywords:
LC/MS/MS, Pesticides Screening, Generic Peak Integration Parameters
Novel aspects:
Pesticides in food matrices were automatically identified by using fast 5 msec dwell time SRM and generic peak integration
parameters
Abstract:
Effective management, use, and disposal of agrochemicals, particularly pesticides, is an increasing important health andenvironment issue in developing countries where economies may be heavily reliant on agriculture. The conventional approach is
to develop highly optimized triple quad MRM methods to achieve the required levels of sensitivity and selectivity whilst still
providing confidence in pesticide identification. The technology developed for fast scanning MRM analysis creates the advantage
of developing multi-screening approaches for environmental target analysis and allows the possibility of a single generic ’universal’
method. In this present study, high speed MRM analysis and a generic parameter set were used for screening 176 pesticides（352SRM transitions）with 5 msec dwell and 1 msec pause times in different food matrices.
Compounds were analysed with a triple quadruple mass spectrometer（LCMS-8040, Shimadzu Corporation, Japan）. Separation
was achieved with UHPLC（Nexera, Shimadzu Corporation, Japan）. 352 MRM transitions were monitored over the entire
chromatographic run, 0 minutes to 20 minutes； therefore retention time data was not required for each compound. The dwelland pause time for each SRM transition was 5 msec and 1 msec, respectively, including one polarity switch during the loop. The
total loop time was 2.058 seconds which allowed approximately 10 data points to be collected for each peak, with a peak width
for studied compounds of about 20 seconds.
Three different challenging matrices were analysed（green tea leaves, leeks and paprika）, with sample preparation carried out
according to methods suggested by Japan’s Ministry of Health, Labour and Welfare. 10 pesticides（Carbofuran, Chlorfluazuron,
Fosthiazate, Hexathiazox, Indanofan, Lufenuron, Mevinphos, Propoxur, Pyrimidifen, and Tricyclazole）were spiked in green tea
leaves, leeks and paprika extract solution, each at a 10 ppb concentration. A 100 ppb spiked sample（green tea leaves）was also
prepared to develop the generic peak integration parameters used for compound identification. For each MRM mass
chromatogram, the largest peak was automatically selected as the target compound to permit automatic identification of target
analytes without retention time data.
The results showed that with regards to leek and paprika extract all 10 pesticides were automatically detected. In the case of agreen tea leaf extract, all pesticides except Indanofan were correctly identified（Indanofan was not automatically identified as alarger peak was also present in the mass chromatogram； a minor change to the method to create a retention window would
resolve this issue）.
The screening of 176 pesticides resulted in eight false positives in leek extract, seven in paprika extract and ten in green tea leaveextract.

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Food safety

PTh-176

13:30 － 14:40

Multi-class pesticide analysis in challenging vegetable matrices using fast
5 msec MRM with 15 msec polarity switching

Yuka Fujito1, Yusuke Inohana2, Kiyomi Arakawa2, Hirano Ichiro2
1

Shimadzu Analytica & Measuring Center, Inc., Kyoto, Japan, 2Shimadzu Co., Kyoto, Japan

Keywords:
Pesticide, QuEChERS, LCMS-8040 triple quadrupole mass spectrometer, Ultra-fast MRM, Ultra-fast polarity switching
Novel aspects:
Over one hundred pesticides spiked into complex vegetable matrices at 5 ppb were successfully detected in a single analysis.
Abstract:
Many regulatory authorities have established multi-class residual pesticides methods for the analysis of vegetables, fruits andother food stuffs. However, there is no global agreement on the provision of a target list of pesticides and this presents a risk withproducts moving between different regulatory requirements.
In order to eliminate this risk, food safety laboratories need to ideally screen as many compounds as possible in a single run
which may reach maximum residual limits（MRL）； typically 10ppb in food matrices. In this study we report the application of ultrafast 5 msec SRM with 15 msec polarity switching for the analysis of 138 pesticides, while still obtaining excellent LOQs for the
majority of compound < 10 ppb.
Leek and Paprika（yellow and red）were purchased from a local Japanese grocery store, with the country of origin Japan（Leek）
and New Zealand（Paprika）. Sample preparation was carried out by the use of a quick, easy, cheap, effective, rugged and safe
（QuEChERs）method. LCMS-8040 triple quadruple mass spectrometer（Shimadzu, Japan）was used to monitor 276 MRM transitions（two MRMs per compound）. Each transition was monitored plus-minus 0.8 minutes of the target analytes retention time. The
maximum loop time was 0.58 seconds and the maximum number of overlapped MRM transitions was 98（Dwell time： 5 msec,
Pause time： 1 msec）.
According to the method published by the European Union Reference Laboratory（EURL）entitled“Multiresidue Method using
QuEChERS followed by GC-QqQ/MS/MS and LC-QqQ/MS/MS for Fruits and Vegetables“, 138 pesticides were selected as target
analytes with sample pre-treatment by QuEChERs methods. Of these 138 pesticides, the EURL analysed and validated 66 pesticides
by GC-QqQ and the remaining 72 pesticides by LC-QqQ. In contrast to the EURL method, in this study all 138 pesticides wereanalysed in a single analysis using MRM after spiking into vegetable matrices. This was achievable due to the fast dwell（ 5 msec）
and pause times（ 1 msec）and rapid polarity switching（15 msec）of the triple quadruple mass spectrometer. Each MRM was
optimised and calibration curves constructed in the range 1 to 1000 ppb. 21 pesticides measured by GC-QqQ in the EURL method
did not reach the necessary LOQ（10 ppb）, but the remaining 45 pesticides achieved excellent LOQs in the range 0.08 to 10 ppb.
5 ppb and 50 ppb matrix matched standards were prepared for recovery studies. 90 % of the pesticides analysed presented
excellent recoveries in the range 70 -120 % in all studied matrices.

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Food safety

PTh-177

11:10 － 12:20

High Throuput Quantitative Analysis of Multi-mycotoxin in Beer-based
Drinks using UHPLC-MS/MS

Masayoshi Tamura1, Keiko Matsumoto2, Jun Watanabe2, Junko Iida2, Yasushi Nagatomi1,
Naoki Mochizuki1
1

Asahi Group Holdings, LTD., Ibaraki, Japan, 2Shimdzu Co., Kyoto, Japan

Keywords:
mycotoxins, carry over, high throughput, beer-based drinks
Novel aspects:
Development of a robust MS/MS method for the analysis of mycotoxins in complex commercial beverages with near zero carryover.
Abstract:
Mycotoxins often exist as contaminants in grains used in beer manufacturing. To maintain consumer needs for food safety, highly
sensitive methods of detection are required by food and beverage manufactures.
UHPLC-MS/MS offers the best combination of selectivity, sensitivity, and speed for detection of these compounds in complex
matrices. A high throughput method for the quantification of 14 mycotoxins in beer-based drinks was developed. While highsensitivity analysis was achieved, the nature of the analysis is characterized by a significant carry over issue as a result of thecomplex matrix. To enhance the robust detection of mycotoxins in beer-based drink analysis and to maintain high sensitivity, anovel autosampler technology was evaluated in this study.
MRM transitions were optimized for a range of mycotoxin standards（patulin, nivalenol, deoxynivalenol, aflatoxin B1, B2, G1, G2,
T- 2 toxin, HT- 2 toxin, zearalenone, fumonisin B1, B2, B3, and ochratoxin A）and analyzed using a Nexera UHPLC coupled to LCMS8030 triple quadrupole mass spectrometer（Shimadzu Co.）. Mycotoxins were separated using ODS column maintained at 40 °C.
Mobile phase： A - water + ammonium acetate, B - methanol + acetic acid. LC/MS was equipped with an electrospray ion source,
and analyzed in positive / negative switching mode.
Nexera autosampler was configured to perform multiple rinse modes with two rinse solvents to eliminate sample carry over.
To optimize MS detection, the MRM method for the 14 mycotoxins took into account positive and negative ion detection；
Aflatoxin B1, B2, G1, G2, T- 2 toxin, HT- 2 toxin, fumonisin B1, B2, B3, and ochratoxin were detected in positive mode while patulin,
nivalenol, deoxynivalenol and zearalenone were detected in negative mode.
UHPLC separation optimization for fumonisin B1, B2, B3 was enhanced with acetic acid as the buffer solution. Conversely, acetic
acid solution in the mobile phase reduced the sensitivity for patulin, nivalenol and deoxynivalenol. By controlling the concentrationof acetic acid and ammonium acetate with gradient elution program, the response and chromatographic behaviour for 14
mycotoxins was optimized using a cycle time of 11 minutes.
However, autosampler carryover was observed for fumonisin B1, B2, B3 and zearalenone using a single rinse mode. To eliminate
this problem completely a novel auto-sampler technology was considered. Using the multi-rinse function of SIL-30AC auto-sampler（Shimadzu Co.）, the interior and exterior surfaces of the autosampler needle were automatically washed with 2 different typesof rinse solutions. In this auto-sampler mode, the dilution series of 14 compound standards resulted in linear calibration curves,
excellent sensitivity near zero carry over. This method was then applied to the routine quantitative analysis of mycotoxins present
in a variety of commercial beer-based drinks following extraction and purification by SPE.

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Food safety

PTh-178

13:30 － 14:40

Molecular Imprinted Polymer@Magnetic Nanoparticles Combined with
Liquid Chromatography Mass Spectrometry for Determination of
Glycoalkaloids in Potato

Cheng-Hsin Yeh, Maw-Rong Lee

National Chung Hsing University, Taichung, Taiwan

Keywords:
molecular-imprinted polymer, magnetic nanoparticles, liquid chromatography-mass spectrometry, food safety, toxic glycoalkaloids
Novel aspects:
MIP@MNPs coupled with LC-MS for determination of trace toxic glycoalkaloids in potato plants.
Abstract:
Some of the glycoalkaloids in food have toxicity to humans. Among the toxic glycoalkaloids； α-solanine and α-chaconine areproduced in all port of the potato plants. In food safety issue, it is important to develop a sensitive and selective analytical methodfor quantification of trace toxic glycoalkaloids in food. In this research, a novel analytical method, molecular-imprinted polymermodified magnetic nanoparticles （MIP@MNPs） as extraction adsorbents combined with liquid chromatography-mass
spectrometry, was developed for determination of trace α-solanine and α-chaconine in potato plants and relative products. The
experimental conditions including the synthesis of MIP@MNPs and extraction parameters were optimized in this study. The
linearity, correlation coefficient, limits of detection（LODs）, and limits of quantitation（LOQs）were also evaluated. The analyticallinear ranges for α-solanine, α-chaconine, and solanidine were obtained at 0.01-10 and 0.05-50 ng/g, respectively. The precision
expressed by relative standard deviation（RSD）were below 8.2%. The LODs of proposed method ranged from 11 to 160 pg/g. The
feasibility of applying the proposed method to determine the trace toxic glycoalkaloids in potato was also examined. The trace
amount of α-solanine, α-chaconine, and solanidine in potato were detected by proposed method at 3.3, 6.0, and 4.2 ng/g,
respectively. The results showed the developed method can be successfully utilized for analyzing the α-solanine, α-chaconine,and solanidine in potato.

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Food safety

PTh-179

11:10 － 12:20

Determination of cyromazine and melamine in chicken eggs by using
QuEChERS coupled with liquid chromatography tandem mass
spectrometry

Pei-Cheng Wang, Ren-Jye Lee, Chi-Chung Chou, Maw-Rong Lee
National Chung Hsing University, Taichung, Taiwan

Keywords:
Cyromazine, melamine, QuEChERS, LC-MS/MS
Novel aspects:
QuEChERS was developed to extract the trace cyromazine and melamine in chicken egg samples by using small volume ofextraction solvent and variety of sorbent.
Abstract:
A rapid and sensitive method has been developed for the simultaneous detection of cyromazine and melamine in chicken eggs byusing the quick, easy, cheap, effective, rugged and safe（QuEChERS）method coupled with a liquid chromatography tandem mass
spectrometry（LC-MS/MS）system. The optimal sample preparation conditions for liquid-liquid extraction were 5 mL ofacetonitrile with a 0.1 M hydrochloric acid aqueous solution（99.5：0.5, v/v）. The extract was cleaned with 0.5 g of anhydrous
magnesium sulfate and 10 mg of graphitized carbon black. The analysis of cyromazine and melamine was accomplished by
combining the use of an anion exchange LC column with tandem mass spectrometry in the positive electrospray ionization withselected reaction monitoring mode（SRM）. The detection limits were 1.6 ng g-1 for cyromazine and 7.5 ng g-1 for melamine, andthe quantification limits were 5.5 ng g-1 for cyromazine and 25 ng g-1 for melamine. The recovery of cyromazine and melamine in
spiked egg sampleswas 83.2 % and 104.6 % , respectively, with an RSD of less than 18.1 % . Intra-day and inter-day precision,
represented as an RSD ranged from 1.5% to 8.8% and 6.8% to 14.3%, respectively. The proposed method was tested by analyzing
chicken eggs from the market and the veterinary medicine laboratory. The results of this study reveal that the proposed method
can be used to adequately analyze cyromazine and melamine in chicken eggs. The concentrations of cyromazine and melamine
detected in these samples were in range of 20 to 94 ng g-1.

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Food safety

PTh-180

13:30 － 14:40

DIFFERENTIATION OF FARMED FISH AND WILD SEAFISH USINGISOTOPIC SIGNATURES

Lian Jie Bay1, Jaime Wong1, Daniel Fliegel2, Sheot Harn Joanne Chan3, Thomas R Walczyk1
1
3

National University of Singapore, Singapore, 2National Institute of Nutrition and Seafood Research, Bergen, Norway,
Health Sciences Authority, Singapore

Keywords:
Strontium Isotopic Ratios, Light Isotopic Ratios, Farmed VS Wild Marine Fish, Atlantic Cod, Food Fraud
Novel aspects:
First report on the use of isotopic tecnniques to differentiate between wild and farmed atlantic cod, particularly the use ofstrontium isotopic ratios which has never been attempted.
Abstract:
Worldwide consumption of fish has risen dramatically over the decades. Overfishing, a consequence of this demand, has led to
decline of fish stocks and depletion of marinefood webs in many parts of the world. To meet the increasing demand for seafood,aquaculture has developed as it offers a cost effective supply of seafood. However, aquaculture employs the use of antibiotics,
dyes, growth hormones,“sustainable fish feeds“ where marine proteins are being substituted with plant based proteins as wellas fish-feed produced from genetically modified raw materials. This may result in increased levels of contaminants in aquacultured
fish but also may change their nutritional value. Wild fish being perceived as the superior product can therefore command a higher
price on retail markets. This opens avenues for food-fraud by mislabeling. It is estimated that the origin of 25 to 70% of seafood isfraudulently declared. This project aims to develop an ultrasensitive method to distinguish wild and farmed cod（Gadus morhua）
by employing strontium（ 87 Sr/ 86 Sr）, carbon（ 13 C/ 12 C）and nitrogen（ 15 N/ 14 N）isotopic ratios in fish meat as a dietary marker.
Techniques involving Thermal Ionisation Mass Spectrometry（TIMS）and Isotope Ratio Mass Spectrometry（IRMS）for measuring
carbon, nitrogen and strontium isotope variations were developed.
Differences in natural strontium isotopic ratios arise in nature due to the decay of 87Rb to 87Sr. This radiogenic decay has produced
distinctly different Sr isotope abundances in different parts of the Earth crust over its history. This isotopic signature isincorporated into plants and will be passed onto fish through feed prepared from the plant material. In contrast, due to the longresidence time of Sr in seawater（millions of years）, compared to the turnover time of the oceans（millennia）, 87 Sr/ 86 Sr ishomogeneous throughout the world’s oceans at any given time. Wild marine fish should display the unique strontium isotopic
ratio of seawater as a result of feeding solely on marine organisms, while farmed fish should exhibit an isotopic ratio moreterrestrial in nature, as fish feed in aquaculture utilizes terrestrial plant based materials. Differences in carbon and nitrogen isotopic
values arise for the same reason, i.e. wild and farmed cod should differ essentially in the isotope signature taken up from feed.
Methods for the analysis of strontium isotopic ratios in cod involved microwave digestion of tissue, separation of the strontium
ions from interfering elements using ion exchange chromatography and finally analysis of the purified fraction using TIMS.
Achievable precisions for 87Sr/86Sr are of the order of parts per million（0.0017% RSD； n= 6 ）. Analysis of both wild and farmed codfrom Norwegian fish farms showed similar strontium isotopic ratios despite differences in feed. This contradicts the initial
hypothesis that both groups of fish should exhibit different strontium isotopic ratios and that cod acquires strontium primarily fromwater and not from feed. Measurement precision was on the order of 0.1‰ for nitrogen and 0.06‰ for carbon. By combining bothsignatures, wild and farmed cod could be distinctly separated from one another which may turn the developed techniques into an
important tool for differentiating farmed from wild cod.

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Food safety

PTh-181

11:10 － 12:20

Evaluation of a new ion source to improve the limit of quantitation forchloramphenicol on a UHR-TOF

Karin Wendt, Thomas Zey, Amalia Apalategui, Carsten Baessmann, Peter Brechlin
Bruker Daltonik GmbH, Bremen, Germany

Keywords:
Heated electrospray source, 15 to 15 fold improvement in sensitivity, quantification of chloramphenicol in different matrices
Novel aspects:
Improved sensitivity for chloramphenicol in different matrices on a UHR-TOF with a newly developed heated electrospray source
Abstract:
Introduction
Chloramphenicol（CAP）is used as a broad-spectrum antibioticum in veterinary medicine. Due to the serious side effects forhumans it has been banned for all food producing animals in various countries. Current law in the European Union（EU）requires aminimum required performance limit（MRPL）of 0.3 ug/kg or 0.3 ppb. We present here a work using an accurate mass target
screening approach to quantify CAP in different typical matrices, such as meat, urine or honey. Heated electrospray sources are
known to be show sensitivity boosts for certain compound classes. A newly developed heated electrospray source will bepresented that is capable of high liquid flows and should help to improve the sensitivity of these samples from complex matrices.
Methods
Chloramphenicol was spiked into homogenized beef-muscle at different concentrations from 0.25 ng/kg to 10 ug/kg. For sample
preparation ethylacetate was added. The sample tubes were vortexed, shaken overhead, centrifuged and the supernatantcollected. The combined ethylacetat-fraction was applied to equilibrated NH 2 SPE（Supelco supelclean NH 2 500 mg, 3 mL）andthe eluate directly collected. The samples were separated with a short 4.5 min gradient on a UHPLC system. Data acquisition was
performed in negative mode using a Ultrahigh Resolution（UHR）-TOF equipped with a new heated electrospray ionization MS
source. For comparison the samples were analyzed with a standard ESI source and the heated ESI source.
Preliminary Data
During the characterization of the source it was identified that important parameters for the heated electrospray are sheath gas
and the vaporizer temperature. For chloramphenicol the optimum conditions were 4 l/min N 2 sheath gas and 320 degree C
vaporizer temperature. With the new heated electrospray a 10 to 15 fold improvement in sensitivity was observed forchloramphenicol in the different matrices, compared to a standard electrospray source. In meat the limit of detection was below
0.001 ug/kg and the limit of quantitation below 0.0025 ug/kg. At 0.0025 ug/kg the average S/N was 15.4 and the RSD 9 % for 6
technical replicates. We found a good linear dynamic range from 0.001 to 10 ug/kg. The limit of quantitation for this zero-tolerance
substance in the different matrices was well below the required MRPL of 0.3 ug/kg. The complete characterization of the source
for the quantification of chloramphenicol in different matrices will be presented.

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Food safety

PTh-182

13:30 － 14:40

MASS SPECTROMETRY IDENTIFICATION AND CHARACTERIZATION OF
GIBBERELLINES IN FRUITSENCARNACION MOYANO, GABINO BOLIVAR-SUBIRATS, HECTOR GALLART-AYALA,MARIA TERESA GALCERAN
Barcelona University, Barcelona, Spain

Keywords:
Gibberellins, UHPLC, Mass Spectrometry, Electrospray
Novel aspects:
A common fragmentation pathway for GAs is established. A strategy for non-target GAs identification using mass spectrometry
data and LC retention windows predicted from estimated log Poct/wat is proposed
Abstract:
Gibberellins（GAs）are an important class of plant hormones that regulate growth and influence some developmental processes.
GAs are used in agriculture and horticulture to break dormancy, to speed up flowering and fruiting and to stimulate theproduction of seedless fruits in the absence of pollination, probably by stimulation of production of endogenous GAs that enhancetheir activity. All known GAs are tetracyclic diterpenoid acids derived via the ent-gibberellane skeleton,but synthesised via entkaurene. Actually, more than 130 GAs have been identified from plants, fungi and bacteria and they are classified in two classes
based on the presence of either 19 or 20 carbons. The 19-carbon gibberellins, such as gibberellic acid（GA 3 ）, possess a fivemember lactone bridge that links carbons 4 and 10. The 19-carbon forms of GAs are the biologically active. These compounds
differ in the number and site of hydroxyl groups. In general, the most biologically active compounds are dihydroxylated
gibberellins, which possess hydroxyl groups on both carbon 3 and carbon 13（e.g. GA 3 ）. The low concentration of some ofthese compounds and the matrix complexity of vegetable samples make indispensable the use of mass spectrometry in
combination with liquid chromatography（LC/MS）for their identification, confirmation and characterization. It must be taken into
account that the few commercial standards available make difficult the development of analytical methods. Additionally, tandem
mass spectrometry information about GAs is limited.
The aim of this work was to develop a sensitive and selective method for the identification and characterization of GAs. For thisgoal, mass spectrometry tools and chromatographic strategies have been combined to propose a method for the analysis of thewhole family of GAs. Ultrahigh performance liquid chromatography on a sub- 2 µm C18 reversed phase column（MeOH： 0.1 %formic acid and gradient elution）provided the separation of available standards（GA1, GA3, GA 4 and GA 7 ）in less than 5 minwith high efficient peaks. Octanol-water constants（log P oct/wat ）were estimated（software： VCCLABS）for all GAs and theirinterpolation in the experimental retention data allowed us to predict retention windows for the searching of non-target GAs.
Electrospray was used as ionization source in negative mode being［M-H］- the base peak. A fragmentation pathway was proposed
for GAs based on the tandem mass spectrometry data obtained in a triple quadrupole mass analyzer and this fragmentation
pathway was used to predict the potential main product ions of non-target GAs. The diagnostic ions were used for the tentative
identification of non-target GAs in fruit samples（apple, pear and peach）collected thorough the whole growth period. The
presence of these GAs was confirmed by using accurate mass measurements on an Orbitrap mass analyzer and product ion scanwith reversed energy ramp（RER）on a triple quadrupole instrument was used to monitor product ions of both low and highmolecular weight. Target GAs（GA1, GA3, GA 4 and GA 7 ）and some non-target GAs（e.g. GA9, GA10, GA21, GA82）were detected
and confirmed in these samples.
References- L.N. Mander, Nat. Prod. Rep., 20, 49-69（2003）.
- M. Kojima, etal., Plant Cell Physiol., 50（ 7 ）：1201-1214.（2009）.
- Y. Izumi, etal., Analytica Chimica Acta, 648215-225（2009）.
- S.D.S.Chiwocha, et al., The Plant Journal,35, 405-417（2003）.
- SofwareVCCLABS for log Poct/watestimation： www.vcclab.org/lab/alogps

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Food safety

PTh-183

11:10 － 12:20

EVALUATION OF ATMOSPHERIC PRESSURE IONIZATION SOURCES FOR
MASS SPECTROMETRIC ANALYSIS OF PHENICOL DRUGSELIDA ALECHAGA, ENCARNACION MOYANO, MARIA TERESA GALCERAN
Barcelona University, Barcelona, Spain

Keywords:
Phenicols, ESI, APCI, APPI, tandem mass spectrometry
Novel aspects:
Comparison of the behaviour of phenicols in API sources（ESI, APCI, APPI）is performed. Mass spectrometry fragmentation
pathway has been established and some product ions has been corretly assignmed
Abstract:
Nowadays, LC-MS has become a robust and well-established analytical technique for the analysis of small molecules in food,biological and environmental matrices. Until recently, the most commonly used atmospheric pressure ionisation（API）sources forcoupling liquid chromatography to mass spectrometry have been electrospray ionisation（ESI）and atmospheric pressure chemical
ionisation（APCI）［1, 2 ］, but atmospheric pressure photoionisation（APPI）has gained popularity in the last few years expanding
the application field of LC/MS［ 3 ］. However, there are only a few studies in which the suitability of the ESI, APCI and APPI
sources for the analysis of veterinary drugs in food matrices has been compared［ 4 - 6 ］.
In this work, the veterinary drug family of phenicols, including chloramphenicol（CAP）, a wide-spectrum antibiotic, and its related
compounds thiamphenicol（TAP）, florfenicol（FF）and florfenicol-amine（FFA）, was studied by LC-MS/MS. Their behaviour in ESI,APCI and APPI under fast liquid chromatographic separation conditions（Fused Core Phenyl-Hexyl column, methanol：acetic
acidammonium acetate buffer, 5 mM and pH 5 ）was compared taking into account both positive and negative ionization modes.
For APPI different dopants（toluene, anisole, tetrahydrofuran, chlorobenzene and acetone）were tested evaluating theirperformance in terms of ionization efficiency and mass spectral data. In all API sources CAP, TAP and FF ionized in both positive
and negative modes, while for the metabolite FFA only positive ionization was possible. In general, in positive mode［M+H］+
dominated the mass spectrum for FFA, while the other compounds, CAP, TAP and FF, with lower proton affinity showed an
additional intense ion［M+NH4］+. Moreover, in-source fragmentation was also observed（loss of H2O）for FFA, FF and CAP at highsource temperature such as in H-ESI and APCI. This water loss was enhanced in APPI and the relative abundance of this fragment
ion depended on the dopant nature. In some cases, it become the base peak of the mass spectrum. In negative mode, ESI andAPPI showed the deprotonated molecule［M-H］-, while APCI provided the radical molecular ion by electron capture. Additionally,
chloride adduct formation［M+Cl］- was observed in ESI and APPI（depending on the dopant）and the loss of HF occurred. All
these ions were characterized by tandem mass spectrometry a common fragmentation pathway was established for this family ofcompounds. This mass spectrometry study allowed us to clarify some discrepancies observed in the literature in relation to theassignment of some product ions commonly used for quantitative and confirmative purposes.
References［ 1 ］Dams, Riet and Huestis, Marilyn A.（2003）J.Am. Soc. Mass Spectrom. 14：1290-1294［ 2 ］Schlüsener, Michael P. and Bester, Kai（2005）Rapid Commun. Mass Spectrom. 19：3269-3278［ 3 ］Robb, Damon B., Covey, Thomas R. and Bruins,Andries P.（2000）Anal. Chem.72：3653-3659［ 4 ］Martínez-Villalba, Anna, Moyano, Encarnación, Martins, Claudia P.B., and Galceran, Maria Teresa（2010）Anal.Bioanal.Chem.
397：2893-2901［ 5 ］Mohamed, Rayane, Hammel, Yves Alexis, Le Breton, Marie Helene, Tabet, Jean Claude, Jullien, Laure, and Guy, Philippe A.
（2007）J. Chromatogr., A 1160：194-205［ 6 ］Takino, Masahiko, Daishima, Shigeki, and Nakahara, Taketoshi（2003）J.Chromatogr., A 1011：67-75

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Food safety

PTh-184

13:30 － 14:40

High-throughput simultaneous analysis of pesticide residues in food bysupercritical fluid chromatography/tandem mass spectrometry

Megumi Ishibashi1, Ando Takashi2, Sakai Miho2, Matsubara Atsuki1, Uchikata Takato1,
Fukusaki Eiichiro1, Bamba Takeshi1
1Osaka University, Osaka, Japan, 2Miyazaki Agricultural Research Institute, Miyazaki, Japan

Keywords:
pesticide residues analysis, supercritical fluid chromatography, mass spectrometry
Novel aspects:
Supercritical fluid coupled with tandem mass spectrometry allowed us to analyze pesticides with a wide range of polarities andmolecular weights simultaneously.
Abstract:
Application of pesticides to agricultural products is an efficient means of mitigating unacceptable loss of agricultural production.
However, pesticide residues in food products may cause health problems ranging from simple skin irritation and nausea to morelife-threatening ones such as acute poisoning and breathing problems. Therefore, the maximum residue limits（MRLs）for
pesticides, veterinary medicines, and food additives have been established in various countries. Hence, many researchers havefocused on the development of a system for the simultaneous and rapid analysis of multicomponent pesticides. Combination
techniques such as gas chromatography/mass spectrometry（GC/MS）and liquid chromatography/mass spectrometry（LC/MS）
are commonly used for pesticide residue analysis, but there is no reported method for the simultaneous analysis of multiple
pesticides in a sample using a single instrument. Supercritical fluid chromatography（SFC）offers high resolution at high flow rates
and various separation modes and hence may aid the rapid simultaneous analysis of pesticide residues. We developed an SFC/
MS/MS method and analyzed 17 pesticides with a wide range of polarities（logPow = 4.6 to 7.05）and molecular weights（112.1 to888.6）within 11 min using a polar-embedded reverse-phase column. This SFC-based system can analyze various pesticides that are
traditionally analyzed by separate processes such as GC/MS and LC/MS. Since the elution power of SFC can be changed by theaddition of a polar solvent, diquat dibromide together with cypermethrin and tralomethrin could be detected in the presence ofvarious other pesticides using a single mobile phase. Usually, diquat dibromide requires a special analysis method such as ion
chromatography and ion-pair LC, while cypermethrin and tralomethrin are analyzed by GC. This result showed that SFC is also
applicable to the analysis of many more pesticides with logPow values ranging from 4.6 to 7.05. Most of the compounds weredetected even at ng/L levels, while diquat dibromide, fosetyl, maleic hydrazide, and cypermethrin were detected at µg/L levels.
The limit of detection（LOD）and limit of quantification（LOQ）of most compounds were higher in agricultural extracts（rice, onion,and spinach）than in standard mixtures. However, the LODs of most compounds except for highly hydrophobic ones such as
tralomethrin, cypermethrin, and etofenprox in onion were much lower than the corresponding Japanese maximum residue limits.
The proposed method can also be used to detect pesticide residues in agricultural product extracts obtained by a suitable
extraction method； the sample purification procedure can be simplified by adopting the standard addition method, and theanalysis does not involve any complex pretreatment process such as derivatization. Therefore, we conclude that SFC/MS is a highthroughput, robust, and highly reproducible technique for rapid and simultaneous pesticide residue analysis. A more practicalscreening system based on SFC/MS for pesticide residues will be constructed in near future.

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Food safety

PTh-185

11:10 － 12:20

Chromatographic Separation of Chrysene and Triphenylene in Smoked
Fish

Mirja L Hokkanen, Marika N Jestoi, Kimmo E Peltonen
Finnish Food Safety Authority Evira, Helsinki, Finland

Keywords:
PAH, Separation, Chrysene, Triphenylene, GC-MS/MS
Novel aspects:
Recently GC columns that allow separation of chrysene and triphenylene were commercialised. A lack of chromatographic
resolution might lead to overestimation of chrysene and to false conclusions of the samples.
Abstract:
Polycyclic aromatic hydrocarbons（PAHs）are organic contaminants formed primarily by incomplete combustion during foodprocesses, such as drying, grilling, smoking and roasting. The European Union（EU）has published a recommendation for themonitoring of 15+ 1 EU priority PAH compounds typically abundant in different kinds of foods. According to the scientific opinion
by the European Food Safety Authority（EFSA, 2008）, benzo［a］pyrene alone is not a suitable indicator for the occurrence andtoxicity of PAHs in food. Based on this fact as of September 2012, in addition to benzo［a］pyrene, the maximum limits will be
determined for the sum of four PAHs（benzo［a］pyrene, benz［a］anthracene, chrysene and benzo［b］fluoranthene）.Typicallythe GC columns used for the separation of PAH compounds include non-polar stationary phases, developed for the determinationof environmentally important PAHs, as reported in the US EPA priority list. However, these phases do not usually provide adequate
separation for certain critical pairs of PAH’s that are important for food contamination. An example of such critical pairs ischrysene（Chr）and triphenylene（Trp）. Chr and Trp are isomeric compounds having exactly the same molecular weight andfragmentation patterns, making their identification a challenging task even when using tandem mass spectrometry（MS/MS）.
Their resulting co-elution, together with the fact that they are undistinguishable in the detector, may cause analytical problems
such as overestimation of contamination levels for Chr, as well as for the PAH 4 compounds. Due to analytical problems for thedetermination of the critical pair of Chr and Trp by using traditional non-polar GC columns, other approaches must be applied toensure consumer safety. Not until recently, did columns that allow separation of Chr and Trp become commercially available. In
this study, GC columns with different chemistries were tested to solve the problem with the co-elution of Chr and Trp. The
analytical columns tested were a Phenomenex Zebron ZB-50 and an Agilent Select PAH. The Zebron ZB-50 column has high polar
phenyl siloxane phase which has higher temperature limits than other widely used stationary phase chemistries. The Select PAH
capillary column offers enhanced selectivity towards PAHs, separating the isomers and overcoming the limitations of other GC
columns. Smoked fish samples were analysed by using both columns. In addition, a sample with known amount of Chr obtained
from an inter-comparison study was analysed. The determination of PAHs was conducted by using an ISO 17025 accredited GCMS/MS method. In the poster presentation the effects of using those two GC column chemistries on the analytical results of Chr infish samples will be presented. Preliminary findings from this study show that successful separation of Chr and Trp was achieved
only with the Agilent Select PAH column.

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Food safety

PTh-186

13:30 － 14:40

Assessing the impact of a novel ion source design in food safety
applications using MS/MS detection

Neil J Loftus1, Simon Hird2, David Baker1, Alan Barnes1
1

Shimadzu, Manchester, UK, 2FERA, York, UK

Keywords:
Food safety, LC/MS/MS, data quality
Novel aspects:
Applying a novel ion source design with turbulent and laminar flow regions to food safety analysis using MS/MS detection
Abstract:
In today’s global marketplace, as foods are produced and distributed throughout the world, food quality and food safety become
increasing concerns for consumers, governments and producers. There is a need to develop residue methods with fast turnaround times and preferably with only minimal sample preparation and clean up. LC-MS/MS is the ideal analytical tool to fulfil therequirements for speed, sensitivity, selectivity and robustness. Recent developments in the design of mass spectrometer and LC
column packing materials allow more components to be analyzed in the same injection within a reasonable short time and thus
make high throughput multi-compound analyses become a reality. Reports of the LC-MS/MS determination of hundreds ofpesticides in various fresh produce and other agricultural commodities and the determination of veterinary drug residues in animal
tissues are now common place［［ 1 ］］.
Difficulties remain when trying to discriminate analytes from co-extracted matrix components that exhibit similar physiochemical
properties. If not addressed this can lead to a reduction in quality of data and can impact a laboratory’s performance andreputation. Matrix components can cause increased chemical noise and ion suppression. When experienced in combination thismakes determinations of residues at low concentrations very difficult. In addition to problems caused by the sample matrix, someanalytes exhibit poor response in electrospray ionisation. Successful analysis of these compounds to the regulatory concentration
limits is difficult. Increasing the amount of sample introduced is impractical as this approach can lead to an increase in ion
suppression. A much more practical solution is to use increased instrument sensitivity to maximise performance at these required
concentrations.
This paper describes the application of a novel ion source design which increases electrospray sampling efficiency using turbulent
and laminar flow gas flows generating ions with well-defined ion energy. Described here are some initial data illustrating theapplication of a novel source design interfaced with a triple quadrupole mass spectrometer to provide suitable sensitivity androbustness for the determination of pesticides and veterinary drug residues in a range of typical foodstuffs and animal tissues,
respectively, for official control purposes.
［ 1 ］A. Malik, C. Blasco and Y. Pico, J. Chromatogr. A 1217（2010）4018.

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Food safety

PTh-187

11:10 － 12:20

Development and applications of a pesticide multiresidue analysis turnkey system utilizing UHPLC-Orbitrap MS and post data processing

James Chang2, Jon Wong3, Kai Zhang3, Serei Thach4, Paul Yang4, Charles Yang2, Kristi Akervik2,
Maciej Bromirski2, Yoko Yamagishi1, Dipankar Ghosh2

Thermo Fisher Scientific K.K., Yokohama, Japan, 2ThermoFisher Scientific, San Jose, CA, 3Food and Drug Administration,
Center for Food Safety and Applied Nutrition, MD, 4Ontario Ministry of the Environment, Ontario, Canada

1

Keywords:
food, pesticide, contaminates
Novel aspects:
UHPLC-Orbitrap based multiresidue method has been shown to have equivalent performance in quantitative and superior ability
in target and unknown identification.
Abstract:
Introduction
Multresidue methods are employed to monitor chemical contaminants in food and environmental samples and quantitate theirconcentrations to ensure that concentrations present do not exceed the tolerance levels established by government regulations.
Many of these multiresidue methods have been developed for pesticides, mycotoxins, veterinary drugs, and polar organic
pollutants employing UHPLC-MS/MS in targeted analysis. These methods are selective, sensitive, cost-effective and can be
optimized to analyze as many target analytes in a single injection. However, optimization of MS/MS parameters is a very time
consuming process； and requires re-optimize the duty cycle, scanning efficiency and MRM transitions whenever new pollutants
are added. Difficulties associated with maintaining MS/MS based method make UHPLC-Orbitrap and cheminformatic based
multiresidue methods an attractive alternative.（119 words in total）

Methods
Samples including fresh produce and other food products such as wheat flour and raisin were purchased locally as organic orconventional products. Incurred samples were obtained from the FDA field laboratories and the EPA Analytical Chemistry
Laboratory, Ft. Meade, MD. QuEChERS procedure was used in the preparation of method validation and incurred samples.
Reversed phase UHPLC separation was achieved using 2.1x100 mm column at various flowrates to characterize and optimize
Orbitrap operation parameters. All data were acquired in positive ionization mode. Full scan, high resolution MS data using asecond generation Exactive were acquired at four different MS resolutions and various concentrations to investigate the selectivity
and sensitivity. Acquired data were transferred and processed off-line using Exact Finder to data mining software to maximize
operation efficiency.（119 words in total）

Preliminary data
Analytical data were obtained from a mixture of > 200 target pesticides prepared in solvent and produce sample matrices. These
data are used for the determination of instrument detection limit（IDL）and matrix specific method detection limit（MDL）.
Analyzed and processed at 5 - 7 levels of concentrations in 5 - 8 replicates analyses, the data shows that optimized Orbitrap
extracted ion chromatogram（XIC）data can be collected at an resolution of 70,000（FWHM at m/z 200）or higher as long as there
is a minimal of 5 - 7 data points to define a specific chromatographic peak. IDLs were determined from solvent analyticalstandards and were in the high part-per-trillion to low part-per-billion range with > 90% of pesticides evaluated showed a MDL of
5 part-per-billion or less. Incurred samples analyzed such as orange, Kale and cabbage showed the presence of various pesticides
that matched patterns of field application and usage very well. Quantitative data obtained from UHPLC-Orbitrap analysis showed
an excellent equivalency compared to those obtained from MS/MS analysis using two MRM transitions. Orbitrap analysis did havean edge over the two MRM based MS/MS analysis in avoiding false-positive identification, especially chemical analogs likespinosyn A, C and D. Post data processing software provided for the identification of targeted unknowns using chromatography
retention time, accurate mass and isotopic pattern； achieving the identification and confirmation requirements in analyticalchemistry. Additional cheminformatic software allowed for the use of ChemSpider of Royal Chemistry Society, greatly increase theconfidence in the identification of unknown.

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Food safety

PTh-188

13:30 － 14:40

Multi-residue method for the confirmation of six avermectin residues infood products of animal origin by liquid-chromatogrphy tandem mass
spectrometry

Myeong-Ae Kim, Jeong Woo Kang, Myung-Sin Lim, Young Hoon Bong, Chae-Mi Lim, Seoung-Wan Son
Animal, Plant and Fisheries Quarantine and Inspection Agency, Anyang, Korea

Keywords:
chromatography, LC/MS： drug residues, ivermectin； animal products, meat； milk
Novel aspects:
The determination and develpement of multi-residue analysis method for the confirmation of six avermectin residues in foodproducts of animal origin using QuEChERS sample preparation and liquid-chromatography tandem mass spectrometry
Abstract:
A confirmation method was developed for the rapid determination abamectin, ivermectin, doramectin, moxidectin, emamectin
and eprinomectin residues in various food products of animal origin, such as pork muscle, pork liver, pork kidney, milk and egg.
Samples were homogenized, extracted by acetonitrile, cleaned via dispersive-clean up procedure using magnesium sulfate andprimary secondary amine（PSA）on based QuEChERS method（quick, easy, cheap, effective, rugged and safe）. All the six
avermectin residues in different animal-food products were simultaneously separated and determined by liquid chromatographyelectrospray ionization tandem mass spectrometry（LC-ESI-MS/MS）within 10 min. Data acquisition under positive ESI-MS/MS was
performed by applying multiple reaction monitoring（MRM）for both identification and quantification, and mass spectrometricconditions were optimized to increase selectively and sensitivity. The matrix-matched calibration curves for different matrices,such as pork muscle, pork liver, pork kidney, milk and egg, were constructed and the interference effect of different sample
matrices on the ionization was effectively eliminated. The LC-MS/MS method was validated with satisfactory linearity, recovery,
precision and stability. Matrix-matched calibration curves of abamectin, ivermectin, doramectin, moxidectin, emamectin andeprinomectin in five different matrices were linear（r20.990）in the range 2.52000 ug/kg. The limit of detection and quantification forthe six avermectins were in the range 0.1-12.2 and 0.2-40.6 ug/kg, respectively. Recoveries were 62.9-120.9 % with good intra- andinter-day precision of 7.0-14.4 and 8.6-15.0%, respectively. The method was rapid, sensitive and reliable, and can be applied to thequantitative analysis of avermectin residues in different animal food products.

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Food safety

PTh-189

11:10 － 12:20

Multi-residue analysis of 59 pesticides in raw bovine milk using GC-MS/
MS and UPLC-MS/MS

JinYoung Shin, Dong-Gyu Kim, Kyung Yuk Ko, MeeKyung Kim, Chae-Mi Lim, Seong-Wan Son
Animal, Plant and Fisheries Quarantine and Inspection Agency, Anyang, Korea

Keywords:
Pesticides； Multi-residue analysis； raw bovine milk； GC-MS/MS； LC-MS/MS
Novel aspects:
Multi-residue analysis of pesticides in raw bovine milk using QuEChERS method with GC-MS/MS and LC-MS/MS
Abstract:
A fast multi-residue method was developed for the screening, quantification and confirmation of 59 pesticides, belonging todifferent chemical families of insecticides, acaricides, fungicides, herbicides and insect growth regulators in raw bovine milk using
QuEChERS method with GC-MS/MS and UPLC-MS/MS. The Optimization of different parameters such as extraction solvents（acetonitrile, acetone, or ethyl acetate）, type of clean-up sorbents（PSA, C18, or GCB）, GC and UPLC separation and MS/MS
parameters were carried out. The developed method which used acetonitrile/ 1 % acetic acid as a extraction solvent and used
PSA/C18 sorbents as a cleanup step was applied. Under the optimized conditions, GC and UPLC-MS/MS gave linear calibrations
over the concentration range from 1 to 100 ng/mL（r > 0.990）. The recoveries were, with a few exceptions, in the range of 61 - 124% at the different spiked levels with satisfactory precision（CV < 16 %）. The quantification of analytes was carried out using themost sensitive transition for every compound and by matrix-matched standards calibration. Limits of detection（LOD）andquantification（LOQ）varied from 0.1-9.2 ng/mL and 0.2-19.5 ng/mL, respectively. The results indicated that the proposed method
can be applied to the determination of 59 pesticides which have been established MRLs（maximum residue levels）in raw bovine
milk.

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Others

PTh-190

13:30 － 14:40

Untargeted Plasma Metabolomics using 13C-Glucose: GlycatedMetabolites as Novel Biomarkers for Aging and Diabetes

Daiki Setoyama, Yoshinori Fujimura, Hiroyuki Wariishi, Daisuke Miura
Kyushu University, Fukuoka, Japan

Keywords:
Glycation, Metabolite, Metabolomics, Plasma, 13C-Glucose
Novel aspects:
With our strategy, some of the glycated metabolites can be identified to be accumulated in aged and diabetic plasma, implying
the potential relationship between the glycation of metabolites and diseases.
Abstract:
Highlights
Protein-glycation has been implicated in the pathogenesis of aging, diabetes, and other angiopathy-related diseases because it
potentially produces advanced glycation end-products（AGEs）to cause intracellular oxidative stress and alterations in proteinfunctions. However, little has been verified for glycation of metabolites in cells and their involvement in cellular dysfunctions. In
this study, we performed LC-MS-based untargeted metabolomics for mouse plasma with an attempt to obtain a library ofglycation-vulnerable metabolites that could react with 13C-glucose in vitro. With the library, it is evaluated whether the glycatedmetabolites would be indeed found（or accumulated）in plasma derived from naturally-aged mice and disease-related mice, suchas senescence accelerated-prone（SAMP 8 ）and diabetic db/db lineages, respectively. Interestingly, we observed that some of theglycated metabolites, including amino acids and peptides； lysine, glutamine, taurine, gamma-glutamyl-lysine and glutathione, wereidentified to be accumulated in db/db plasma as compared with control plasma. We could also detect two kinds of AGEs,
carboxymethyl lysine（CML）and 3 -deoxyglucosone（ 3 DG）in diabetic plasma. Although the causal relationship between theglycated metabolites identified and their productivity of AGEs awaits further investigation, this study will provide some potential
biomarkers for early diagnosis of aging and diabetes and also offer a useful annotating strategy for previously unregistered（indatabase）metabolites having specific chemical modification, like glycation.
Rationale
Glycation, a chemical reaction in which a reducing sugar is added to an amino group of proteins, occurs non-enzymatically in highglucose conditions or hyperglycemia. It results in the accumulation of deleterious advanced glycation endproducts（AGEs）, someof which accelerate cross-linking reaction between proteins, thereby causing their aggregation, degeneration, and dysfunction.
Since accumulation of AGEs is suggested to be involved in aging and deterioration of diabetes-related complications, it is criticalto regulate generation of AGEs for maintaining intracellular homeostasis. Previous studies, however, have mainly focused onprotein-glycation as an AGEs-generator, underestimating the significance of the glycation of small-weight molecules, metabolites,in cells. Given that some of the metabolites have amino group in their side chain and high reactivity with many biomacromolecules such as proteins, nucleic acids, and lipids, it would be reasonable to say that metabolites can be glycated as well
to serve as a source generating harmful AGEs or per se as a noise or trigger for intracellular signal transductions. Here, we perform
a mass spectrometry-based metabolomics in combination with 13C isotope labeling strategy to explore the glycated metabolites
in mouse plasma. To our knowledge, this kind of comprehensive study would be unprecedented and we therefore believe thatthe knowledge obtained in this study would not only be beneficial for a better understanding of diseases but also lead to uncovernovel aspects of molecular mechanism underlying intracellular homeostasis.

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Others

PTh-191

11:10 － 12:20

The examination of high-throughput, high-sensitive and more-ecologicalmeasurement by micro flow UHPLC/MS/MS system

Seiji Horie1, Kazuhide Inoue1, Masazumi Yasumoto1, Kayoko Hagiwara2, Shigeru Yamada2,
Yasutoshi Kawase1
1

TAKARA BIO INC., Shiga, Japan, 2K.K. AB SCIEX, Tokyo, Japan

Keywords:
Micro-flow-UHPLC, LC/MS/MS, High-throughput, Needle-spray-voltage
Novel aspects:
High-throughput and high-sensitive LC/MS/MS analysis by using micro flow UHPLC
Abstract:
The measurement by LC/MS/MS is used at various fields, e.g., development of drugs, examination of food safety and so on. The
sensitivity of the MS has been improving according to demand of the user year by year. However, further high sensitive device isrequired, and besides, high-throughput method is regarded as important. Recently high flow rate HPLC, UHPLC, is used very widelyfor LC/MS/MS analysis. But there is a demerit from viewpoint of disposal and environment load because of using a lot of organic
solvent as mobile phase.
On the other hand, Nano LC is used in high-sensitive detection of small amount of biological substances. But operation of Nano
LC is complicated and it is difficult to obtain stable results. So, it is not spread widely in the field using traditional HPLC methods.
In this report, we present the examples of high-throughput and more-ecological analysis by using“Micro flow UHPLC“ specialized
in the flow rate between Nano LC and traditional HPLC.
As Micro flow UHPLC, Eksigent ekspertTM microLC 200 was used. This system can analyze by flow rate range 5 to 50ul/min or 20
to 200ul/min respectively by changing the flow modules. Microfluidic Pump Control（MFC）technology in this system allows for
rapid gradients to be run with excellent accuracy and reproducibility.
All experiments were run on an AB SCIEX QTRAP（R）5500 LC/MS/MS system. To reduce band broadening at low flow rate, Hybrid
electrode which has small diameter more than normal electrode was set to AB SCIEX QTRAP（R）5500 LC/MS/MS system.
We also report the examination of the suitable voltage applied to the spray needle of ESI for various flow rate.

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Others

PTh-192

13:30 － 14:40

Screening analysis for drugs of abuse by LC-MS/MS enables fast polarity
switching MRM triggered product ion scanning on the fly

Toshikazu Minohata1, Ichiro Hirano1, Junko Iida1, Keiko Kudo2, Noriaki Ikeda2, Kei Zaitsu3,
Noriaki Shima3, Munehiro Katagi3, Hitoshi Tsuchihashi4, Koichi Suzuki4

Shimadzu Co., Kyoto, Japan, 2Kyushu University, Fukuoka, Japan, 3Osaka Prefectural Police, Osaka, Japan, 4Osaka
Medical Collage, Takatsuki, Japan

1

Keywords:
LC-MS/MS, forensic, drug screening
Novel aspects:
A new approach of drugs of abuse screening using simultaneous analysis method by LC-MS/MS
Abstract:
Introduction. In recent years, the needs of forensic, toxicological and clinical analysis are becoming increasingly challenging as aconsequence of a growing trend in the use of illicit drugs and the non-medical use of prescription drugs. Screening applications
require rapid and unambiguous results that can be achieved using a generic analysis method designed for a large number oftarget compounds. To meet this need a universal high speed MRM triggered product ion scanning method with fast polarity
switching was applied to simultaneously screen, quantitate and confirm（by reference to a MS/MS data base）the majority ofdrugs of abuse available in Japan. Methods. Samples were measured using a Nexera UHPLC system and LCMS-8030 triple
quadrupole mass spectrometer（Shimadzu Corporation, Japan）with MRM triggered automatic MS/MS data acquisition. Samples
were separated using a standard clinical protocol on a Shim-pack FC-ODS column（150 x 2 mm, 3 um）. A flow rate of 0.3 mL/min
was used together with a gradient elution［A； 10mM ammonium formate in water, and B； methanol］. Results. A high speed LC/
MS/MS data acquisition system was applied to drug screening in forensic, toxicological and clinical analysis. To achieve a highly
specific and sensitive detection in screening and quantitation a MRM triggered product ion scanning method using a polarity
switching speed of 15msec and a scan speed of 15,000u/sec was applied to 111 components including illicit drugs, psychotropics,
hypnotics, pesticides and other substances. As the MRM acquisition time was very fast, this enabled product ion spectra to begenerated in both positive and negative ionization mode which could be matched against a user library of compounds as an
automated aid to screening and compound identification. Conclusions. By bringing together optimized detection Synchronized
Survey Scan™ parameters（product ion spectral data acquisition parameters based on the MRM intensity as threshold）and library
matching into a single integrated method package helps to generate high quality data without the need for a complex start-up.

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Others

PTh-193

11:10 － 12:20

HPLC method scouting system using ultra high performance liquid
chromatography coupled to single quadrupole mass spectrometer

Yusuke Inohana, Taku Tsukamoto, Hidetoshi Terada, Kiyomi Arakawa, Ichiro Hirano
Shimadzu Co., Kyoto, Japan

Keywords:
Method scouting, UHPLC, sensitivity（LC/MS）, chromatographic separation
Novel aspects:
Within a day, high sensitive LCMS methods were developed for geno-toxic impurities, several amines and acidic compounds.
Abstract:
Introduction
Traditionally, the process of LC/MS amenable separation method development has been time consuming as well as labor
intensive task. UHPLC based method scouting system will dramatically shorten total run time comparing to the conventional
system and enables to output multiple results based on the various combination of different mobile phase and multiple columns
allow you to make the comprehensive investigation of analytical conditions. And also, it is well known the sensitivity of mass
spectrometric detection will be varied with the type of mobile phase. In this study, the UHPLC based method scouting system
coupled to single quadrupole mass spectrometer was applied to develop the LC/MS amenable method for the simultaneous
analysis of geno-toxic impurities, several amines and acidic compounds.
Methods
UHPLC based method scouting system（Nexera Method Scouting System, Shimadzu Corporation, Japan）is configured by Nexera
UHPLC modules. For the detection, single quadrupole mass spectrometer（LCMS-2020, Shimadzu Corporation, Japan）was used.
The system can be operated at maximum pressure 100MPa and enables to automatically select up to 96 unique combinations of
8 different mobile phases as well as 6 different columns. To control the system, a dedicated control software was newly
developed. This software（Method Scouting Solution, Shimadzu Corporation, Japan）allows you to visually configure the different
type of columns and mobile phases. Thanks to the seamless integration with workstation software for LC/MS（LabSolutions,
Shimadzu Corporation, Japan）, batch queue can be created automatically.
Preliminary Data
Some geno-toxic compounds such as benzene sulfonic acid ethyl, toluene sulfonic acid butyl and toluene sulfonic acid ethyl aswell as amines and acidic compounds were selected as experimental samples. Those geno-toxic compounds have low polarity
and are supposed to be retained by ODS column. Therefore, Shim-Pack XR-ODSII was selected. Aqueous phase must be volatilefor LCMS, 4 kinds of aqueous phase were selected as following： 1 ）water, 2 ）0.1 % formic acid / water, 3 ）0.1 % formic acid +5 mmol/L ammonium formate water and 4 ） 5 mmol/L ammonium formate - water. For organic phase, acetonitrile, methanol andacetonitrile / methanol（50 / 50）were selected. Totally, 12 conditions were investigated by using Nexera Method Scouting System.
In case of geno-toxic compounds, the combination of 5 mmol/L ammonium formate / water and methanol was selected.
Furthermore, the gradient program was optimized using above mobile phase condition and analytical method for geno-toxic
compounds were successfully developed. In case of amines, due to the high polarity, those could not be retained amply by ODS
column. For amines, PFP column was selected. As the sensitivity is the primary concern for LC/MS method development, thecombination of 0.1 % formic acid and methanol was selected even though there was other HPLC condition has shown better
chromatographic separation. Finally, the analytical method of amines was established productively after the investigation ofmultiple gradient programs. In case of acidic compounds, those needed acidic condition for better chromatographic separation.

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Others

PTh-194

13:30 － 14:40

Detection of Emerging Legal Highs and Common Drugs of Abuse in Drug
Seizures by Fast LC-MS/MS Method

Kheng Aik Lim1, Yuan Yuan Cheryl Yeo1, Peiting Zeng2, Lijie Yu1, Mei Ching Ong1,
Tiong Whei Angeline Yap1
1

Health Sciences Authority, Singapore, 2Shimadzu (Asia Pacific) Pte Ltd, Singapore

Keywords:
LC-MS/MS, Synthetic Cannabinoids, Synthetic Cathinones, Neutral Loss, Precursor Ion
Novel aspects:
This study is to establish the identification of legal highs via rapid LC-MS/MS scan method and their analogues via neutral loss andprecursor ions scan modes.
Abstract:
One of the biggest challenges faced by forensic drug laboratories nowadays is the advent of legal highs - drugs designed tocircumvent drug laws. They are usually analogues of existing controlled substances, with some structural modifications created tofall outside of legislation and to avoid identification using traditional testing methods. The analysis of legal highs can be very
problematic due to the lack of drug reference standards for these new substances.
In this paper, we expand upon an existing Liquid Chromatography with Tandem Mass Spectrometry（LC-MS/MS）assay forcontrolled drugs of abuse to include about 20 new synthetic cannabinoids and cathinones that have been encountered by ourlaboratory or recently reported in literature. The updated assay was developed with a reduced analysis time to offer fast andselective detection of these legal highs along with current controlled drugs of interest. Drug standards of these synthetic
cannabinoids and cathinones were analysed and the optimized Multiple Reaction Monitoring（MRM）for each compound wasdetermined.
Based on the MRM data obtained as well as a study of the drugs’ fragmentation patterns, we have developed screening methods
for the two most common groups of legal highs： synthetic cannabinoids and cathinones. As each of these groups share a similar
scaffold, the methods utilize precursor ion and neutral loss scans that allow screening for novel analogues even without theavailability of drug standards. This technique is extremely useful for detecting the presence of any new legal high with no known
MRM transitions or reference MS spectrum that would otherwise have been overlooked.

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Others

PTh-195

11:10 － 12:20

Untargeted detection of novel abused substances using high-resolution
time-of-flight mass spectrometry with comprehensive collision-induced
dissociation

Michiko Kanai1, Kevin Siek2, Joe Binkley2, Jeffrey S Patrick2, David Alonso2
1

LECO Japan Corporation, Tokyo, Japan, 2LECO Corporation, MI, USA

Keywords:
High-resolution TOFMS, Comprehensive In-source CID, Forensic analysis
Novel aspects:
Coupling comprehensive In-source CID with high-resolution TOFMS enables advanced signal processing algorithms and datainterrogation tools to rapidly detect and confirm novel abused substances.
Abstract:
To accurately map the rapidly evolving landscape of chemical substance abuse requires analytical technologies that can detecttargeted and untargeted analytes with equal facility. High-resolution time-of-flight mass spectrometry can offer the forensic
scientist such capabilities, but in practice, extracting signals of untargeted drugs from volumes of high-resolution spectrometricdata proves challenging. Coupling comprehensive collision-induced dissociation（CID）with high-resolution time-of-flight mass
spectrometry enables advanced signal processing algorithms and data interrogation tools to rapidly detect and confirm novel
abused substances present in urine or seized materials.
The mass spectrometric technology and the data processing strategies discussed in this presentation were used to identify adesigner psychostimulant and its metabolites present in human urine, and to identify an unknown drug and an unknown
metabolite present in racehorse urine. High-resolution time-of-flight technology aided the identification of structurally diagnostic
fragment ions for the synthetic cannabimimetic JWH-018. Extending high-resolution time-of-flight mass spectrometry withcomprehensive CID to detection of designer steroids is also discussed briefly.

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Others

PTh-196

13:30 － 14:40

Identification of triazolam, etizolam and their metabolites byliquidchromatography tandem mass spectrometry

Mayumi Matsui1, Toshikazu Minohata1, Noriko Shoji2, Naohiro Kuriyama2, Chie Yokoyama2,
Keiko Matsumoto1, Jun Watanabe1, Junko Iida1
1

Shimadzu Corporation, Kyoto, Japan, 2YMC Co., LTD., Komatsu, JAPAN

Keywords:
LC-MS/MS, forensic, etizolam, triazolam, metabolites
Novel aspects:
Simultaneous analysis of Triazolam, Etizolam and their metabolites usinghigh resolution hybrid silica-based UHPLC column coupledwith LC-MS/MS
Abstract:
Benzodiazepines are among the most frequently prescribed drugs due to their sedative, hypnotic, anxiolytic, muscle relaxant andantiepileptic properties. Because of the high consumption of benzodiazepines worldwide, this class of drugs and their metabolites
are frequently present in both clinical and forensic cases. For these reasons, the analysis ofbenzodiazepines and their metabolites in biological fluids is of great interest to clinicians and forensic toxicologists. It’s been hard
to identify Etizolam, Triazolam, and their metabolites（alpha-Hydroxyetizolam, 8 -Ethylhydroxyetizolam, alpha-Hydroxytriazolam
and 4 -Hydroxytriazolam）mixture because of a similar chemical structure, molecular weight and mass spectra of these drugs.
Here, we report a high resolution separating method for simultaneous determination of 2 benzodiazepine drugs and their
metabolites developed using LC-MS/MS.
As samples, three samples were prepared；（A）mixture sample of standards of alpha-Hydroxytriazolam, 4 -Hydroxytriazolam,
triazolam and etizolam,（B）metabolized sample of triazolam and etizolam and（C）metabolized sample of blank. Then samples
were loaded to LC-MS/MS system with a Nexera UHPLC system and LCMS-8030 triple quadrupole mass spectrometer（Shimadzu
Corporation, Japan）. Separation occurred on YMC-Triart C18 column, 1.9um, 12nm（150 mm x 2 mm）at column temperature 40 C.
Samples were eluted at flow rate 300uL/min with a binary gradient system； the mobile phase consisted of（A）10mM formic acidand（B）mixture of 10mM formic acid / acetonitrile（ 1 / 1 ）. LC-MS/MS with electrospray ionization（ESI）source was operated in
multiple-reaction-monitoring（MRM）mode and MRM triggered automatic MS/MS data acquisition.
The analysis about drugs of abuse in clinical and forensic laboratories requires a highly specific detection for the simultaneous
determination of several drugs. The method for forensic toxicology that we developed contains not only MRM transition
parameters for quantitatibe analysis but analytical conditions and compounds information for MRM triggered automatic MS/MS
parameters（product ion spectral data acquisition parameters based on the
MRM intensity as threshold）optimized for screening analysis. For typical hypnotics, we developed simultaneous analysis methods
for 35 benzodiazepines and corresponding metabolites including etizolam, triazolam, and their known metabolites. In thisexperiment, 3 samples were prepared as mentioned above. Sample A was standard, sample B and C were metabolized samples
using in human liver S9. First of all, sample A was acquired with the method of 6 MRM transition, which was etizolam, triazolam,and their known metabolites.
As a result, an excellent separation was obtained, and all 4 compounds were detected. Sample C（blank sample）was acquired
and it resulted that no peak was found. Then, analysis of sample B found new three peaks detected, so 7 peaks were detected
and all peaks, of which some had same MRM transition, were excellently separated. Two of the new three peaks were easily
confirmed 8 -Ethylhydroxyetizolam and alpha-Hydroxyetizolam because they were known metabolites. However, last one wasdifficult to be confirmed. Next, sample C was re-acquired with MRM triggered automatic MS/MS method and then product ion
scan spectra of all peaks were acquired. These spectra were searched by typical hypnotics MS/MS library, so 6 peaks was able
to assign with high hit score to corresponded compounds. It is expected that this method is applicable to the determination ofdrugs in forensic biological materials.

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Others

PTh-197

11:10 － 12:20

Determination of Prohibited Components in Hair Dyes by Solid Phase
Extraction Coupled with Liquid Chromatography-Tandem Mass
Spectrometry

Chung-Yu Chen, Tsai-Jung Lin, Maw-Rong Lee
National Chung Hsing University, Taichung, Taiwan

Keywords:
Hair dye, solid phase extraction, liquid chromatography, tandem mass spectrometry, prohibited component
Novel aspects:
SPE-LC-MS/MS for determination of prohibited components in hair dye.
Abstract:
In this study, solid phase extraction coupled with liquid chromatography-tandem mass spectrometry（SPE-LC-MS/MS）wasdeveloped for determination of prohibited components, including 2,4-diaminodiphenylamine, 2,4-diaminoanisole, HC Yellow No.12,
2,3-naphthalenediol and 1 -methoxy-2,5-diaminobenzene, in hair dye. The optimum extraction conditions were using SPEC MP 1 asextracting cartridge and 1 mL methanol containing 0.1 % potassium hydroxide as eluting solvent. Under optimal conditions, thelinearity of proposed method ranged from 50 to 5000 ng/mL for all analytes expect 2, 3 -naphthalenediol（100-2000 ng/mL）. The
limit of detection and limit of quantitation were 6.7-16.8 ng/mL and 22.3-56.1 ng/mL, repsectively. The intra-day and inter-day
precisions were 0.6-7.1 % and 9.0-17.3 % , resprctively. The feasibility of applying the proposed method to analyze the five illegal
components in commercial hair dye was also examined. The results showed the method proposed is useful for determination ofprohibited components in hair dye.

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Others

PTh-198

13:30 － 14:40

Doubly-Etched Microfabricated Gas Chromatography Columns for High
Resolution Mass Spectrometry

Sanggoo Kim, Sung Min Lim, Sejong Yong
Korea Basic Science Institute

Keywords:
Microfabrication, Chromatography chip, High Resolution MS
Novel aspects:
Novel structure of microfabricated columns for GC/MS and HR/MS
Abstract:
Gas chromatography columns in borosilicate have been microfabricated for high resolution mass spectrometry. The
microfabricated columns were made using a wet chemical etching process that created a 100-mm wide 50-mm deep semi-circularcross section channel. The photoresist SU- 8 （both patterning and etching mask）was applied on a 500-mm thick 4 -inch wafer
which was subsequently immersed in a HF etching solution for 3 ~ 5 minutes. Using an aligner/exposure, the process was
repeated on a single wafer. The three-dimensional etched profile of the microfabricated columns illustrated that they werecomparable to fused-silica capillary columns. The inlets of the microfabricated columns were made using dry film patterning andsandblasting（0.5 MPa）. Anodic bonding was applied as bonding of the borosilicate wafer to a silicon wafer after the photoresists
were selectively removed. At 500 °C for 2 hr and an applied
voltage of 900V, it offered high bond strength and effective annealing. The microfabricated columns were connected with atransfer line of the mass spectrometer. The detailed mass spectrometry using the microfabricated columns and high resolution
GC/MS will be presented.

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Others

PTh-199

11:10 － 12:20

Coupling of Capillary Electrophoresis with Solvent Assisted Inlet
Ionization-Mass Spectrometry Using a Poly(dimethylsiloxane)-based
Sheath Liquid Interface

Che-Wei Wang, Guor-Rong Her

National Taiwan University, Taipei, Taiwan

Keywords:
capillary electrophoresis, mass spectrometry, poly（dimethylsiloxane）, solvent assisted inlet ionization, sheath liquid interface
Novel aspects:
This is the first attempt to couple CE with solvent assisted inlet ionization MS.
Abstract:
Solvent assisted inlet ionization（SAII）, developed recently by Trimpin et al., produces mass spectra similar to electrosprayionization（ESI）. The analyte/solvent solution could be introduced into the inlet tube due to a pressure drop from atmosphericpressure to vacuum and ionized without the use of voltage or nebulizing gas. This characteristic was utilized to simplify the
implement of CE/MS. In this work, a Poly（dimethylsiloxane）（PDMS）-based CE/MS interface using SAII as the ionization method
was developed. This interface consists of a CE separation capillary, a sheath liquid delivery capillary, a PDMS substrate, and asprayer. Because the CE eluents could be drawn directly into the inlet tube and ionized without the ESI process, both the sprayer
coated with conductive materials and the MS inlet were grounded. Sheath liquid was used to reach the flow rate at which a stable
spray was achieved. At a flow rate of 0.5- 2 μL/min, the sensitivity of CE/SAII-MS was comparable to that of low-sheath-flow CE/
ESI-MS interface. Because of the use of no fragile nano-ESI tip, this interface provides the advantage of robustness and easy-to-use,
and thus facilitates the operation of CE/MS analysis.

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Others

PTh-200

13:30 － 14:40

The Development of a Sheathless CE/ESI-MS interface Based on Thin
Conducting Liquid Film

Ju-Li Huang, Ren-Yu Hsu, Guor-Rong Her
National Taiwan University, Taipei, Taiwan

Keywords:
Capillary electrophoresis - mass spectrometry, sheathless interface, conducting liquid film.
Novel aspects:
A simple and robust two capillary sheathless CE/MS interface was constructed.
Abstract:
A simple two column sheathless CE/MS interface was constructed using polydimethylsiloxane to fabricate a microdevice allowing
facile column alignment and electrical connection. A liquid reservoir and a hole with a depth of &#65374； 1 mm were fabricatedon the microdevice at the edge of the conducting reservoir. The ESI sprayer was inserted through the reservoir to the hole soallowing it to be aligned with the separation column. The reservoir was filled with a conductive liquid prior to inserting the ESI
sprayer. Electrical connection was established through the thin liquid film formed in the space between the ESI sprayer and liquid
reservoir. The interface design presented was both easy to fabricate andoperate and demonstrated good performance. The deadvolume did not significantly affect operation as indicated by a demonstrated preservation of separation integrity. The repeatability
and reproducibility observed using this interface ranged from 3.3 % to 8.0 % and 5.2 % to 9.6 %, respectively.

Poster Session
Thursday, 20th September

Event Hall

Core Time : 11:10 - 12:20 (Odd number), 13:30 - 14:40 (Even number)

Others

PTh-201

11:10 － 12:20

Proteome analysis with molecular simulation can reveal the TTR
amyloidogenesis

Kenji Miura1, Naoya Hatano1, Mika Ohta1, Hirotaka Sato2, Aki Sugano1, Eiichi Maeda1, Yoshiyuki Sakaki3,
Ken-ichi Yamamura4, Yutaka Takaoka1
1
Kobe University Graduate School of Medicine, Kobe, Japan, 2Iwate Medical University School of Dentistry, Iwate,
Japan, 3Toyohashi University of Technology, Aichi, Japan, 4Kumamoto University, Kumamoto, Japan

Keywords:
transthyretin, hemopexin, amyloidogenesis, MD simulation
Novel aspects:
Proteome analysis with molecular simulation can reveal the TTR amyloidogenesis
Abstract:
7.2-hMet30 mice is a disease model of familial amyloidotic polyneuropathy（FAP）, that consists of human amyloidogenic mutant
transthyretin（TTR V30M）［ 1 ］. We previously found that 60% of this model showed amyloid depositions. In addition, nonfibrillar
TTR depositions were found in 2 of 12 Amyloid-negative 7.2hMet30 mice. These results suggest that the TTR amyloidogenesis iscaused not only by mutant TTR but also additional factor（s）such as etiologic molecule（s）.
To find the differences between amyloid-positive and negative in 7.2-hMet30 models, we analyzed the sera of these mice models
by proteome analysis. We firstly analyzed by SDS-PAGE. Secondly, in-gel digestion with trypsin was performed and the resulting
protein digests were analyzed by MALDI-TOF mass spectrometry. Among differentially displayed protein bands, we foundhemopexin and transferrin which were showed only in amyloid-positive mice, but not in the amyloid-negative mice. We also
confirmed these molecules by Western blotting. These two proteins were not detected in the nonfibrillar-TTR deposition byimmunohistochemical staining. These results suggest that the two molecules may accelerate amyloidogenesis of TTR V30M.
We then simulated the docking of native TTR or TTR V30M to hemopexin or transferrin and analyzed the binding affinity of thetotal four pairs. Finally, molecular dynamics（MD）simulations of the TTR-transferrin or TTR-hemopexin complex were performed
to elucidate the involvement of hemopexin or transferrin in amyloidogenesis. The docking simulation showed that binding affinity
of TTR V30M and each of the two proteins - hemopexin and transferrin - were both higher than that of native TTR to each protein.
In addition, MD simulation analysis showed that hemopexin and transferrin may facilitate the degradation of beta-strand in TTRhemopexin and TTR-transferrin complex. This result suggests that these molecules accelerate fibrillogenesis of TTR because
preceding studies revealed that TTR amyloid is triggered by beta-strand degradation.
In conclusion, our results suggest that hemopexin and/or transferrin associate with TTR amyloidogenesis in vivo.

［ 1 ］Takaoka, Y., Ohta, M., Miyakawa, K., Nakamura, O., Suzuki, M., Takahashi, K., Yamamura, K., and Sakaki, Y., Cysteine 10 is a
Key Residue in Amyloidogenesis of Human Transthyretin Val30Met, The American Journal of Pathology, 164（ 1 ）： 337345, 2004.

Plenary Lecture
Friday, 21st September

Main Hall

08:00 － 08:45

Plenary Lecture 5: Richard M Caprioli
Chair:

Dominic M Desiderio（University of Tennessee Health Science Center, USA）

PL5-0800
08:00 － 08:45

Molecular Imaging of Tissues by Mass Spectrometry: Looking Beyond theMicroscope

Richard M Caprioli

Vanderbilt University, Nashville, TN, USA

Keywords:
Imaging MS
Novel aspects:
Technological advances both in sample preparation and instrumental performance to achieve images at high spatial resolution（ 1 -10 microns）and at high speeds.
Abstract:
Imaging MALDI MS produces molecular maps of peptides, proteins, lipids and metabolites present in intact tissue sections. It
employs desorption of molecules by direct laser irradiation to map the location of specific molecules from fresh frozen andformalin fixed tissue sections without the need of target specific reagents such as antibodies. Molecular images of this nature areproduced in specific m/z（mass-to-charge）values, or ranges of values, typically covering the MW range 200-100,000. We have also
developed a similar approach for the analysis of targeted areas of tissues by integrating mass spectrometry and microscopy,
termed histology-directed molecular analysis, whereby only selected areas of cells in the tissue are ablated and analyzed.
We have employed Imaging MS in studies of a variety of biologically and medically relevant research projects, such as
developmental studies of embryo implantation is mouse and studies of disease including kidney disease, cancers, andneurodegenerative disease. Molecular signatures have been identified that are differentially expressed in diseased tissuecompared to normal tissue and also in differentiating different stages of disease. These signatures typically consist of 10-20 or moredifferent proteins； each identified using classical proteomics methods. In addition, Imaging MS has been applied to drug targetingand metabolic studies both in specific organs and also in intact whole animal sections following drug administration.
This presentation will focus on recent technological advances both in sample preparation and instrumental performance toachieve images at high spatial resolution（ 1 -10 microns）and at high speeds so that a typical sample tissue（e.g., a whole mousebrain section）can be imaged in less than 10 min. Some selected examples will include studies of diabetic nephropathy, tumor
bearing tissues and normal developmental processes. Other aspects of the technology, such as 3 -D imaging, will also be
considered. Finally, new biocomputational approaches will be discussed that deals with the high data dimensionality of Imaging
MS and our implementation of ’image fusion’ in terms of predictive integration of MS images with microscopy and other image
modalities.

Oral Session
Friday, 21st September

Main Hall

09:00 － 11:00

Session 41: Chemistries of Trapped Ions and their Applications to Bio- logical Mass
Spectrometry
Chair:

Gavin E Reid（Michigan State University, USA）

S41-0900
09:00 － 09:40[Keynote Lecture] Bioconjugation in the Gas Phase: New Chemistry for
Tandem Mass Spectrometry

Scott A McLuckey
Purdue University

Keywords:
Tandem MS, ion/ion reactions, bioconjugation
Novel aspects:
Novel specific gas phase chemistries for selective modification of primary amines, quanidinium groups, and carboxylate groups.
Abstract:
The advent of ionization methods that form multiply charged ions from biomolecules has allowed for the exploration of ion/ion
reactions as a new class of chemical reactions in analytical mass spectrometry. Attractive features common to all ion/ion reactions
are that they are highly exothermic for virtually any anion/cation combination and that the overall reaction kinetics are
independent of the chemical identities of the reactants. The ion/ion reactions of multiply charged peptide, protein, andoligonucleotide ions involving proton transfer, electron transfer, or metal ion transfer have been extensively studied. Recent results
have shown that much more complex types of reaction that involve bond formation and bond cleavage can also take place during
the course of an ion/ion reaction. These results demonstrate that selective chemistries can be effected between stages of mass
spectrometry. Some of these reactions are those normally associated with bio-conjugation chemistries in solution.
The ability to conduct bioconjugation chemistries in the mass spectrometer enable new approaches to derivatize analyte species
of interest for structural characterization. This presentation will summarize what is currently known about the underlying factorsthat govern the outcomes of ion/ion reactions that involve selective chemistries. Several examples will be given to demonstrate
the utility of gas-phase derivatization for primary and tertiary structure determination of peptide and protein ions.
All of the data underlying the findings related in this talk have been collected in either three-dimensional quadrupole ion traps or
linear ion traps operated in the presence of a background gas at 1 -10 mtorr.
This presentation will summarize current understanding of the dynamics of gas-phase bio-ion/ion reactions with emphasis on thefactors that determine the major observed pathways. Phenomena common to all such reactions will be described first followedby the implications for particular reaction types. Of particular emphasis in this presentation will be electron transfer, single protontransfer, multiple proton transfer is a single ion/ion encounter, metal transfer, and attachment. Essentially all of these outcomescan be dominant for a given analyte, depending upon the form of the analyte ion and the identity of the reagent. This presentation
will draw on data, both old and new, that illustrate the underlying factors that make reagents ions either particularly well-suited or
poorly suited for a particular analyte ion transformation of interest.

Oral Session
Friday, 21st September

Main Hall

09:00 － 11:00

Session 41: Chemistries of Trapped Ions and their Applications to Bio- logical Mass
Spectrometry
Chair:

Gavin E Reid（Michigan State University, USA）

S41-0940
09:40 － 10:00

Trap that Fat: Structure elucidation of ionized lipids by selective ionmolecule reactions and radical directed dissociation

Huong T Pham1, Alan T Maccarone1, Tony Ly2, Adam J Trevitt1, Larry J Campbell3, Todd W Mitchell1,
Stephen J Blanksby1
1

University of Wollongong, Wollongong, Australia, 2University of Dundee, UK, 3AB Sciex, Concord, Canada

Keywords:
Ion activation, Ozone induced dissociation, Radical directed dissociation, Lipidomics, Ion-trap mass spectrometry
Novel aspects:
Ozone induced dissociation and radical directed dissociation in ion trap mass spectrometers provide novel and complementary
capabilities for lipid structure elucidation that are compatible with high throughput shotgun lipidomics
Abstract:
Molecular structure elucidation in the fast emerging field of lipidomics presents some unique challenges to the traditional ion
activation methods employed in contemporary mass spectrometers. Notably, isomeric lipids that occur frequently in nature exhibit
quite distinct biochemical and biophysical behaviour but can be difficult to differentiate by mass spectrometry alone. For example,
the conjugated linoleic acid, CLA（10E,12Z）has been associated with atherosclerosis while its isomer CLA（ 9 Z,11E）- differing
only in the positions of carbon-carbon double bonds - was shown to inhibit the disease. Conventional strategies for modern
lipidomics rely on electrospray ionisation（ESI）to form even-electron ions with subsequent low energy collision induced
dissociation（CID）for structural characterisation. Under such conditions however, product ions arising from dissociation of carboncarbon bonds within the hydrophobic chains of lipids are typically absent or of low abundance. As a result, ESI-CID spectra
obtained from isomeric lipids are often indistinguishable particularly where the molecular structures differ in motifs such as： thepositions and stereochemistry of carbon-carbon double bonds； the positions and size of carbocyclic structures； or positions ofchain branching. In contrast simple lipids, such as fatty acid methyl esters, have been analysed by electron ionisation（EI）for
decades and the resulting radical cations generated in this process lead to extensive fragmentation of the hydrocarbon chain.
Unfortunately the high energies imparted by EI also lead to extensive rearrangements with a consequent loss of structural
specificity and here too differentiation of isomers（of the types described above）can be ambiguous.
In this presentation we will outline two alternative activation methods that rely on the selective reaction of unsaturated lipids withozone（ozone induced dissociation or OzID）and the generation and dissociation of radical ions generated by photolysis（radical
directed dissociation or RDD）. Recent studies of the gas phase reactions of mass-selected even-electron ions with ozone on atandem linear ion-trap mass spectrometer（QTRAP2000, AB Sciex, Concord, Canada）reveal that lipids with conjugated double
bonds have a significant enhancement in reaction rate（up to 120 times）relative to non-conjugated isomers. Furthermore,ozonolysis of conjugated double bonds gives rise to unique radical dissociation pathways that are diagnostic of the presence andlocation of this bonding motif. The speed and selectivity of this ion-molecule chemistry is such that the triple quadrupole geometryof the QTRAP can be exploited to undertake sensitive neutral loss surveys of complex lipid extracts to search for specific isomersof conjugated linoleic acids that are commonly present at low abundance with respect to non-conjugated variants. In parallel, we
have applied RDD to the study of lipids for the first time. In this approach, bifunctional molecules that contain a photo-caged
radical initiator and a lipid-adducting group, such as 4 -iodoaniline and 4 -iodobenzoic acid, are used to form non-covalent
complexes（i.e., adduct ions）with a lipid during electrospray ionization. Laser irradiation（266 nm）of the mass selected complexes
within a single-stage linear ion-trap mass spectrometer（LTQ, Thermo Fisher, San Jose, CA）cleaves the carbon-iodine bond toliberate a highly reactive phenyl radical. Subsequent activation of the nascent radical ions results in RDD with significant
fragmentation along the hydrocarbon chain. This approach provides diagnostic product ions that are associated with the doublebond position and the positions of chain-branching in phospholipids, sphingomyelins and triacylglycerides and has thus been used
to differentiate isomeric lipids differing only in such motifs.

Oral Session
Friday, 21st September

Main Hall

09:00 － 11:00

Session 41: Chemistries of Trapped Ions and their Applications to Bio- logical Mass
Spectrometry
Chair:

Gavin E Reid（Michigan State University, USA）

S41-1000
10:00 － 10:20

Harvesting High-Level Structural (Stereo- and Anomric-) Information from
Oligosaccharides with Single Sugar Resolution - A Tandem Mass
Spectrometric Approach

Chiharu Konda1, Frank A Londry2, Brad Bendiak3, Yu Xia1

1
Purdue University, West Lafayette, IN, USA, 2AB Sciex, Concord, Ontario, Canada, 3University of Colorado Denver,
Aurora, CO , USA

Keywords:
tandem mass spectrometry, oligosaccharide
Novel aspects:
Determining the stereochemistry and anomeric configuration of each sugar units within an oligosaccharide via multiple stage
beam-type CID on a triple-quadrupole/liner ion trap instrument.
Abstract:
Carbohydrates are ubiquitous in cells. In addition to providing energy and being structural components in cell walls, they play
important roles in cell-cell interactions, cell signaling, self and non-self recognition events. Understanding these roles requires that
their structures be determined with confidence. Due to the structure complexity of carbohydrates, four levels of structure
information need to be determined, including： 1 ）the stereochemistry of each sugar（subunit identity）, 2 ）anomericconfigurations, 3 ）linkage positions between connecting sugars, and 4 ）any modifications on substituents. Tandem mass
spectrometry（MS n ）has been widely applied in structural analysis of carbohydrates for determining the sequence, branching,linkage type, and positions of substituents. Recently, an MS3-CID method has been developed to pinpoint the stereochemistry andanomeric configuration of the non-reducing sugar of hexose-containing disaccharides having any of the 16 possible stereo-chemicalvariants. This approach was based on the distinct CID fragmentation patterns of m/z 221 product ions, having the structure of a
non-reducing sugar glycosidically linked to a glycolaldehyde（GA）, derived from CID of deprotonated disaccharides. In thispresentation, we further applied this methodology to small oligosaccharides by using MSn - CID（n= 4 and 5 ）to sequentiallybreak oligosaccharide anions down to dimmers and finally perform CID on m/z 221 diagnostic ions. By comparing the CID patternsof m/z 221 ions to that of the standards（monosaccharide-GA）, the identity and anomeric configuration of each sugar unit within
the oligosaccharide could be determined for the first time. A series of tri-, tetra-, and penta- saccahrides were used todemonstrate the capability of this method. All experiments were performed on a modified triple quadrupole linear ion trap mass
spectrometer（QTRAP 4000）. This instrument allowed high efficiency bi-directional ion transfer between quadrupole arrays, wheremultiple-stage（two or three）beam-type CID could be enabled to produce the ladder of fragment ions within a MS4 or MS 5
experiment.

Oral Session
Friday, 21st September

Main Hall

09:00 － 11:00

Session 41: Chemistries of Trapped Ions and their Applications to Bio- logical Mass
Spectrometry
Chair:

Gavin E Reid（Michigan State University, USA）

S41-1020
10:20 － 10:40

Measurement of the Peptide Ion Temperature by Using N-Acylated
Dipeptide Tags as Internal Standards

Seung Koo Shin, Jongcheol Seo, Min-Soo Suh, Hye-Joo Yoon
Pohang University of Schence and Technology, Pohang, Korea

Keywords:
ion temperature measurement, N-acylated dipeptide tag, internal standard, tandem mass spectrometry, peptide fragmentation
Novel aspects:
N-acylated dipeptide tags, isobaric tags originally developed for protein quantification, enable precise measurement of the ion
temperature in peptide fragmentation, regardless of the peptide sequence and the charge state.
Abstract:
Peptide fragmentations into the b- and y-type ions are useful for identification of proteins. The b ion that dissociates to the a-type
ion with loss of CO has the structure of N-protonated oxazolone. The kinetics of this unimolecular CO-loss process allows
measurement of the temperature of the oxazolone ion. Herein, we report a use of N-acylated dipeptide tags, isobaric tags
originally developed for protein quantification, as internal standards for measurement of the ion temperature in peptide
fragmentation. Amine-reactive dipeptide tags were attached to the N-termini of sample peptides and collision-induced dissociationof the tagged peptides yielded the b-type quantitation signal（bS）from the tag. The bS ion subsequently dissociated into the aS ion
upon CO-loss and the yield of aS ion was obtained from the mass spectra. As the length of alkyl side chain on the dipeptide tag
was extended from C1 to C8, the aS-ion yield gradually increased for the 4 -alkyl substituted oxazolone ion but decreased for the
2 -alkyl substituted one. To gain insights into the unimolecular dissociation kinetics, we obtained the potential energy surface fromab initio calculations. Resulting potential energy surfaces were used to calculate the microcanonical and canonical rate constants
as well as the aS-ion yield. Arrhenius plots of canonical rate constants provided the activation energy and pre-exponential factor
for the CO-loss process in the 500-900 K range. Comparison of the experimental aS-ion yield with the theoretical one led to precise
determination of the temperature of the bS ion. Thus, the bS-ion temperature of any peptide can be measured simply by combining
kinetic parameters and the experimental aS-ion yield, regardless of the peptide sequence and the charge state. Significantly, when
the bS-ion temperatures were nearly the same with only a few K difference, the y-type fragment patterns were almost identical toeach other. Our results demonstrate a novel use of N-acylated dipeptide tags as internal standards to measure the bS -ion
temperature that can be a good parameter to obtain reproducible peptide fragment spectra.

Oral Session
Friday, 21st September

Main Hall

09:00 － 11:00

Session 41: Chemistries of Trapped Ions and their Applications to Bio- logical Mass
Spectrometry
Chair:

Gavin E Reid（Michigan State University, USA）

S41-1040
10:40 － 11:00

Gas phase reactions of trapped bioorganic ions

Gianluca GIORGIUniversity of Siena

Keywords:
nuclephilic additions； ion trap； DFT calculations
Novel aspects:
Study of nuclephilic additions to fluorinated triazine and imidazole cations that show interesting aspects of their gas phasereactivity and adds to the very few examples reported in the literature
Abstract:
The study of properties and reactivity of organic and bioorganic ions in the gas phase is an interesting challenge in several fieldsof research, ranging from drug design to biotechnology and nanomaterials, to optoelectronics.
Many kinds of gas phase reactions can occur in ion traps. These can involve ion-molecule, ion-ion interactions, even- or oddelectron positive or negative species, and can occur through different mechanisms, such as nucleophilic or electrophilic additions,substitutions, eliminations, etc.
In our research activity, aimed at investigating gas phase ion chemistry and reactivity of bioorganic ions, we studied different kindsof ion-molecule reactions, mainly consisting in nucleophilic additions, produced by trapped ions. Those occurring in two series ofheterocyclic compounds are shown in this work.
Triazines represent a class of interesting heterocyclic compounds with applications in several fields, such as pharmaceuticalchemistry, agrochemistry, and biology. A new series of fluorinate triazines have been ionized by electron ionization or electrospraythus producing radical cations, protonated or deprotonated molecules. When submitted to low-energy collision-induced
dissociations, their protonated molecules show interesting elimination/hydration reactions consisting in consecutive losses of HF
followed by nucleophilic additions of water that produce puzzling ions in their MS n spectra. DFT calculations carried out on
different stable species and transition states, have allowed us to elucidate sites and mechanisms of these reactions and to
propose a reaction pathway.
Imidazoles are an important class of heterocycles that play several roles not only in biology, but in also emerging disciplines, suchas nanotechnology and molecular engineering, due to their capability to produce nanostructured materials.
A series of diphenylimidazole derivatives protonated by ESI, show addition of water and alcohols to their acylium cations thus
producing diol and carboxylic acid derivatives. The kinetics of these reactions has been studied by selecting the precursor ions,varying the activation time, and monitoring the abundance of product ions. Two different pathways yielding elimination of benzene
have been also found and studied by isotopic labeling.
Collision induced decompositions involving losses of radical species from even-electron cations and anions produced by thesetwo classes of compounds, as exceptions to the“even-electron rule“ will be also presented.
DFT theoretical calculations have been carried out on different neutral and charged species by using the B 3 LYP density functional
method with the triple-zeta basis set 6 -311+G（d,p）for evaluating their structure, stability and to propose reaction pathways.

Oral Session
Friday, 21st SeptemberRoom A

09:00 － 11:00

Session 42: New Developments in Instruments and Detectors
Chair:

Takaya Sato（JEOL, Japan）

S42-0900
09:00 － 09:40[Keynote Lecture] From supercomputer modeling to highest mass
resolution in FT ICR

Evgeny N Nikolaev1,2

The Institute for energy problems of chemical physics Russian Academy of Sciences, 2The Institute of biochemical
physics Russian Academy of Sciences

1

Keywords:
FT ICR,cell harmonization,superhigh resolution
Novel aspects:
New FT ICR detector cell disign. Superhigh mass resolution. High mass accuracy. Fine structure of peptide mass spectra. Isotopic
resolution for near 200 kDa proteins at 7 Tesla
Abstract:
Since its opening by Alan Marshall and Melvin Comisarow in 1974, FT ICR Mass Spectrometry was always attracting special
attention in mass spec community because of the extremely high mass resolution and high mass accuracy it demonstrated. The
early attempts to explain high mass resolution were limited by analyses of two factors： mathematical - the longer is time domain,
the narrower is the mass peak, and physical -collisions with residual gas molecules are reducing synchronous motion of ions andthus decreasing time domain signal longitude. The first attempts to analyze the nature of pick broadening in FT ICR spectra using
supercomputer, undertaken by Nikolaev’s group in 1995, have shown that there is mass resolution limitation caused by electricfield distribution in FT ICR measuring cell. Electric field deviation from ideal hyperbolic distribution causes losses of ion cyclotron
motion synchronization, transformation of ion clouds into comet like structures and signal degradation. This phenomenon depends
only on FT ICR cell design. The other phenomena - peak coalescence, which strongly influences ion cloud behavior（initiallyobserved experimentally）, has been reviled by supercomputer simulations based on Particle in Cell（PIC）algorithm first
introduced for FT ICR simulations by Dale Mitchell and further developed by Nikolaev’s group. PIC algorithm permitted to observe
the new phenomenon in ion cloud behavior - absence of comet formation at large number of ions in the cloud. We called thiseffect“condensation“. The critical number of ions for occurrence of condensation is independent of magnetic field strength.
Currently available software for simulation of ion cloud behavior in FT ICR cell during all stages of mass spectra measurement
procedure includes different methods of solving Laplace and Poisson equations such as capacity matrix method for ICR cell ofarbitrary geometry. Understanding of behavior of ion ensembles inside FT ICR cell gives rise to the new ideas of cell designs.
Among them is the idea of dynamic harmonization of the cell. The recently introduced novel FT-ICR cell based on a Penning ion
trap with specially shaped excitation and detection electrodes prevents distortion of ion cyclotron motion phases（normallycaused by non-ideal electric trapping fields）by averaging the trapping DC electric field during the ion motion in the ICR cell. Thiseffect results in mass resolving powers which exceed any values obtained up to now. The performance of the novel FT-ICR cellwith shaped electrodes has been characterized by isotopically resolved mass spectra of proteins close to 200kDa as well as finestructure of resolved isotopic peaks of peptides and small proteins. Accordingly, detection times of 5 minutes resulting in
resolving power close to 40,000,000 have been reached for reserpine at m/z 609 at a magnetic field of only 7 Tesla. Furthermore,fine structures of resolved 13Cn isotopic cluster groups could be measured for molecular masses up to 5.7 kDa（insulin）withresolving power of 4,000,000 at 7 Tesla. Based on resolved fine structure patterns atomic compositions can be directly determinedusing a new developed algorithm for fine structure processing. Extremely high mass accuracy below 100 ppb（RMS mass error）
could be achieved in complex mixtures. As example for high mass species, proteins and multimers of proteins could be measured
reaching masses up to 186 kDa（enolase tetramer）with isotopic resolution. For instance, at 7 Tesla resolving power of 800,000
was achieved for enolase dimer（96kDa）and 500,000 for molecular masses above 100 kDa. Experimental data indicate that there ispractically no limit for the resolving power of this ICR cell except by collisional damping in the ultrahigh vacuum chamber.
Acknowledgements：Ivan Boldin,Anton Grigor’ev,Oleg Harybin,Gleb Vladimirov,Igor Popov,and Bruker Daltinics-Roland Jertz,Jens
Fuchser,Claudia Kriete,Matthias Witt,Jochen Friedrich,Gokhan Baykut

Oral Session
Friday, 21st SeptemberRoom A

09:00 － 11:00

Session 42: New Developments in Instruments and Detectors
Chair:

Takaya Sato（JEOL, Japan）

S42-0940
09:40 － 10:00

A New magnetic TOF Detector with 1.5 ns Pulse Width and Very HighDynamic Range

Dick Stresau, Yair Benari, Kevin Hunter

ETP Electron Multipliers, Melrose Park (Sydney), Australia

Keywords:
TOF detector, magnetic detector, electron-multiplier
Novel aspects:
A new TOF detector delivers 5 volt linear pulses with 1.5 ns pulse width, and floatable to 15 kV.
Abstract:
During the past several years major advances in operational life and dynamic range have been achieved for very high performancemagnetic TOF detectors（<0.5 ns pulse width）, as reported by this group at previous conferences. The concepts employed toachieve these results have now been applied to detectors which are smaller and have been designed for use in instruments withless demanding timing requirements. The result is a detector with a 1.5 ns pulse width（for multiple ions）which exhibits
exceptional dynamic range and operational life.
Simulations indicate that the multiple ion pulse width of the new detector includes a ~350 ps contribution from jitter across its 17.5x 25mm aperture. When the aperture is masked to 12 x 25 mm, this jitter is reduced to ~150 ps. For this measurement jitter isdefined as the contribution to the detector’s pulse width resulting from variations in transit times from different portions of thedetector’s input aperture.
The new detector has been designed to have nearly the same form factor as a widely used discrete dynode TOF detector,
enabling a simple replacement which will result in a significant performance boost.
As well as a standard non-floating detector, a version is being developed with built-in electrical isolation that allows the detector
to be floated up to 15kV while still providing a ground-referenced output signal.
Pulse dynamic range is the common term used to describe a TOF detector’s linearity. This refers to the ratio of largest linear pulse
to smallest measurable pulse in a single shot spectrum. Because the level of the smallest measurable pulse is determined by thedetails of a specific TOF system, the most meaningful measurement for a detector will be the level of the maximum linear pulsedelivered by the detector（generally expressed as millivolts（mV）into a 50 ohm load）. Expressed in these terms, the newdetector’s linear pulse height limit exceeds 5 volts（5000 mV into 50 ohms）.
An orthogonal TOF system（with an electron impact ion source）and a 12 bit, 2 Gsample/second digitizer will be utilized to help
evaluate the new detector. The digitizer has ~1000： 1 useful dynamic range for a single shot spectrum. In reality a single ion
should be digitized with an average pulse height of ~ 10 LSB’s（least significant bits）to efficiently detect all incident ions.（Single
ion collection efficiency is critical in dynamic range/ linearity studies , as multiple ion events will always have near 100% efficiency,
so any single ion inefficiencies will inappropriately appear to be a non-linearity.）Therefore this system is capable of accurately
collecting single shot spectra with an ion abundance ratio（dynamic range）of ~100：1. In previous work we have made use of amuch smaller digitizing range and merely looked for distortions in abundance ratios（typically ~10： 1 ）at high input ion flux levels
to establish the detector’s dynamic range capability. The 12 bit digitizer enables collection of more realistic spectra and its 100： 1
dynamic range enables the use of carbon isotopes which provide a convenient series of markers for this study.
Work is progressing towards generating spectra demonstrating accurate abundance ratio measurements of 100,000+： 1
accumulated within practical averaging times（minutes）for multiple spectra. This data will be shown in the presentation alongwith diagrams detailing the detector’s design and operational characteristics.

Oral Session
Friday, 21st SeptemberRoom A

09:00 － 11:00

Session 42: New Developments in Instruments and Detectors
Chair:

Takaya Sato（JEOL, Japan）

S42-1000
10:00 － 10:20

Novel Compact Double Focusing Mass-Analyzer for Biochemical andElemental Analysis.

Viacheslav D Sachenko, L N Gall, R N Gall
Institute for analytical Instrumentation of RAS

Keywords:
double focusing mass-analyzer, mass resolution, line of focuses, spectrographic mode
Novel aspects:
a novel ion optical scheme of the double focusing mass-analyzer for biochemical and elemental analysis, and SIMS devices
Abstract:
Static mass-analyzers（MA）loose their positions during last 20 years, and are being replaced by dynamic MA： by quadrupole
mass filters for tasks requiring a low resolution, by TOF’s for a wide mass range, and by ion traps for high sensitivity. The main
reasons are traditional drawbacks of static MA： low scanning speed, limited mass range, large size and heavy weight. However,dynamic mass analyzers have brought their own drawbacks, and the main is the bad quality of mass-spectrometric line. For
example, a modern multipass TOF showing resolution higher than 100,000 at half-height will show less than 5000 at 5 %, and willloose addiyionally about 3 orders of magnitude at 0.5 %（!）.This makes practically impossible to register minor peaks locatednear the major ones, and necessary to use a sample pre-separation. However, a Nier-Jones static MA shows the half-heightresolution nearly the same magnitude as the best TOF, but has a very good line quality characterized by the isotopic sensitivity of
10- 6 - 10-7, the resolution remains high down to the lowest intensity levels. A lot of native mixtures can be analyzed for ecological,
elemental and biochemical purposes without chromatographic pre-separation as minor peaks are well defined near the major
ones.
The spectrometric mode, i.e. mass analysis in scanning mode, is available for any MA. On the contrary, the spectrographic mode
having the highest theoretical duty time can be realized only by a static MA when a mass-spectrum or its part is recorded
simultaneously by a position-sensitive detector or a number of collectors. Up to now, this mode was used only for isotopic andelemental analysis, and the MA of Mattauch-Herzog type were ideal for that as they do not require scanning. Unique features ofthese MA result in their long life（being proposed by authors in 1934, this solution is still in use!）. The most attractive feature of
their optics is the direct line of focuses with a double focusing for all ion masses. This feature allows to register simultaneously awide mass-spectrum of ions. However, the MA of Mattauch-Herzog type have no opportunity to filter the ion beam in energy. This
limits their resolution and makes these mass-analyzers ineffective with ESI and ICP ion sources, and for biochemical applications.
We present a novel scheme of static MA which combines the best features of both known double focusing ion-optical schemes,
and possesses additional advantages. The new optics has the direct line of focuses by double focusing for all masses, and has apossibility to filter the ion beam in energy, and to make fast electrical scanning of the ion beam without interfering with operationof ion source. On the contrary to the Mattauch-Herzog scheme having S-shaped geometry, the new optics is C-shaped, andelectrostatic lenses are added with the aim to compensate the energy dispersion coefficient, and to provide a device adjustment.
The new MA has been simulated and optimized within geometrical and chromatic 2 -nd order aberrations. It shows the theoreticalresolution magnitude about 3500 at 5 % of the peak height for the mass range 12-1800, energy spread of 40 eV, and maximal radius
200 mm. The permanent magnet weight does not exceed 45 kg, the MA dimensions are 600x500 mm. This allows to combine the
new MA with ESI or ICP ion source without a cooling cell and additional pumping stage, and to register the near-located minor
and major peaks.
The novel ion-optical scheme of the double focusing mass-analyzer suits ideally for SIMS devices too.
The authors dedicate this report to the 100-anniversary of R. Herzog and A.O. Nier, made an outstanding contribution to thetheory and practice of high precision mass-spectrometry.

Oral Session
Friday, 21st SeptemberRoom A

09:00 － 11:00

Session 42: New Developments in Instruments and Detectors
Chair:

Takaya Sato（JEOL, Japan）

S42-1020
10:20 － 10:40

Penning traps and ICR MS in physics research: From highly-charged atomic
ions to metal-clusters to short-lived atomic nuclei

Lutz Schweikhard

University of Greifswald, Greifswald, Germany

Keywords:
Penning trap, ICR, multi-reflection time-of flight MS, polyanionic metal clusters, short-lived nuclei
Novel aspects:
Several MS techniques recently introduced： implementation of MR-ToF mass separator to Penning/Paul-trap system（with new
focusing）, switching of trapping voltage for increased electron attachment, SIMCO excitation, ... and corresponding results
Abstract:
Penning-trap ion storage and ion-cyclotron-resonance mass spectrometry are applied in a wide range of investigations in physics
research. These methods are used for（a）the containment of ions,（b）the selection of particles with respect to their mass-over-charge ratio,（c）the analysis of reactions（CID, photodissociation, electron-impact ionization and electron attachment）as well as（d）the high-accuracy mass determination of exotic nuclear species.
The presentation will give recent examples of new techniques and applications. In particular, the following aspects will be
addressed based on developments at ClusterTrap［ 1 ］, ISOLTRAP［ 2 ］and SHIPTRAP［ 3 ］as well as the Berlin-EBIT［ 4 ］recently
re-installed at Greifswald：（a）Delicate particles such as atomic ions in very high charge states can only survive if they are in no contact with any othermaterials. They are produced in electron beam ion sources or traps（EBIS/EBIT）which use some of the properties of“standard“
ion-cyclotron-resonance devices.
（b, c）Penning traps are valuable tools in metal-cluster research. They allow a selection of the cluster sizes such that the cluster
properties can be determined for one size at a time. Multiple selection, reaction and analysis steps may be applied. Furthermore,
simultaneous storage of several species as different as very heavy cluster anions and electrons is possible and their reactions suchas the production of multiply negatively charged clusters can be followed as a function of time. Recently, electron attachment up
to a charge state of z = - 5 has been achieved［ 5 ］by changing the trapping potential during the storage period［ 6 ］.
（c, d）By measuring the cyclotron frequency of ions with even very short-lived atomic nuclei（i.e. in the sub-second range）thenuclear masses and thus their nuclear binding energies can be determined with very high accuracy. This includes nuclides thatshow exotic decay modes（proton decay or pairs of nuclides between which neutrino-less double-beta decays may occur）andisotopes of very heavy elements（such as nobelium［ 7 ］and lawrencium［ 8 ］, Z= 102 and 103, respectively）.
To address more and more exotic particles, ever new techniques are developed. For example, a multi-reflection ToF MS has
recently been added to the ISOLTRAP setup, complementing the already existing three-story combination of one linear Paul trap
and two Penning traps to a system of in total four ion traps, each with dedicated functions. In addition, new methods of ion
injection and ejection as well as time focusing have been developed with this MR-ToF MS［ 9 ］and for the Penning traps a new
excitation mode has been introduced（the simultaneous application of both dipolar and quadrupolar rf fields［10］）.
As much as the research areas vary as much do the techniques that are thought of to answer the corresponding challenges. As
outlined above the presentation will give specific examples of very recent developments and results.
［ 1 ］F. Martinez et al., Eur. Phys. J. D 63, 255（2011）, DOI： 10.1140/epjd/e2011-10528- 3［ 2 ］M. Mukherjee et al., Eur. Phys. J. A 35, 1 （2008）, DOI： 10.1140/epja/i2007-10528- 9［ 3 ］S. Rahaman et al., Int. J. Mass Spectrom. 251, 146（2006）, DOI： 10.1016/j.ijms.2006.01.049［ 4 ］C. Biedermann et al., Physica Scripta T73, 360（1997）, DOI： 10.1088/0031-8949/1997/T73/118［ 5 ］F. Martinez et al., submitted［ 6 ］F. Martinez et al., Int. J. Mass Spectrom. 313, 30（2012）, DOI： 10.1016/j.ijms.2011.12.009

［ 7 ］M. Block et al., Nature 463, 785（2010）, DOI： 10.1038/nature08774［ 8 ］in preparation［ 9 ］R.N. Wolf et al., Int. J. Mass Spectrom. 313, 8 （2012）, DOI： 10.1016/j.ijms.2011.12.006［10］M. Rosenbusch et al., submitted

Oral Session
Friday, 21st SeptemberRoom A

09:00 － 11:00

Session 42: New Developments in Instruments and Detectors
Chair:

Takaya Sato（JEOL, Japan）

S42-1040
10:40 － 11:00

A multi-reflection time of flight mass spectrograph for short-lived nuclei
and heavy moleculesPeter Schury1, Michiharu Wada2, Yuuta Ito1,2, Sarah Naimi2, Hermann Wollnik3, Tetsu Sonoda2,
Sousuke Nakamura1,2
1

Tsukuba University, Ibaraki, Japan, 2RIKEN, Wako, Japan, 3Geissen University, Geissen, Germany

Keywords:
Time of Flight Mass Spectrometry, Nuclear Masses, Trans-Uranium Nuclei, Biomolecules
Novel aspects:
A mass spectrograph capable of quickly achieving high-resolution with high-accuracy. It is efficient and able operate at in a highdynamic range of ion intensity.
Abstract:
We present the status of a new multi-reflection time of flight mass spectrograph（MRTOF-MS）. The spectrograph comprises apair of electrostatic ion mirrors connected by a field-free drift tube. Within the spectrograph ion bunches reflect between the
mirrors for an extended flight path of as much as 1 km or more. By properly tuning the electric potential within the mirrors, it ispossible to compensate for the initial energy spread of the ion pulse such that the time of flight becomes independent of theinitial energy of the ion. We have achieved mass resolving powers of more than 100,000.
Using techniques such as Penning trap mass spectrometry（PTMS）, it is possible to achieve mass resolving powers far exceedingthat which can be achieved by the MRTOF-MS. However, the MRTOF-MS overcomes certain drawbacks of such a technique. In
particular, it is especially well-suited for application to short-lived（t 1/2 < 100 ms）and heavy（A/q > 100）ion species. This isbecause the observation time required for a given resolving power scales with the square root of the mass, as opposed to scaling
linearly with the mass as in PTMS. As a result, while MRTOF-MS cannot compete with PTMS in maximum achievable resolving
power, it is capable of achieving respectable resolving powers（Rm> 100,000）much more quickly. For heavier species, it is a factorof ten or more faster.
Additionally, the measurements are not affected by large amounts of contaminants. On the contrary, the MRTOF-MS exhibits nosystematic shifts for even large amounts of simultaneous contaminants, it in fact benefits by using such contaminants as
simultaneous reference measurements. In this way, the device is a true spectrograph, capable of simultaneously analyzing many
species.
Fast mass measurements are particularly useful for short-lived radioactive nuclei such as are of interest e.g. for nucleosynthesis via
rapid neutron capture（r-process）in stellar supernovae. Such species tend to be both heavy（A>100）and short-lived（t1/2< 100ms）. In such cases nature imposes a maximum observation time by way of the short half-lives, creating an imperative for speedy
measurements such as is achievable with the MRTOF-MS. In such cases, the contaminant tolerance is also of great value, as timeneed not be spent on purification.
It is similarly well suited for measuring trans-uranium nuclei. Such nuclei are produced at incredibly low rates via heavy ion nuclear
fusion reactions. They are rather heavy（A > 250）and in many cases have half-lives of only a few milliseconds. These qualities are
all well aligned with the characteristics of the MRTOF-MS. A planned campaign to measure such exotic nuclei is briefly discussed.
Finally, since the achievable resolving power does not diminish for heavier species, the MRTOF-MS may also be of interest for
precise identification of very heavy（A/q > 1,000）molecules. The fast measurement cycle combined with the high resolving power
may make it ideal as a high-throughput spectrometer for biomolecules. Results demonstrating the high resolving power for suchvery heavy molecules will be presented.

Oral Session
Friday, 21st SeptemberRoom B-1

09:00 － 11:00

Session 43: Novel Proteomics Methodologies
Chair:

Yasushi Ishihama（Kyoto University, Japan）

S43-0900
09:00 － 09:40[Keynote Lecture] Shifting Proteomics from a Hypothesis Generating
Workflow to a Hypothesis Testing Workflow

Michael J MacCoss1, Jarrett Egertson1, Michael Bereman1, Richard Johnson1, Christine C Wu2,
Brendan MacLean1
1

University of Washington, 2University of Pittsburgh

Keywords:
proteomics, data independent acquisition, Skyline, software, quantitation
Novel aspects:
Development of data independent acquisition strategies for quantitative proteomics.
Abstract:
Proteomics technology has improved dramatically over the last decade. The technology developments have largely been directedaround instrument hardware, where instruments have been developed that scan faster, are more sensitive, and have greater massmeasurement accuracy. However, the basic workflow has remained largely unchanged -- mass spectrometers are directed toward
the acquisition of tandem mass spectra on the most abundant molecular species eluting from a chromatography column. Morerecently, efforts have been focused on the acquisition of mass spectrometry data on target peptides of interest. With
improvements in instrument hardware and instrument control software, the practical experimental difference between a targeted
and discovery proteomics is beginning to become blurred. These analyses are a significant change from the traditional proteomics
workflow and have required the development of novel computational strategies to analyze, visualize, and interpret these data. We
will present work illustrating our efforts in the development of targeted proteomics and provide a vision for challenges that stillneed to be overcome before these analyses become routine and replace more traditional discover proteomics methodology.

Oral Session
Friday, 21st SeptemberRoom B-1

09:00 － 11:00

Session 43: Novel Proteomics Methodologies
Chair:

Yasushi Ishihama（Kyoto University, Japan）

S43-0940
09:40 － 10:00

Profiling of Human Kinome Using In Vitro Kinase Assay in Combination
with Quantitative Phosphoproteomics

Naoyuki Sugiyama1, Masaki Gouda2, Haruna Imamura3, Masaru Tomita1, Yasushi Ishihama1,3

Institute for Advanced Biosciences, Keio University, Tsuruoka, Japan, 2Carna Biosciences, Inc., Kobe, Japan, 3Graduate
School of Pharmaceutical Sciences, Kyoto University, Kyoto, Japan

1

Keywords:
proteomics, signal transduction, protein kinase, phosphorylation, LC-MS
Novel aspects:
Novel kinome tree based on substrate specificities was generated and enabled deep analysis of phosphoproteome data.
Abstract:
Reversible protein phosphorylation regulated by protein kinases and phosphatases is a key event for signal transduction in
eukaryote cells. Recently, large-scale phosphoproteomics based on LC-MS and phosphopeptide-selective enrichment has becomea powerful tool to reveal the signaling mechanism. However, it still remains difficult to map all phosphoproteome data to known
signaling pathways due to the incomplete data for the kinase-substrate relationship. In this study, we profiled 385 recombinanthuman kinases, including 21 mutants and 10 lipid kinases, by using in vitro kinase assay combined with quantitative
phosphoproteomics approaches.
HeLa cell lysates were pretreated with alkaline phosphatase, and then reacted with or without individual recombinant protein
kinase. The reaction mixtures were subjected to reductive alkylation followed by in-solution digestion with trypsin. After stable
isotope dimethyl labeling using formaldehyde, phosphopeptides were enriched with hydroxy acid-modified metal oxide
chromatography（HAMMOC）using titania. The phosphopeptide fractions were analyzed with LC-MS/MS, and then thephophorylated sites by kinases and the phosphorylation motifs were determined using relative peak areas with respect to thoseof control sample as criteria.
As a result, 175,574 kinase-substrate relationships were obtained from the in vitro assay using HeLa cell lysate as the substratesource. Using the kinase-substrate combination data, we extracted a total of 1,525 phosphorylation motifs targeted by 303 kinases
and classified the kinases to generate a novel phylogenic kinome tree from the in vitro substrates specificities instead of the
traditional tree based on the sequence similarity between their kinase domains. Finally, two approaches based on in vitro
substrates and phosphorylation motifs were developed to predict kinases which are activated or inactivated by particular stimuli.
Using phosphoproteome data obtained from epidermal growth factor（EGF）-stimulated cancer cells, we successfully predictkinases belonging to EGF receptor pathways by both approaches. These novel kinome profiling using in vitro substrates,
phosphorylation motifs and phylogenic tree provides invaluable information to unveil the entire map of cellular signaling pathways.

Oral Session
Friday, 21st SeptemberRoom B-1

09:00 － 11:00

Session 43: Novel Proteomics Methodologies
Chair:

Yasushi Ishihama（Kyoto University, Japan）

S43-1000
10:00 － 10:20

In-depth analysis of human proteome by High Resolution Isoelectric
Focusing (HiRIEF)-LC-MS/MS

Kie Kasuga, Rui M Branca, Henrik J Johansson, Janne Lehtiö

Karolinska Institutet / Science for Life Laboratory, Stockholm, Sweden

Keywords:
HiRIEF, meter-long monolithic capillary column, quantitative proteomics, in-depth analysis
Novel aspects:
High-resolution isoelectric focusing（HiRIEF）approach successfully increases the coverage of proteome. This method provides
greater possibilities for in-depth analysis of complicated biological samples, such as clinical materials.
Abstract:
Mass spectrometry（MS）based proteomics can make a valuable contribution in clinical research by providing phenotypic
information. Since clinical materials are limited in amount and diverse, highly sensitive and high resolution analysis is appreciated.
To gain higher coverage of proteome, currently two distinct approaches are used； reduce of complexity of biological samples by
pre-fractionation or enrichment, or usage of high-resolution chromatography to separate complicated samples without prefractionation. In clinical proteomics research, it is vital to employ an approach that allows an adequate analysis of proteome using
small amount of materials. Such methods can be applied to small number of cells such as circulating tumors cells（CTC）in blood.
Proteome analysis of CTC is of interest in diagnosis and cancer progression. Since sample loss is considerable in small amount ofmaterials, high-resolution chromatography becomes the preferable to pre-fractionation.
Here, we will show results comparing two distinct approaches to analyze human cellular proteome： HiRIEF-LC-MS/MS and themeter-long monolithic capillary column.
［Method］
Quantitative proteomics by HiRIEF-LC-MS/MS1,2：
MCF- 7 cells（Human breast cancer cell lines）were stimulated with endogenous ligands or vehicle, then lysed by Urea-based
lysis buffer. Following tryptic digestion, peptide samples were labeled by iTRAQ, and dissolved in rehydration solution（ 8 M
Urea）. IPG strip（pI 3.7-4.9, 24 cm, linear gradient）was re-swelled with 8 M urea with 1 % IPG phamalyte（pH 2.5-5.0, GE Healthcare）overnight. Peptide mixture sample was focused and passively eluted into 72 fractions. Each fraction was analyzed on NanoLC-MS/MS（LTQ-Orbitrap Velos）with 15 cm long C18 Pico frit column（100 µm internal diameter, 5 µm bead size, Nikkyo TechnosCo., Tokyo, Japan）.
The aliquot of labeled samples were analyzed by Nano-LC-MS/MS equipped with 2 m-long monolithic capillary column
MonoCap® C18 High Resolution 2000（100 µm internal diameter, GL-Sciences, Japan）
All MS/MS data were searched by MASCOT with Percolator（FDR 1 %）against SWISSPROT human database. Quantitation of
iTRAQ reporter ions was conducted by Proteome Discoverer（version 1.3）. The relative abundances between the sample sets
were calculated by normalizing each peptide signal to the corresponding pooled internal standard.
During the session, we will also discuss about proteome analysis from small amount of cells（“Cent Cells“）by 2 m-longmonolithic capillary column .
References：
1 ）Pernemalm M. etal., Proteomics, 2009, 13： 3414-3424.
2 ）Sandberg A. et al., Mol. Cell Proteonics. 2012 Apr.12
3 ）Masuda T. et al., J Proteome Res. 2008, 7 ：731-740.
Acknowledgments：
KK is the recipient of VINNMER Marie Curie Chair co-founded by VINNOVA and PEOPLE Marie Curie Action.

Oral Session
Friday, 21st SeptemberRoom B-1

09:00 － 11:00

Session 43: Novel Proteomics Methodologies
Chair:

Yasushi Ishihama（Kyoto University, Japan）

S43-1020
10:20 － 10:40

Enrichment of plasma membrane proteins using cationic nanoparticle
pellicles

Waeowalee Choksawangkarn1, Sung-Kyoung Kim1, Joe Cannon1, Nathan Edwards2, Sang Bok Lee1,3,Catherine Fenselau1

Department of Chemistry and Biochemistry, University of Maryland, College Park, Maryland 20742, United States,
Department of Biochemistry and Molecular & Cellular Biology, Georgetown University Medical Center, Washington, D.
C. 20074, United States, 3Korea Advanced Institute of Science and Technology, Daejeon 305-701, Korea

1
2

Keywords:“plasma membrane proteins“,“pellicle enrichment“,“nanoparticles“,“spectral counting“
Novel aspects:
Various cationic nanoparticles with different density, size, shape, and surface modification have been evaluated for efficientenrichment of plasma membrane proteins using the pellicle technique.
Abstract:
Unbiased mass-spectrometry based proteomic profiling of plasma membrane（PM）proteins reveals biological processes involvedin communication and signaling between cells and/or environment. Understanding qualitative and quantitative changes in
expressed PM proteins at certain conditions, e.g., disease state, is a key for biomarker discovery and therapeutic development.
The identification of PM proteins is made difficult by their low abundance, high hydrophobicity, and fluid population. We copewith an underrepresentation in LC-MS/MS analysis by enriching PM proteins using cationic nanoparticle pellicles and optimizing
the proteomic workflow for analysis of highly hydrophobic proteins. The cationic nanoparticles increase the density of plasma
membrane sheets and thus enhance separation from other lysed cellular components by centrifugation. We have synthesized
silica, iron oxide, and gold nanoparticles and nanowires, followed by positive charge modification with Al 2 O 3 , aminopropyl
triethoxysilane, or aminopropyl thiol. Effects of shape, density, size, and surface modification of the nanoparticles on PM
enrichment efficiency have been investigated using RPMI 8226 multiple myeloma cells grown in suspension. We have confirmed theattachment of nanoparticles/nanowires to the cell surface by SEM and EDX analysis. Three workflows have been compared thatallow tryptic digestion of PM proteins solubilized in SDS prior to LC-MS/MS analysis of tryptic peptides and their parent proteins（J.
Proteome Res., 2012, DOI： 10.1021/pr300188b）. Using an optimized workflow, the enrichment efficiency of each type ofnanoparticle/nanowire has been compared, based on fractions of the proteins assigned to the plasmamembrane/transmembrane/cell surface categories in the UniProt KnowledgeBase. In addition, quantitative comparison of PM proteins andPM markers has been assessed based on spectral counting. Overall, we have identified 1,415 PM proteins（based on one or more
peptides）, which represents about 35% of the human plasma membrane proteome listed in UniProt.

Oral Session
Friday, 21st SeptemberRoom B-1

09:00 － 11:00

Session 43: Novel Proteomics Methodologies
Chair:

Yasushi Ishihama（Kyoto University, Japan）

S43-1040
10:40 － 11:00

Informatics-Assisted Label-free EDC-MRM Quantitation for Targeted
Signaling Pathway Analysis

Yi-Ting Wang2,3, Chia-Feng Tsai1,4, Pei-Yi Lin1, Hsin-Yi Wu1, Szu-Hua Pan6, Sung-Liang Yu5,
Pan-Chyr Yang3,7, Yu-Ju Chen1,2,4

1
Institute of Chemistry, Academia sinica, Taiwan, 2Chemical Biology and Molecular Biophysics Program, Taiwan
International Graduate Program, Institute of Chemistry, Academia Sinica, Taipei, Taiwan, 3Institute of Biochemical
Sciences, National Taiwan University, Taipei, Taiwan, 4Department of Chemistry, National Taiwan University, Taipei,
Taiwan, 5Department of Clinical Laboratory Sciences and Medical Biotechnology, College of Medicine, National Taiwan
University, Taipei, Taiwan, 6Institute of Biomedical Science, Academia Sincia, Taipei, Taiwan, 7College of Medicine,National Taiwan University, Taipei, Taiwan

Keywords:
Lung cancer, phosphoproteomics, MRM, label-free, pH/acid control IMAC
Novel aspects:
A rapid and sensitive label-free quantitation strategy for high throughout over-activated pathway discovery and verification
Abstract:
Despite the promise of quantitative phosphoproteomics, analytical challenges still remain for its utility in clinical application due
to its low throughput in discovery and verification stages. Due to the complexity of proteome and incompatible low acquisition
speed of MS, additional time-consuming fractionation step is a critical prerequisite to enable profiling protein at low abundantlevels. Furthermore, site specific verification for protein phosphorylation is most limiting bottleneck because of the lack ofavailability of good antibody required by conventional immunoassays.
Based on alternative long and short LC-MS/MS for peptide reference database and fast acquisition, respectively, here, we present
a high speed label-free quantitation strategy for personalized phosphoproteomic profiling and verification of targeted signaling
pathways in human tissues. The strategy utilizes long LC gradient to construct a reference database of protein identification, which
will serve as a peptide positioning system for cross-assignment and quantitation of data obtained from short LC gradient. On thepilot study of Raji B cell, nearly 100 % of the identified peptides in long（210-min）LC-MS/MS can be informatically matched to
peptides that were not selected for MS acquisition in short（40-min）LC-MS/MS based on the information of peptide sequence, m/z and aligned elution time. After elution time alignment and cross-assignment, a total of 5678 peptides were quantified in 40-min
LC-MS/MS runs（SD < 0.3）in biological replicate experiments（by Q-TOF MS）. High linearity on quantitation working curve wasachieved（r2=0.991）over 200-fold dynamic range using a standard phosphoprotein β-casein. At proteome scale, high quantitative
accuracy（log2 ratio= 0 ±0.27）and precision（CV=13.6%）were routinely obtained in 40-min LC-MS/MS. Compared to the precursor
ion-based quantitation strategy, the high speed EDC-MRM HR strategy increased 10-fold sensitivity of fragment ion detection anddemonstrated superior quantitation linearity, high quantitation accuracy（<20%）and precision（<10%）. On the pilot analysis of 12
pairs of cancerous and adjacent normal human lung cancer tissues, the high speed quantitation approach first identified theconstituently over-activated signaling pathways. Furthermore, the high speed EDC-MRM HR was applied to verify the targeted
singling pathway on Iressa resistant lung cancer cell. This strategy holds promise to translate the discovery of in vitro models toclinical specimens for analysis of targeted signaling pathway unique to a specific subtype of cancer and drug responsive targets.

Oral Session
Friday, 21st SeptemberRoom D

09:00 － 11:00

Session 44: Ambient Ionization
Chair:

Jentaie Shiea（National Sun Yat-Sen University, Taiwan）

S44-0900
09:00 － 09:40[Keynote Lecture] Ambient desorption/ionization mass spectrometry forminimal samples

Kenzo Hiraoka1, Mridul K Mandal1, Lee C Chen1, Hideki Fujii2, Kentaro Yoshimura2, Sen Takeda2
1

University of Yamanashi,Kofu,Japan, 2University of Yamanashi, Chuo, Japan

Keywords:
DBDI, Leidenfrost, PESI, sheath-flow PESI
Novel aspects:
New desorption/ionization methods have been developed for minimal gas, liquid, and solid samples.
Abstract:［Introduction］： Ambient mass spectrometry is growing very rapidly as reviewed by Fernández et al.（Anal. Chem. 2011, 83, 4508）.
In this paper, ambient desorption/ionization methods for minimal gas, liquid and solid samples developed in our laboratory will bepresented. The sample amounts should be as small as possible for valuable small samples and also for least contamination of theion source. For this purpose, we made efforts to develop high-sensitive ionization methods that need samples with minimal
amounts. Desorption methods for non-volatile samples will also be presented.
［Low-pressure helium dielectric barrier discharge ionization (He DBDI)］： This was designed for high-sensitive detection of vapor
samples. He DBDI detected 25 pg of desorbed HMTD（explosive）. LOD of H2O2 in air was 800 ppt. H2O2 leaking from aqueous
solution of H2O2（30%）contained in a plastic bottle with tightly sealed cap could be detected. This method, however, is difficult toapply to nonvolatile samples. In order to circumvent this problem, we developed desorption methods based on the Leidenfrost
phenomenon and flash heating by using a hot filament.
［Leidenfrost desorption/ionization］： A pencil size cartridge heater at temperatures in the range of 250-350 °C was used. 20 mL
liquid solution was dropped on the heater. The liquid floated on the heater for 10s seconds due to the Leidenfrost phenomenon.
At the moment of total evaporation of liquid droplet, non-volatile molecules being concentrated in the tiny droplets weredesorbed instantly induced by the last-stage explosive solvent evaporation. The vaporized samples were ionized by He DBD, dc
corona, or electrospray. The LODs of cocaine, codeine, morphine, and cyclosporin in methanol solutions were found to be ppb or
sub-ppb levels.
［Flash heating/desorption］： For the analysis of organic solids such as synthetic polymers, flash-heating desorption method wasdeveloped. A stainless steel（0.4 mm o.d.）filament at 100-800 °C moved up and down along the vertical axis. At the lowest
position, the filament slightly touched the sample surface（~10s mm in depth）. The pyrolyzed sample vapor was ionized by He
DBD. Due to the instant heating of subsurface, secondary thermal decomposition reactions should be suppressed. In fact, theprotonated monomer units of polylactic acids up to 10 mer were observed.
［Probe electrospray ionization (PESI)］： To mimic FD, PESI was developed. It used a metal needle with the tip diameter of <   1
mm. The needle was driven up and down by using a linear actuator. At the lowest position, the needle captured about ~ 1 pL
liquid samples. At the highest position, a high voltage was applied to generate electrospray. By PESI, sequential electrospray ofcomponents in the order of their surface activities was realized and thus suppression effect inevitable for capillary-based
electrospray was minimized. PESI was successfully applied to the cancer diagnosis.
［Sheath-flow PESI］： PESI is applicable only to wet samples. To apply PESI to dry samples, sheath-flow PESI was developed. By
using a coaxial glass or plastic capillary with a fine metal needle through it, the solvent liquid was supplied to the tip of the needle
that acted as a PESI probe. The liquid flowing out of a capillary extracted the components on the sample surfaces. By scanning a
US dollar bill, cocaine was detected near the edge of the bill.

Oral Session
Friday, 21st SeptemberRoom D

09:00 － 11:00

Session 44: Ambient Ionization
Chair:

Jentaie Shiea（National Sun Yat-Sen University, Taiwan）

S44-0940
09:40 － 10:00

Advances in Ambient Ionization and Imaging using DART and Microplasma
Ion Sources

Facundo M Fernandez1, Joel Keelor1, Rachel Bennett1, Prabha Dwivedi1, Joshua Symonds2,
Reuben Gann1, Thomas Orlando1,2Georgia Institute of Technology/School of Chemistry and Biochemistry, Atlanta, GA, USA, 2Georgia Institute ofTechnology, School of Physics

1

Keywords:
ambient ionization, direct analysis, DART, microplasmas, imaging
Novel aspects:
Novel applications and instrumentations for plasma and microplasma ion generation techniques are discussed
Abstract:
Ambient or“open air“ surface sampling techniques are a group of ion generation approaches that can be readily coupled tomass spectrometric detectors for MS analysis of target compounds. These technologies have been coupled to a variety of mass
spectrometers equipped with atmospheric pressure interfaces with only minor modifications, enabling unknown identification via
fragmentation pattern matching to databases, elemental formula determination via accurate mass measurements, multi-analyte
quantitation, spatially-resolved measurements, and selective ionization enhancement for target compounds of interest. Ambient
MS sampling/ionization techniques such as Direct Analysis in Real Time（DART）and Desorption Electrospray Ionization（DESI）
have grown in popularity because they enable the sampling of analyte under atmospheric pressure conditions from both liquidand solid phases remotely from the mass analyzer, have been used to investigate objects or surface features of a wide range ofshapes, sizes and textures, can perform qualitative or quantitative analysis with no or minimal sample preparation such as
dissolution, grinding, extraction or pre-concentration, and can conduct all these operations in real time with high sensitivity andminimal unwanted ion fragmentation.
Plasma-based ambient sampling techniques such as DART, FAPA, LTP, DBDI, DCBI and DAPCI involve the generation of a DC or RF
electrical discharge between a pair of electrodes in contact with a flowing support gas such as N2 or He, generating a constrained
flux of ions, radicals, excited state neutrals, and electrons which trigger the ionization process. Some or all of these plasma species
can be directed towards the surface being sampled, inducing desorption and ionization in a single step. Optional resistive heatingof the support gas, can further enhance desorption of neutrals which are then ionized by plasma species or species derived fromthose. Plasma-based ambient MS instrumentation tends to be fairly simple and rugged, and can be coupled to a variety of mass
spectrometers, including quadrupole ion traps, linear ion traps, and quadrupole time-of-flight, providing both MS/MS and/or
accurate mass capabilities. Plasma source mass spectra tend to be relatively simple, as most of the time the analytes are ionized
as one or two adduct types, simplifying peak assignment in the case of unknowns. Their applicability is generally limited tomolecules below 1 KDa.
DART uses a point-to-plane atmospheric pressure glow discharge to generate metastable species in a chamber that is physicallyseparated from the ionization region. The discharge support gas, containing metastables, is heated and directed through a gridelectrode that filters ions and electrons to mitigate ion-ion and ion-electron recombination of species generated within the DART
ionization source. DART can be used to sample gases, liquids and solids. In laboratory settings, gases are directly injected into theionization region following the grid electrode, whereas liquids are generally sampled by dipping a glass capillary in them andplacing it in the ionization region. Solids can be directly analyzed by holding them with tweezers and exposing them to the ionizing
gas, or in transmission mode geometry（useful for transmissive samples, such as meshes）. Powders can be mixed with metal
particles, adhered to a permanent magnet and exposed to the DART gas. Foam swabs, solid-phase extraction materials, andPDMS coated stir bars, can also be directly placed within the DART ionization region.
In this presentation we will discuss advances from our group in（a）using commercial DART sources to screen falsified artemisinincombination therapy drugs found in Africa,（b）development of an electrothermal vaporization interface for DART,（c）developing
an imaging laser-ablation/DART MS system for imaging 2 D-HPTLC plates, and（d）development of microplasma ion sources forambient ionization as los gas flow DART-alternatives.

Oral Session
Friday, 21st SeptemberRoom D

09:00 － 11:00

Session 44: Ambient Ionization
Chair:

Jentaie Shiea（National Sun Yat-Sen University, Taiwan）

S44-1000
10:00 － 10:20

Monitoring intra- and inter-day human breath metabolic signatures
supports the existence of individual phenotypes

Pablo M-L Sinues1, Lukas Meier1, Christian Berchtold1, Noriane Sievi2, Giovanni Camen2,
Malcolm Kohler2, Renato Zenobi1
1

ETH, Zurich, Switzerland, 2University Hospital Zurich, Zurich, Switzerland

Keywords:
Breath analysis；secondary electrospray ionization； metabolomics
Novel aspects:
Unraveling the existence of individual breath metabolic signatures.
Abstract:
Introduction
The study of changes in the human metabolome towards diseases is a promising area of metabolomic research［ 1 ］. However,
the reliability of such studies may be obscured by the day-to-day individual metabolic variability. Groundbreaking urine
characterization with NMR has suggested the existence of strong individual phenotypes［2, 3 ］which, moreover, are stable andstatistically“recognizable“ even after years［ 4 ］. These studies emphasize the importance of defining individual metabolite
phenotypes for future personalized medicine［ 3 ］. The analysis of breath is an attractive metabolomics technique because it iscompletely non-invasive. Moreover, if conducted on-line, it is rapid and circumvents artifacts due to sample storage/manipulation.
In addition, it covers different metabolites than NMR does. Therefore, it is an ideal approach to confirm the existence of suchindividual metabolic signatures.
Method4 subjects were asked to breathe at a flow rate of 3.8 L/min through a tube connected to the curtain gas line of a commercial
MS. The breath samples encountered an electrospray of water, whereby some compounds in breath were ionized and readily
detected. A total of 341 mass spectra collected during four days were analyzed statistically.
Preliminary data
We observe strong intra-day variations of metabolic signatures presumably due to individual circadian cycles. However, in fullagreement with prior NMR work［ 2 - 4 ］, we also observe a clear inter-individual separation due to the existence of a strong
individual“core“ signature.

［ 1 ］J.K. Nicholson, J.C. Lindon, Systems biology： Metabonomics, Nature, 455（2008）1054-1056.
［ 2 ］M. Assfalg, I. Bertini, D. Colangiuli, C. Luchinat, H. Schäfer, B. Schütz, M. Spraul, Evidence of different metabolic phenotypes inhumans, Proceedings of the National Academy of Sciences, 105（2008）1420-1424.
［ 3 ］S.S. Heinzmann, C.A. Merrifield, S. Rezzi, S. Kochhar, J.C. Lindon, E. Holmes, J.K. Nicholson, Stability and Robustness of Human
Metabolic Phenotypes in Response to Sequential Food Challenges, Journal of Proteome Research, 11（2011）643-655.
［ 4 ］P. Bernini, I. Bertini, C. Luchinat, S. Nepi, E. Saccenti, H. Schafer, B. Schutz, M. Spraul, L. Tenori, Individual Human Phenotypes
in Metabolic Space and Time, Journal of Proteome Research, 8 （2009）4264-4271.

Oral Session
Friday, 21st SeptemberRoom D

09:00 － 11:00

Session 44: Ambient Ionization
Chair:

Jentaie Shiea（National Sun Yat-Sen University, Taiwan）

S44-1020
10:20 － 10:40

Alternative Desorption/Ionization Processes of Low-Temperature Plasma
Probe Ambient Mass Spectrometry

Joshua S Wiley, Jobin Cyriac, Jacob T Shelley, R Graham Cooks
Purdue University, West Lafayette, IN, USA

Keywords:
LTP, Ionization Mechanisms, Ambient MS
Novel aspects:
This is the first evidence of non-thermal desorption with a plasma-based ambient ionization source.
Abstract:
Ambient ionization mass spectrometry has been an emerging field since its onset in 2004. Of the many different ambient ionization
techniques, which desorb and ionize samples from their native environment with little to no sample preparation, nearly half use an
atmospheric-pressure discharge for desorption and/or ionization. While little work has focused on elucidation of desorption
mechanisms for the various plasma-based ambient ionization sources, many studies have suggested that plasma-based sources
rely on thermal desorption as the primary mechanism with no apparent evidence for non-thermal-desorption mechanisms.
Furthermore, it is widely believed that thermal desorption is followed by commonly known atmospheric-pressure chemical
ionization（APCI）reactions, such as proton and charge transfer from capable reagent ions（formed from ambient air molecules inthe discharge）.
The present work demonstrates that non-thermal desorption processes take place with an ambient plasma ionization source inaddition to the widely accepted thermal desorption. In this study, a room-temperature plasma, the low-temperature plasma（LTP）
probe, was used in conjunction with a Thermo LTQ ion trap mass spectrometer to examine various non-volatile samples. Samples
ranged from thermally stable self-assembled monolayers（SAMs）to extremely low volatility organic salts to long-chain polymers.
One observed desorption pathway involved breaking of chemical bonds through a high energy transfer from reagent species. For
example, a heavily fluorinated decanethiol which forms a monolayer commonly called F-SAM, revealed numerous peaks
corresponding to breaking of carbon-carbon bonds in various positions as well as varying degrees of oxidation. The nature of thisinteraction of high-energy plasma species with surfaces will be further probed by examining the modification of surfaces after
exposure to the LTP. Upon LTP-MS analysis of organic salts, the intact molecular cation was almost never observed. Quaternaryamines typically revealed loss of the most favorable R-group with subsequent protonation. An additional category of compounds
included low-volatility alkyl halides（e.g. thyroxine）, which often revealed only the negatively charged halide in the mass
spectrum.
A potential explanation for the responsible desorption process includes electron transfer to the non-volatile samples analyzed,
which each have high electron affinities and undergo excessive fragmentation as shown in the mass spectra（common withelectron transfer dissociation）. While volatile perfluorinated compounds have been readily shown to ionize via electron transfer
under ambient conditions, a significant difference with the present research stems from electron transfer to a solid phase sample,
providing the energy necessary to break chemical bonds and release a fragment into the gas phase. Many reagent ions producedby LTP are capable of electron transfer to samples of higher electron affinity, including O2 - which is abundant in the LTP samplingregion. O2 - has been listed as the likely candidate in other atmospheric-pressure ETD processes and evidence for large amountsof oxidation to the fragments obtained in this study further substantiate this hypothesis. The role of O2 - as an ETD reagent will be
investigated by controlling the ambient environment and limiting the amount of oxygen that can reach the discharge/samplingregion. The effect of sample（or substrate）temperature on analyte-ion signal will also be determined to better understand therole of plasma-gas temperature in the desorption of these species.

Oral Session
Friday, 21st SeptemberRoom D

09:00 － 11:00

Session 44: Ambient Ionization
Chair:

Jentaie Shiea（National Sun Yat-Sen University, Taiwan）

S44-1040
10:40 － 11:00

Analysis of Polar Components in Heavy Oils by Gas Chromatography
Electrospray Ionization Mass Spectrometry (GC-ESI/MS) and Multivariate
Analysis

Chu-Nian Cheng1, Yi-Tzu Cho2, Hung Su1, Jentaie Shiea1

1National Sun Yat-Sen Univrsity, Kaohsiung, Taiwan, 2Yuh-Ing Junior College of Health Care & Management, Kaohsiung,TaiwanKeywords:
Gas chromatography； Electrospray ionization； Heavy oil
Novel aspects:
Gas chromatography electrospray ionization mass spectrometry（GC-ESI/MS）and multivariate analysis were used to analyze polar
components in heavy oils.
Abstract:
In the coupled atmospheric electrospray ionization source with gas chromatography system for mass spectrometry（GC-ESI/MS）,after injecting samples into GC injector, volatile compounds underwent GC separation and then individually flew out from theoutlet of GC column through carrier gas. These separated analytes mixed with the plume that was generated from an electrospray
emitter spraying acidic methanol solution continuously. In this process, ion-molecule reaction carried out that assisted polar
analytes to be ionized and formed protonated ions, which was later detected by quadrupole time-of-flight mass spectrometer.
In this study, GC-ESI/MS was demonstrated for the detection of polar components, following with using a multivariate analysis,
principle component analysis（PCA）, to differentiate the polar chemicals in heavy oils. The heavy oils used in this study were theengine oils collected from vehicles at different mileage. In the analysis of heavy oils using conventional GC/MS, it was found thatthe ions of hydrocarbon and other relative compounds suppressed the ions of polar component even after the pretreatment of
GC separation. In contrast, our developed GC-ESI/MS reduced interferences of hydrocarbon and other relative compounds due tothe feature of the ionization source ESI, which is distinct from electron impact that is commonly used in the conventional GC/MS.
For assessing the applications of GC-ESI/MS in analyzing heavy oils, we collected engine oil samples from vehicles at different
driving distances of 0, 100, 300, and 500 kilometers. In sample pretreatment, the samples were mixed and extracted with methanol.
The extractants were then diluted with methanol for subsequent GC-ESI/MS analysis, and a series of mass spectrometric results
were acquired. In the ion chromatographic diagrams and mass spectral profiles, because the ion signals of hydrocarbon
compounds were eliminated, the specific ion information of polar compounds in engine oil samples was obtained. The GC-ESI/MS
analyses on each engine oil sample were performed in triplicate, and the results were further processed using PCA. In thegrouping of samples, oils collected at 0, 100, 300, and 500 kilometers clearly formed different groups from each other, reflecting
their different degrees of polar component generations at varied driving distances.

Oral Session
Friday, 21st SeptemberRoom E

09:00 － 11:00

Session 45: Cell Biology / Cellular Pathways
Chair:

Renato Zenobi（ETH Zurich, Switzerland）

S45-0900
09:00 － 09:40[Keynote Lecture] Future of Cell Analyses and Mass Spectrometry

Tsutomu Masujima

Hiroshima University, Hiroshima, Japan/RIKEN QBiC, Suita, Japan

Keywords:
future analyses of cells, cell biology, mass spectroscopy
Novel aspects:
The future analyses of cells are foreseen by standing on recent development in mass spectroscopic analyses of cells in the world.
Abstract:
When you search the Web-of-Knowledge with the words“Cell“ and“Mass Spectrometry“, you can find more than 400,000
papers published since 1964 in which“Biochemistry Molecular Biology“ is 11,300papers,“Biochemical Research Methods“ 7,300
papers,“Chemistry Analytical“ 6,500 papers, and“Cell Biology“ is 2,400 papers. The number of papers for methodology oriented
and one aiming for biology may be in similar percentage. In any decades, methodological development and its application areusually coupled in idea. Total number of papers shows that biology or cell biology has been one of the big driving force fordevelopment of mass spectrometry in the past half century. Here, by reviewing the past researches in biology or cell biology usingmass spectrometer, I would like to foresee the future analyses of cells or cell molecular dynamics with my little knowledge.
I would like to foresee the future analyses from another view point, i.e. desires of cell biologist. I think they would like to perform：
1 ）Direct and exhaustive molecular analysis of cells with easy identification, quantitation and its localization.
2 ）Cross correlations of gene expressions, transcription, translation and small molecules, including molecular chain correlating or
depending reactions and its mapping.
3 ）Morphological evidence and direct relating of it to above mentioned aspects.
4 ）Molecular mechanism in macro, micro or even single molecular scale.
Against to these desires, mass spectrometry has been powerful but the power is decreasing in this order. In addition, I made self
question“Is it real way of strategy or desire for future cell biology?“ Because this kind way of thinking is all standing on
substance. Actually, the body, fluid（e.g. cytosol）, and partitions（e.g. Nucleus）etc. of cells are all constructed by molecular
substances and are designed very purposefully. However, I still think something is missing for understanding the“life“. It may be
one which is not deterministic or dynamic or unstable.
Mass spectrometry is very powerful for substance quantitation and identification, because the mass is a deterministic aspect of
substance. However, it may be possible to know the molecular interaction or molecular arrangement force through direct
ionization process of whole cell sample, for example. I think mass spectrometry has many potentials which are not considered to
be of use for future analyses of cells.
In this keynote, I would like to discuss these potential future analyses of cells by standing on recent development mass
spectroscopic analyses of cells in the world.

Oral Session
Friday, 21st SeptemberRoom E

09:00 － 11:00

Session 45: Cell Biology / Cellular Pathways
Chair:

Renato Zenobi（ETH Zurich, Switzerland）

S45-0940
09:40 － 10:00

Telling the Biological Story - Post Processing of -Omics Data

Michael Kohl1, Hagen Meckel1, Maike Ahrens1, Helmut E Meyer1, Martin Hofmann-Apitius2,
Martin Eisenacher1

Medizinisches Proteom-Center, Ruhr University, Bochum, Germany, 2Fraunhofer Institute for Algorithms and Scientific
Computing SCAI, Sankt Augustin, Germany
1

Keywords:
bioinformatics, data processing, integrating -OMICS techniques
Novel aspects:
The delineated workflow utilizes the result of the SCAIView text mining software and integrates different -OMICS techniques.
Abstract:
The development of new diagnostic markers and therapeutic targets that aim at both early diagnosis and novel treatment
opportunities, are very important tasks of current medical research.
Hence, the PROFILE consortium（www.profile-project.de）addresses these challenges with respect to the most important acuteand chronic liver diseases. Liver diseases have extraordinary socio-economic significance, because they are characterized by both
high mortality and morbidity. Additionally, due to the central role of the liver, regarding for example metabolism and detoxification,
liver dysfunction often has highly significant effects on the function of other organs（e.g. brain, kidneys or the immune system）.
The consortium uses a broad range of state of the art OMICS techniques including Genomics, Proteomics and Transcriptomics,
aiming at a more rapid and efficient development of both therapeutic and diagnostic approaches.
However, the huge amount of acquired multidisciplinary data needs to be integrated in a structured manner. Data processing
within PROFILE requires the application of adequate algorithms in order to compare and to relate the findings obtained from theparticipating working groups. Additionally, there is need to develop approaches to extract biologically relevant meaning startingfrom the pre-processed data.
Here, a software named PROFILE commander（ProfCom）is presented, which facilitates the data analysis of several different
OMICS experiments. The software is modularly organized in order to support easy integration of further analysis opportunities.
Currently, the ProfCom comprises five modules that can be used independently. However, when connecting in series, the usage ofthe modules represents a centralized work flow for the data obtained within the PROFILE consortium.
The first ProfCom module deals with evaluating the proteins found by mass spectrometry regarding the reliability of the underlying
set of identified peptides.
The second module facilitates conversion into the data formats used by several tools that are applied within the PROFILE
consortium. Furthermore, missing information is retrieved from internet resources and then added to the experimental data.
The next module involves some statistical analysis, i.e. the considered biomolecules（i.e. genes or proteins）are ranked in order todetect promising candidates for further experimental validation. Furthermore, multivariate methods are used to differentiate subpopulations within a given data set.
Scientific standards call for relating the own experimental results with the knowledge found in the literature. To this end, ProfComuses the output of a web based text mining tool（SCAIView）in order to reveal associations between the considered disease andthe relevant citations, in a structured way.
Finally, the last ProfCom module relates the findings of the studies, i.e. regulated proteins and genes, to knowledge given bypathway information stored in databases that are available online. The software produces a final output, which can be useddirectly as input for Cytoscape. Cytoscape is one of the most widely used network analysis and visualization software and can beutilized for the production of publication-ready visualizations and for further analysis（e.g. GO term enrichment analysis via the
BinGO plugin）.
The ProfCom is further linked to ProLiC, software developed by the Medizinisches Proteom-Center. ProLiC is a web based tool thatenables comparison of an arbitrary number of data sets and calculates the overlap as well as groups the members of the data
sets according to several identity criteria.
In summary, the delineated work flow covers a directed data processing, starting with the application of quality criteria to the preprocessed data, up to scientific presentation. Here, performance of the software is demonstrated in the context of hepatocellular
carcinoma, the most prevalent cancer of the liver.

Oral Session
Friday, 21st SeptemberRoom E

09:00 － 11:00

Session 45: Cell Biology / Cellular Pathways
Chair:

Renato Zenobi（ETH Zurich, Switzerland）

S45-1000
10:00 － 10:20

Realtime Molecular Analysis of Single Cell State by Fluorescence-assisted
Live Single-cell Mass Spectrometry

Naohiro Tsuyama1, Hajime Mizuno1, Takanori Harada1, Sachiko Date2, Tsutomu Masujima1,2

Hiroshima University Graduate School of Biomedical & Health Sciences, Hiroshima, Japan, 2Quantitative Biology Center(QBiC), RIKEN, Osaka, Japan

1

Keywords:
single cell, nanospray, direct ionization, fluorescent probe
Novel aspects:
Fluorescent probe assisted Live Single-cell MS can clarify the exact nature of cellular state of targeted cell such as redox ormolecular diffusion.
Abstract:
Improvement of mass spectrometry allows the volume of samples of biological analysis smaller. Widely used conventional
molecular analyses use large numbers of cells which allow detection of rare molecules even with low content, however, give
averaged result. As a consequence, phenomena with cell variation or asynchrony are hardly analyzed by these methods. To
resolve this issue, we developed Live Single-cell MS which enables targeted cell recovery with a nanospray tip and direct mass
spectrometric analysis to detect molecular content of the cell. In this paper we will show two different approaches using
fluorescent probes-combined Live Single-cell MS to show cell state related molecular change which cannot be achieved using
conventional large scale analyses.
Analyses of cell state related molecular composition
Ordinary microscopic observation only gives morphology or morphological change of target cells. To know cell conditionalvariation related molecular change such as cellular redox state, we used fluorescent probes. Human EB transformed B lymphocyte
apoptosis induced by betamethasone was monitored with dichlorofluorescein（DCF）to clarify cellular level of reactive oxygen
species. Cells of high or low oxidative stress were selectively recovered under a fluorescent microscope（OLYMPUS）and analyzed
by this method. Conventional LC-MS analysis was also performed to show the advantage of single cell analysis. Among decreased
peaks in treated cells, glutathione and its metabolites were gradually decreased after treatment： the intensities of these ionswere half of non-treated cells at 1 hour. Whentreated cells were observed under a phase contrast microscope, cell shape wasvarious； some cells died, and others showed round live shape. Then, cells stained with DCF with bright and dark fluorescent
strength were recovered separately with a nanospray tips and applied to SIM analysis in a LTQ-Orbitrap（Thermo Scientific）mass
spectrometer. Peaks monitored were m/z 308.1,［M+H］+ for reduced form of glutathione, and m/z 402.2,［M+H］+ for DCF as acontrol. Comparison of glutathione level between oxidative stress-high cells and -low cells showed that more than one fifth
decrease of glutathione level in stress-high cells was observed whereas the LC-MS data was half. These data indicated thatconventional LC-MS method gave an averaged result of heterogeneous cells, whereas our fluorescence assisted-single cell MS
gave sharp exact results.
Analysis of dymanic molecular diffusion
Live normal cells have pores called“gap junction“ which allow substances including membrane impermeable compounds to passthrough between two adjacent cells. Existence and activities of gap junction are usually monitored with fluorescent dyes, forexample, Lucifer Yellow CH（LY）loaded into cells. However, no method which can clarify non-fluorescent molecules to diffuse
through the junction is existed. We applied the Live Single-cell MS to detect non-fluorescent molecules diffusing through gap
junction in normal human fibroblasts in parallel fluorescent staining. Human fibroblasts were microinjected with LY andfluorescence transport through gap junction was monitored periodically. Non-fluorescent molecules, such as homovanillic acid
（HVA）and vanillylmandelic acid（VMA）, were co-loaded. A targeted cell was sucked with a Cellomix tip and analyzed similarly.
Peaks obtained with SIM mode were extracted and evaluated. LY fluorescence were detected initially in an injected cell andgradually diffused to the adjacent cells. Then, a cell adjacent to the dye-injected cell was analyzed. LY peak of m/z 221.5 detected
as［M- 2 H］2- were clearly observed. A cell without LY fluorescence separated from the dye loaded cell was sucked simultaneously,
and no LY peak were indicated. Molecules without fluorescence could be also monitored. HVA peak（m/z 181.0,［M-H］-）, VMA
peak（m/z 197.0,［M-H］-）were detected accompanied with LY peak. These results indicated that the Live Single-cell MS can beused to study gap junction mediated molecular diffusion using non-fluorescent molecules.

Oral Session
Friday, 21st SeptemberRoom E

09:00 － 11:00

Session 45: Cell Biology / Cellular Pathways
Chair:

Renato Zenobi（ETH Zurich, Switzerland）

S45-1020
10:20 － 10:40

Multi-omics Approach to study the effect of rapamycin treatment on ahuman cell line

Sudha Rajagopalan, Siji Joseph, Guha Nilanjan, Syed S Lateef, Yugandhar Reddy
Agilent Technologies India Pvt.Ltd

Keywords:
multi-omics, pathway, mass spectrometry, microarray
Novel aspects:
Integration of data from multi-omics study to understand pathways affected by a drug treatment
Abstract:
Rapamycin is an immunosuppressant drug that specifically inhibits mTOR activity and cellular hyperproliferation in many cells
types resulting in G 1 growth arrest. In this study we illustrate a multi-omics approach that uses genomics, transcriptomics,
proteomics, and metabolomics to understand the effects of rapamycin on a human embryonic kidney cell line（HEK293）. HEK293
cells were harvested at 16hrs post treatment of rapamycin or vehicle alone. RNA, miRNA, proteins and metabolites extracted infive replicates from million cells each were pooled and analyzed. The complete genomic status of the cell was interrogated using
CGH arrays. CGH analysis showed that HEK293 cells possess large amount of aberrations at the genome level. Eighteen miRNAs
were found to be affected by rapamycin treatment. The target entity list of these miRNAs showed good overlap with differentiallyexpressed gene list from the gene expression microarray. For the proteomics analysis, digested peptides from five replicate
extracts were pooled and analyzed in five replicates on a QTOF coupled to HPLC chip MS system. Statistically significant
differential features between the rapamycin treated and control samples were identified by targeted MS/MS analysis followed bydata base search. Metabolites were extracted using Methanol： water： chloroform mixture. Organic portion was analyzed on GCMS and aqueous portion was analyzed on LC-MS.
Pathway analysis of differentially expressed mRNA, miRNA, proteomics and metabolomics data indicates rapamycin affects several
biological processes including translation, protein synthesis, DNA repair, purine metabolism, and stress response. Many of these
individual biological responses have been corroborated by observations in other cell lines. This study demonstrates the utility ofapplying an integrative approach to investigate cellular responses to environmental change by co-analyzing data acquired fromcomplementary technologies.

Oral Session
Friday, 21st SeptemberRoom E

09:00 － 11:00

Session 45: Cell Biology / Cellular Pathways
Chair:

Renato Zenobi（ETH Zurich, Switzerland）

S45-1040
10:40 － 11:00

Screening and identification of sulfur mustard metabolites from in vitro
samples using different LC-MS experiments for new biomarker
development

Mia Halme1, Maija Pesonen2, Marja Hagström3, Markku Pasanen2, Paula Vanninen1

1
VERIFIN, Department of Chemistry, University of Helsinki, Finland, 2School of Pharmacy, Faculty of Health Sciences,
University of Eastern Finland, Finland, 3Center of Drug Research, Faculty of Pharmacy, University of Helsinki, Finland

Keywords:
Sulfur mustard, metabolism, biomarker, identification, LC-MS
Novel aspects:
This study represents method for studying sulfur mustard metabolism without animal experiments or samples from humancasualties. With these in vitro assays it is possible to trace specific metabolic reactions.
Abstract:
A biomarker is a distinctive biological or biologically derived indicator（e.g. a metabolite）of a process, event or condition. To
verify exposure and to link the exposure with the putative health effects of harmful agents, good validated biomarkers of exposure
are essential. For the validation of a biomarker, both in vitro and in vivo studies are needed. Validated analytical methods are alsoessential for the identification, characterization and quantification of a novel biomarkers.
Sulfur mustard（bis（ 2 -chloroethyl）sulfide, HD）is a potent chemical warfare agent and a Schedule 1 chemical on the Chemical
Weapons Convention（CWC）［ 1 ］. Metabolism of sulfur mustard is complex. It acts as a vesicant and biological alkylating agent
causing erythema, edema, blistering, chronic obstructive pulmonary diseases, bronchiectasis, pulmonary fibrosis, and lung andpleural cancer. The immediate tissues affected are the eyes, skin and respiratory tract.
Based on the earlier animal studies, acute urine metabolism of HD includes hydrolysis, oxidation, and conjugation with glutathione
followed by b-lyase catalyzed reactions［ 2 ］. These b-lyasemetabolites are considered as unequivocal biomarkers of exposure to
HD since they have been found both in animal and human samples. A quantitative and fast LC-MS/MS（SRM）method for thedetermination of two known b-lyase metabolites 1,1’-sulfonylbis-［ 2 -（methylsulfinyl）ethane］（SBMSE）and 1 -methylsulfinyl- 2 -［ 2 （methylthio）ethyl-sulfonyl］ethane（MSMTESE）from real human urine was validated during this project［ 3 ］.
Our objective is to study the metabolism of HD by exposing hepatic cell fractions（e.g. microsomes, cytosol or whole
homogenate）to develop a platform for finding new possible biomarkers and to confirm the b-lyase pathway in vitro. Incubations
were performed using cell fractions, 0.1 M potassium phosphate buffer containing 3.3 mM MgCl2（pH 7.4）, 5 mM glutathione and0.25 mM NADPH at 37°C. The reaction was started by addition of HD in dimethyl sulfoxide. The reactions were terminated after
specific incubation times by addition of cold acetonitrile, followed by vortex mixing. Precipitated proteins were removed bycentrifugation and the supernatants were analyzed by LC-MS.
As there are no reference chemicals for the new metabolites, the structural characterization has to be made using different MS
techniques. LC-MS analyses of in vitro samples were performed using three different MS detectors： ion trap, triple quadrupole
and time-off-flight（Q-TOF）. These provided whole range of different data acquisition methods e.g. full scan MS/MS, MS 3, SRM,
parent ion scan, neutral loss scan and exact mass measurement for screening and identification of HD metabolites.
It was observed that the metabolism of HD occurs partially differently in vitro than in human or animal studies. For example twodifferent glutathione conjugates were detected（both mono and bi-conjugates）. Results and conclusions will be presented.

References［ 1 ］http：//www.opcw.org/chemical-weapons-convention/［ 2 ］R.M. Black, K. Brewster, R.J. Clarke, J.L. Hambrook, J.M. Harrison and D.J. Howells, Xenobiotica22（1992）405-418.
［ 3 ］M. Halme, M. Karjalainen, H. Kiljunen, P. Vanninen, J. Chrom. B 879（2011）908-914.

Plenary Lecture
Friday, 21st September

Main Hall

11:45 － 12:30

Plenary Lecture 6: R Graham Cooks
Chair:

Yoshinao Wada（Osaka MCHRI, Japan）

PL6-1145
11:45 － 12:30

Through a Glass Darkly: Glimpses into the Future of Mass Spectrometry

R Graham Cooks

Purdue University, West Lafayette, IN/USA

Keywords:
Mass analysis； Ionization； Instrumentation； Ion reactions； Surfaces； Chirality； Miniaturization； Diagnostics
Novel aspects:
A review and preview of the scope of mass spectrometry
Abstract:
Mass spectrometry may be defined, broadly, as the science and technology of ions. As such, it is a subject of enormous scope.
The achievements of 20 th century mass spectrometry now have come into focus. In rapid succession these include themeasurement of mass/charge ratios of ions, the discovery of isotopes, measurements of mass defects, fundamental understandingof ionization and fragmentation mechanisms and the relationship to bond energies. All this was achieved in the first half of the
century. In the second half-century, organic mass spectrometry began with applications to petroleum distillates and moved quickly
to natural products, steroids, and small-molecule drugs. Gas chromatography /mass spectrometry was a key to early studies of
biological systems. A preoccupation with improving mass resolution and mass range became an established theme and different
types of mass analyzers were introduced for this purpose. This quest drove biological mass spectrometry and propelled thedevelopment of ionization methods that were increasingly successful in handling solid samples（desorption methods）andsolutions（spray methods）. Complex environmental and biological samples led to the coupling of liquid chromatography to mass
spectrometers and the development of the tandem mass spectrometry for complex mixture analysis. The highpoint of theseapplications in chemical analysis was the emergence - largely from within mass spectrometry - of the new subject of proteomics.
The explicit discovery of ion/molecule reactions at mid-century saw the emergence of a major subject which had affinities withsolution-phase reactivity. The study of solvation effects on thermochemistry and reaction kinetics was one important result.
Unimolecular reaction kinetics, energy partitioning during unimolecular dissociation, and the dynamics of ion/molecule and laterion/surface collision events opened new fundamental areas of research with wide relevance to physical chemistry.
The likely preoccupations and achievements of current and later 21st century mass spectrometry can only be glimpsed....“througha glass darkly“. Will the established role of mass spectrometry in chemical analysis be complemented by a significant role insynthesis whether by soft landing of ions onto surfaces or reactive scattering? MS already has a prominent role in surface
characterization but is surface tailoring by molecular modification a major future topic? Is there a role for mass-selected ion
beams in transfection and other medical processes? Certainly it is already clear that MS has a role in diagnostics. Direct
quantitative analysis of whole blood by the ambient ionization method of paper spray is a demonstrated capability for therapeutic
drugs at biologically relevant levels. Diagnosis using characteristic lipid profiles allows diseased tissue to be distinguished fromhealthy tissue. Will such experiments be used intrasurgically? The requirements include automated correlations of the MS datawith libraries and reduction in the size of the instrument, but the fact that the measurements can already be made on unmodified
tissue makes an affirmative answer likely. If one can make ions in air can one detect them in air, mass-analyze them in air? Can theworst performance parameter of the mass spectrometer the ionization efficiency be significantly improved? Can atomic probe
measurements be coupled with mass spectrometry to characterize biological ions? Alternatively, instead of examining ions after
landing on surfaces, can the physical and chemical properties of trapped ions be characterized? A beginning has been made using
fluorescence resonance energy transfer, electron diffraction and precise thermochemical measurements based on a desolvation
enthalpy scale. Surely MS could make a larger contribution to chiral analysis through ion/molecule reactions or the kinetic
method. Will array format mass spectrometers take their place beside single-channel instruments? Finally, perhaps mostimportantly, does the mass spectrometer have a role in the everyday life of everyday people as a personal and householdmeasurement device?

Workshop 1
Monday, 17th SeptemberRoom B-1

17:15 － 19:15

Mass Spectrometry of Polymers and Industrial Materials
Organizer: Hiroaki Sato（AIST, Japan）

Keywords:
Structural characterization, High-resolution TOF, Ion mobility, Polymer degradation
Scope of Session:
Mass spectrometry is commonly used as a technique for the characterization of polymeric industrial materials. This session will
discuss the recent progress in the structural characterization of polymers (especially for degraded materials) by advanced mass
spectrometry such as high-resolution techniques and ion separation techniques.
Abstract:
Polymeric materials have been requested to have versatile physical properties and special functions depending on their utilization.
Nowadays the chemical structures of functional polymeric materials become even more complex based on sophisticatedmolecular design. Just like new instrumentation of MS technology make a significant contribution to the development of biologicalsciences, the characterization of industrial materials should also be promoted by the MS technologies. In this workshop, we want
to discuss the recent progress in the structural characterization of polymers and industrial materials by advanced mass
spectrometry.
One of the important topics of polymer characterization by MS is the development of high resolution time-of-flight mass
spectrometry (HRTOFMS). A mass of different combinations of polymer constituents (comonomers, end-groups, branching pointetc.) increases the likelihood of isobaric interference. The peaks of different chemical compositions with the same nominal mass
overlap can be separated by high-resolution MS. In this workshop, the applications of matrix-assisted laser desorption/ionization(MALDI)-HRTOFMS and gas chromatography (GC)-HRTOFMS will be presented and discussed.
Industrial materials are usually mixtures of homologues or isomers, and off course the isomers having same mass cannot distinct
even by high resolution mass spectrometry. The application of ion mobility MS (IMS) may be powerful tool for the characterizationof such complex materials. Because the ion separation by IMS is based on the differences in three-dimensional conformation, IMS
provides another view to the structural characterization of complex industrial materials. In this workshop, the IMS topic with a fineanalysis of a mobiligram for petroleum products will be presented and the applicability of IMS to polymer characterization will be
discussed.

Workshop 2
Monday, 17th SeptemberRoom D

17:15 － 19:15

Hydrogen/Deuterium Exchange Mass Spectrometry
Organizers: Yoshitomo Hamuro（ExSAR, USA）
Rachel A. Garlish (UCB, UK)

Keywords:
Hydrogen/deuterium exchange, Higher order structure, Protein-ligand interaction, Protein-protein interaction, Epitope mapping,
ETD fragmentation, Automation, Software
Scope of Session:
Hydrogen/deuterium exchange mass spectrometry (HDX-MS) is an increasingly popular protein characterization method. Thissession covers both the application and new method development of HDX-MS. The application of HDX-MS includes higher order
structure analysis, protein-ligand interaction, and protein-protein interaction, particularly epitope mapping. Method development
includes opportunities for higher resolution analysis using ETD fragmentation, data generation automation, and data extracting
software.

Workshop 3
Tuesday, 18th SeptemberRoom B-1

17:15 － 19:15

Careers in Mass Spectrometry
Organizer: Anthony W.T. Bristow (AstraZeneca, UK)
Chair: John Langley (University of Southampton, UK)

Confirmed topics and presenters:
Academic Career – Rainer Cramer (University of Reading, UK)
Industrial Career – Caroline Pritchard (LGC, UK)
Government Career – Simon Hird (FERA, UK)
Instrument Manufacturer Career – Takaya Satoh (Jeol, Japan)
Preparing for interviews, career options for mass spectrometrists and the global job market in MS – Dave Jones (VRS, UK)
Scope of Session:
The session is designed to outline a variety of career paths that begin and stay within mass spectrometry and also expand beyond
a starting point in mass spectrometry. Several international speakers will briefly describe their career paths (for example:
academia, industry, instrument manufacturer). These short presentations will outline the choices made during career development,
any challenges encountered and the opportunities available in the different career pathways. In addition, there will be apresentation that describes the current job market for people with a background in mass spectrometry and more broadly inanalytical science. The workshop will be very interactive, and we actively encourage attendees to question the speakers about
their careers and the choices made.

Workshop 4
Tuesday, 18th SeptemberRoom D

17:15 － 19:15

Mass++ and MassBank: Tools for Data Processing and Database on PC
Organizers: Satoshi Tanaka (Shimadzu, Japan)
Takaaki Nishioka (NAIST, Japan)

Keywords:
Freeware, Proteomics, Metabolomics, Multi-vendor, Chemical identification
Scope of Session:
Mass++ (mass plus plus) is freeware for viewing and manipulating various types of mass spectrometric data. Mass++ 2.1.0, which
was released recently, is easy to use as search engines such as Mascot and MassBank. MassBank is a public repository forsharing mass spectra of small molecules among research community. A bottleneck in metabolomics is to submit a large amountof mass spectra from LC-MS as the queries to database searches manually. Mass++ and MassBank projects have collaboratively
developed a tool that relieves the users from the repeated manual works. Two projects will give tutorials of their tools.
Topics 1:“Mass++: A Visualization and Analysis Tool for Mass Spectrometry”
Satoshi Tanaka (Koichi Tanaka Laboratory of Advanced Science and Technology, Shimadzu Corporation, 1, NishinokyoKuwabaracho Nakagyo-ku, Kyoto 604-8511, Japan)
Abstract: Mass++ (mass plus plus) is freeware for viewing and manipulating various types of mass spectrometric data. Its primaryobjectives are: 1. To provide essential functionality for proteomics and metabolomics analysis. 2. To support a wide range ofvendors’ data file formats. 3. To be easily extendible using plug-in technology.
This year we have released a new version of Mass++ (Mass++ 2.1.0), which contains new functionality such as AXIMA filereading, Batch Processing, De novo sequencing, a new peak detection algorithm, statistical analysis, and overlapping trace
settings. The new version of Mass++ also makes it easy to identify substances with search engines such as Mascot and MassBank.
Previously, MassBank could be used directly from Mass++, but Mascot could not; to use Mascot search, users had to extract peakdata and search databases using other software, which was very time-consuming. In the latest version of Mass++, users can easily
perform Mascot search on raw data. Mass++ can be downloaded from http://www.first-ms3d.jp/english/achievement/software.
Topics 2:“Constructing MassBank Database on Laboratory PCs”
Takaaki Nishioka (Nara Institute of Science and Technology, Ikoma, Nara 630-0192, Japan)
Abstract: MassBank (http://www.massbank.jp) is a public repository for sharing mass spectra among research community. Data
shared on MassBank are not placed on the MassBank site, but distributed on the contributor’s data servers. Currently 24 researchgroups contribute from nine data servers. Contributors prepare their mass spectral data in“MassBank Record Format”, provide
PCs as data servers, and manage their data on their own data servers. Users are able to access to all the distributed MassBankdata from the access point,“www.massbank.jp”. Thus MassBank project has provided various user’s tools to search the data andto prepare and manage MassBank records on their data servers. Recently we transferred these technologies to“In-House
MassBank”on Windows PCs. Users are able to build their in-house MassBank databases by depositing their own mass spectral
data and the copied MassBank data from the MassBank download site. One of the applications of in-house database is to searchuser’s in-house database with a large set of query data that LC-MS output. Mass++ imports and submits the query data to thespectral search against the in-house database by using MassBank batch SOAP-API. This relieves users from tedious, repeatedmanual works for the identification of the small molecules detected by LC-MS.

Workshop 5
Wednesday, 19th SeptemberRoom B-1

17:15 － 19:15

Path to Next-generation IMS: New Concepts, Advanced Instrumentation, and Leveraging
the Ion-molecule Chemistry
Organizers: Toshiki Sugai (Toho Univ., Japan)
Alexandre A Shvartsburg (PNNL, USA)

Scope of Session:
Ion mobility separations coupled to mass spectrometry have become a mainstream analytical tool, and both conventional IMS
and FAIMS are available in commercial systems now applied in various areas. This workshop will look beyond establishedproducts and methods into qualitatively new approaches that would take IMS to the next level. Topics of presentations anddiscussion will include multi-stage platforms of much greater specificity and peak capacity, use of ion-molecule chemistry to
expand the separation space and modify resolution of specific targets, and various integrative measurements incorporating the
IMS dimension.
Abstract:
Over the last decade, IMS coupled to MS has grown into a mainstream analytical tool that now rapidly expands into diverse
applications in proteomics, metabolomics, characterization of natural products, and industrial and environmental monitoring. In
another major shift, differential or field asymmetric waveform IMS (FAIMS) that exploits the difference between ion mobilities athigh and low fields has appeared as a broadly useful approach that is competitive and often superior to conventional IMS basedon absolute mobilities at moderate fields. These trends have largely been due to introduction of conventional IMS/MS and FAIMS/
MS systems by Waters, Thermo, and AB Sciex. On that trajectory, most high-end commercial MS instruments would in a few years
include an (optional) IMS stage. This situation signifying successful integration of IMS into the repertoire of MS and thus analyticalscience, it is timely to think beyond the current paradigm to take IMS to the next level. Such emerging work will be the focus of
our workshop.
The workshop will be appropriately opened by David Clemmer (Indiana Univ.) who had launched the transformation of IMS/MS
from a niche physical chemistry technique of academic interest to a widely employed analytical method in 1990-s and forcefully
pushed the frontier of this field since. His groundbreaking development of multistage IMS platforms and cyclotron IMS has enabled
unprecedented resolving power that would be central to future IMS advances.
Since the invention in early 1990-s and until recently, conventional IMS (in both drift-tube IMS or DMA variants) employed static
electric fields only. An innovative direction is use of dynamic fields, notably in the traveling-wave IMS implemented in the Synapt
series. Here, Dr. Vidal de Miguel (SEADM) will present a novel IMS configuration employing orthogonal oscillatory and fixed fields.
The first prototype already provides reasonable resolution in a compact footprint.
Next, Toshiki Sugai (Toho Univ.) will talk on the development of an ion trap/IMS system that enables one to monitor structural
evolution of ions for hours. In particular, evaporation of water droplets and slow thermal deformation of polystyrene particles havebeen observed. Applications to nanomaterials and the new system with much higher resolving power will be discussed.
The quest for more specific ion separation and identification drives use of IMS to pre-separate isomers for various spectroscopic
and chemical selectivity studies. Fuminori Misaizu (Tohoku Univ.) will lecture on the spectroscopy and reactions of isomerselected clusters. His versatile apparatus coupling drift-tube IMS to ToF MS enables performing photodissociation, collisioninduced dissociation, ion-molecule reactions, and negative-ion photoelectron spectroscopy for mobility-selected isomers,
exemplified by carbon and silicon cluster cations and anions.
A major aspect of next-generation IMS will be buffer gases comprising vapors that interact with ions specifically rather than inert
media that merely retards the ion motion. While such“shift reagents”have helped in some conventional IMS applications, they
appear of most value for FAIMS where they generally expand both the overall peak capacity and resolution of targeted analytes.
Utilization of vapor dopants is a key feature of the SelexION technology, and the latest highlights in this fast-evolving area will be
communicated by Brad Schneider (AB Sciex).
The FAIMS resolving power scales roughly as the cube of field strength that was limited by electrical breakdown threshold. This
constraint is essentially lifted in the FAIMS microchips by Owlstone, where fields up to 75 kV/cm permit ion filtering in <100 μs.
Richard Yost (Univ. of Florida) will show first findings on the use of these chips with solvent vapors. While resolution gains
resemble the effect in full-size FAIMS, the extreme fields in microchips cause intense ion heating that enables rich new chemistryleading to additional separation mechanisms.
A raising MS approach to structural elucidation of proteins and their complexes is chemical cross-linking, but enormous number
and diversity of products greatly challenge their detection and identification. Separations by IMS have a major potential here, andTara Pukala (Adelaide) will describe the use of novel negative-ion cleavable cross-linking reagents in this context.

Workshop 6
Wednesday, 19th SeptemberRoom D

17:15 － 19:15

Mass Spectrometry for Food Safety
Organizer: Jentaie Shiea (Nat'l Sun Yat-Sen Univ., Taiwan)

Keywords:
Food safety, Mass spectrometry, Foodborne microorganisms, Mycotoxins, Chemical residues, Pesticides, Food packaging andprocessing
Scope of Session:
Recent food incidents have drawn the public’s awareness of how food supply is regulated and inspected to be deemed safe for
consumption. The common food harmful substances include foodborne microorganisms, mycotoxins, chemical residues,
pesticides, melamine, plasticizers, and chemical contaminants from food packaging and processing. Only trace amount of theseharmful substances is sufficient to cause harmful health effects. Thus, mass spectrometry plays a critical role as an excellent
analytical tool to identify food contaminants accurately and efficiently. This workshop explores the recent advances andapplication of mass spectrometry to food safety, and challenges in food sample preparation.