MSP2018 Late-Breaking Abstracts
Presenter

Affiliation

Title

ID

Development of Ion Trap Ion Mobility Measurement System and Observation of
Toshiki Sugai

1P-52 (LB)

Toho Univ.
Observation of Nanomaterials
Detection of Intact Biomolecular Ions by inTrap MALDI and Charge-Sensing

Yu-Cen Sun

Acromass

1P-53 (LB)
Particle Detector
Comparison of Metabolites in Saliva and Gingival Crevicular Fruid for Searching

Moe Okuyama

1P-54 (LB)

OSAKA UNIV.
Periodontal Disease Biomarkers
Comparison of CO2 and N2 drift gas for the separation of isobaric isomer

Yoshiteru Iinuma

OIST

2P-63 (LB)compounds in a travelling wave ion mobility mass spectrometry
Development of Sample Preparation Method of Mass Spectrometry Imaging

Daisuke Miura

SUNTORY

2P-64 (LB)
toward Metabolite Localization Analysis with Higher Sensitivity and Reproducibility

Alejandro Villar-

LC-MS-Compatible Salt-free High-Resolution Microfractionation of Tryptic
OISTBriones

2P-65 (LB)
Peptides at Nanogram Levels
Improved iST workflows for the streamlined analysis of tissues and high-Garwin Pichler

3P-51 (LB)

PreOmics
throughput preparation of samples using isobaric labeling
Taipei Medical

A Oncoproteogenomics Strategy for Multiplexed Screening of EGFR Mutations in

Univ.

Non-small-cell Lung Cancer

Radoslaw

A*STAR,

High Resolution - Mass Spectrometry Cellular Thermal Shift Assay (HR-MS-

SobotaSingapore

CETSA)-post-translational modifications impact on thermal protein stability

Yi Wang

BPRC

Urine proteome profiling predicts lung cancer from control cases and other tumors

HEE-SUB

Novel interaction of RSK3 and IκBα induced cell growth and survival in breast

YUN

cancer cells

Chia-Li Han

3P-52 (LB)

3P-53 (LB)

Hee-sub Yun

3P-54 (LB)

3P-55 (LB)
Quantitative analysis of ERBB protein complex assembly as a diagnostic tool for

Shujuan Wang

BPRC

4P-41 (LB)
abnormal signaling in cancer cells
Urinary metabolomics study of the therapeutic mechanisms of Ding-Zhi-Xiao-Wan

Hui ZhouJilin University

4P-42 (LB)
against Alzheimer's disease rat model using UPLC-Q-TOF-MS

Tongji

Large-scale Identification and Visualization of N-Glycome with Primary Structures

University

Using GlySeeker

3A-02-P-

Zhixin TIAN
1555

1P-52 (LB)
Development of Ion Trap Ion Mobility Measurement System and Observation of
Observation of Nanomaterials(Department of Chemistry Toho University)
Y. Hoshino, R. Miyamoto, F. Uchiyama, N. Terada, ○T. Sugai
Keywords: Nanomaterials, Mobility, Ion Trap
Ion mobility spectrometry (IMS) has been utilized toreveal novel information on nano materials1,2). However, nolong-term successive measurement has been performed with
IMS usually because of the lack of trap functions, which
limits the resolution and the varieties of information.
Recently ion trap ion mobility system has been developed toenhance the resolution and long-term evolution of
biomolecules. The trap time, however, is restricted to lessthan 2 seconds and no other measurement such as optical
spectroscopy is combined3). Here we present long-term
successive IMS measurements with optical measurements
on nano materials such as graphene quantum dots4) (GQD)
and metal clusters as single molecules.
Fig. 1 shows TEM images of GQD with few layers ofgraphene sheets and with an average diameter around 20 nm.
The sample was measured with our system. Fig. 2 shows thelong-term time dependence of the observed velocities ofeach GQD molecule with various charges. Single GQD
molecules can be observed and the structures and charge
states are conserved for up to 7 hours. The diameter
evaluated to be 20 nm from the velocity and the estimatedcharge state, which is in good agreement with that from theTEM observation. We have also succeeded to observe noble
metal clusters. Optical spectroscopy on each molecule arealso in progress by utilizing thus realized long-term trap
functions.

References
1) T. Sugai et al., J. Am. Chem. Soc. 123, 6427- 6428
(2001).
2)

T. Sugai, J. Mass. Spectrom. Soc. Jpn. 58, 47-73(2010).

3)

F. Fernandez-Lima et al., J. Ion Mobil. Spec. 14, 93-98(2011)

4)

R. Sekiya et al., Chem. Eur. J., 22, 8198-8206 (2016).

20 nm

Fig. 1 TEM images of GQD

Fig. 2 Observed velocity of GQD withvarious charges

1P-53 (LB)
inTrap MALDI と電荷センシングでの粒子検出器によって構造完全な生体分子イオンを直接検出する方法（譜光儀器株式会社 1）
スンユーツェン
1ヂョウスーウェイ

リーイークン

ゾンイャォシンヤンシージェ

ヂォンジュンイェン

孫 宇 岑 ・○ 周 思 瑋 1・李 怡 坤 1・曾 耀 興 1・ 楊 世 頡 1・ 鄭 俊 彥 1

Detection of Intact Biomolecular Ions by inTrap MALDI and Charge-Sensing Particle
Detector(AcroMass Technologies, Inc.1)
Conan Sun1, ○Szu-Wei Chou1, Yi-Kun Lee1, Yao-Hsin Tseng1, Shin-Chien Yang1, Chun-Yen Cheng1
Keywords: charge detection, ion trap mass spectrometry, MALDI, intact protein
We have developed a novel ion cloud detector called charge-sensing particle detector (CSPD, Figure 1), which
is used in mass spectrometry to unbind the detectivity limitation of high-mass biomolecular ions, such as intact
proteins. Whenever an ion cloud suddenly approaches the Faraday tray on the CSPD, the weak yet-fast charging
along with incoming event is nonlinearly converted into to a sharp pulse signal.
The setup of mass spectrometer (Figure 2), includes inTrap MALDI as a highly efficient ion source and aquadrupole ion trap (3D Paul Trap) as a mass-to-charge analyzer. Two phase-synthesized RF waveforms (main &auxiliary) are sent to the trap electrodes for intact-molecular mass spectrum, via ion trapping, adiabatic cooling,phase synchronization and unstable ejection. The ions are directly inTrap prepared, as depicted in Figure 3, withno ion guide for ion transmission.
One example is the mass spectrum of biomolecule Cytochrome C, via unstable ejection and synchronization aid+
from auxiliary pulses (shown in Figure 4). Beside the primary peak of pure Cyto C , CSPD finesse responses every+
finely-resolved matrix-modified Cyto C cloud in its tail, and goes without ambiguous overshooting. CSPD is abipolar device, right for the general case with both positive and negative ions.

Fig.1. The charge-sensing particle detector

Fig.3. inTrap MALDI

Fig.2. The brief functional chart

Fig.4. Uncalibrated MALDI ion trap mass spectra of Cytochrome C

References
1) Raymond E. March, Journal of Mass Spectrometry, Vol. 32, 351E369 (1997).
2) Urs P. Schlunegger et al., Rapid Commun. Mass Spectrom. 13, 1792–1796 (1999).

1P-54 (LB)
歯周病バイオマーカー探索に向けた唾液と歯肉溝滲出液内代謝物の比較（阪大院理）
おくやまも えお お す が じゅんいちと よ だ みちさと

○奥山萌恵・大須賀 潤 一 ・豊田岐聡

Comparison of Metabolites in Saliva and Gingival Crevicular Fruid for Searching
Periodontal Disease Biomarkers(Osaka University)○M. Okuyama, J. Osuga, M. Toyoda
Short Abstract: Periodontal disease is one of the most concerned threats to oral health. Therefore, it isdesired to establish new method of on-site analysis to diagnose periodontal disease conditions. In the previous
research, nineteen metabolites in gingival crevicular fluid (GCF) were identified. Additionally, it was suggested
that periodontitis progression could be observed with a changing metabolite profile. In this study, saliva withpretreatment was analyzed using Gas Chromatography/ Mass Spectrometry (GC/MS). As a preliminary result,metabolites in saliva and GCF were compared. It seems that concentrations of metabolites in saliva are lower
than those in GCF.
Keywords: Periodontal disease, GC/MS
本研究では，歯周病オンサイト診断法開発を目標としたバイオマーカー探索をおこなう．歯周病は歯を支える歯周組織が細菌に侵され破壊される感染症であり，進行に伴って歯周組織の破壊が高度化することから，成人が歯を喪失する第一位の原因である．また，糖尿病や心臓病などの全身疾患との相互関連性も近年報告されている．しかしその病態の的確な評価法はなく，歯周病の進行度合いも個人差があるため，オンサイトで迅速かつ簡便に評価ができる新規診断法の確立が望まれている．先行研究において，歯周ポケットから採取可能な歯肉溝滲出液を用いたバイオマーカー探索が行われ，進行度合いに応じて有意差が見られるバイオマーカー候補化合物が 19 種類特定された 1．歯肉溝滲出液
は患部から直接採取できるが，一方で採取は歯科医しか行えない．そこで，非侵襲性で場所を選ばず容易に採取可能な唾液による診断法の開発を行えないか考えた．ここでは歯周病診断の試料に唾液を用いるための基礎的な検討として，唾液と歯肉溝滲出液を分析し検出される代謝物がどの程度共通しているか調べた．
前処理として，参考文献 2 にならって実試料から親水性化合物を抽出，濃縮，乾燥させ，誘導体化した．その後，GC/MS 法で一斉分析をおこなった．内部標準試料として 2-イソプロピルリンゴ酸を用い，相対面積値による半定量解析をおこなった．
健常者 1 名の唾液 3.0μL と歯肉溝滲出液 0.20μL を分析した．計 94 成分が検出されたうち，両試料から 59 成分，歯肉溝滲出液のみから 16 成分，唾液のみから 19 成分の代謝物が検出された．この結果から，62.8%は唾液と歯肉溝滲出液双方で検出されていることがわかる．しかしながら，検出された化合物のうち，バイオマーカー候補として挙がっている成分に注目すると，13 成分が歯肉溝滲出液
試料で検出されており，標準品を用いて保持時間とスペクトルを確認した．これらの化合物について，各化合物のベースピークと各化合物と内標との相対面積値をまとめたものを Table.1 に示す．ただし，比較のため分析試料の同体積あたりの相対面積値とした．この結果から，両試料で検出された化合物はいずれも唾液試料の方が低い値を示しており，バイオマーカー候補化合物の唾液内代謝物濃度は歯肉溝滲出液と比べて 12 倍から 600 倍程
度低いことがわかった．本発表では，唾液のサン
プル量を増やした時の検出ピーク数や分析法の堅牢性についても議論したい．
Table.1 Relative intensity of identified metabolites
参考文献
1) M. Ozeki et al., Mass Spectrometry 5 : A0047(2016)2) S. Nishiumi et al., Metabolomics 6 : 518-528 (2010)

2P-63 (LB)
Comparison of CO2 and N2 drift gas for the separation of isobaric isomer compounds ina travelling wave ion mobility mass spectrometry(Okinawa Institute of Science and Technology1, National Institute for Environmental Studies2, Tianjin University
Japanese Mass Spectrometry Co.3)○Y. Iinuma1, E. Hayakawa1, S. Inomata2, K. Sato2, H. Ren3, S. Yue3, P. Fu3
Keywords: Ion Mobility, Travelling Wave Ion Mobility, Environmental Samples, Aerosol, PM
Isobaric isomer compounds are ubiquitous occurrence in ambient atmospheric aerosols. They originate fromoxidation processes of anthropogenic and biogenic volatile organic compounds (VOCs) such as aromaticcompounds and monoterpenes. For example, monoterpenes are known to produce a series of isobaric isomercompounds such as C9H14O4, C8H12O4, C10H18O5S, and C10H17NO7S. Among these isobaric isomer compounds,
C10H17NO7S compounds are still not conclusively structurally characterized to date owing to the lack ofknowledge about their formation mechanisms and the difficulty associating with the synthesis of authentic
standard compounds1). An ion mobility mass spectrometry (IMS-MS) is useful for the structural analysis of suchisobaric isomer compounds, as their structural differences lead to their separation in IMS-MS and their mobility
data can be used to construct potential structures for a mobility modelling study2).
In this study we compare the effects of drift gas types on the separation of C10H17NO7S compounds in a
travelling wave ion mobility mass spectrometer (Waters SYNAPT G2-S). These isomeric compounds produce
very similar fragment ions that are difficult to associate with the positions of functional groups. A particular focus
here is to use CO2 gas to separate isobaric MS2 fragment ions that are not well separated with N2 drift gas, andinfer the structures of C10H17NO7S compounds. One of PM2.5 samples collected in Chengdu, China in June 2016
was selected for the detail investigation.
A commercially available travelling wave ion mobility MS system using the N2 drift gas tends to work well forthe separation of larger ions such as proteins. However, the separation of smaller molecules is challenging as theresolving power is low in comparison to their differences in collisional cross section values (CCS). Figure 1
shows an example of drift-time plots using CO2 and N2 for the m/z 247 (C10H15O5S-, [M-H]-) MS2 fragment of
C10H17NO7S compounds. As can be seen the CO2 drift gas showed a much better separation for the m/z 247 MS2
fragments compared to N2 drift gas, especially for the peaks eluting between 8.5 and 9 minutes. Although thisinformation alone does not lead to the conclusive structural elucidation of the precursor or fragment ions, thisdata can be useful in the elucidation of the substructures of C10H17NO7S compounds. In this case, the peakdetected at 9 min has a longer drifting time than the peak at 9 min, suggesting that either the sulfate group is
attached at the terminal position or it has a larger monoterpene skeleton. The result here demonstrates that the
separation power of a commercially available travelling wave ion mobility instrument can be improved for small
ions with similar CCS when we use a more polarizable drift gas such as CO2, and provide more structural
information than the N2 drift gas.

Fig.1. Drift time as a function of chromatographic retention time using CO2 (left) and N2 (right) for the m/z 247
MS2 fragment [M-H]-.
References
1) B Noziere et al., Chem. Rev., 115(10), 3919-3983, doi:10.1021/cr5003485 (2015).
2)

M F Mesleh, et al., J Phys Chem, 100(40), 16082-16086 (1996).

2P-64 (LB)
高感度かつ再現性の高い代謝物局在解析をめざした質量分析イメージングの試料調製法の開発（九大農学研究院 1・島津製作所 2）
いちのせともみふじむらよしのりやまざきゆうぞう

みうらだいすけ一瀬智美1・○藤村由紀1・山崎雄三2・三浦大典1

Development of Sample Preparation Method of Mass Spectrometry Imaging toward
Metabolite Localization Analysis with Higher Sensitivity and Reproducibility(Fac.Agric., Kyushu University1 & Shimadzu Co. 2)
T.Ichinose1, ○Y.Fujimura1, Y.Yamazaki2, D.Miura1
Keywords: MALDI-MS, Metabolite imaging, Matrix Application, Sublimation, Sensitivity
Introduction: Matrix-assisted laser desorption/ionization (MALDI)-mass spectrometry imaging (MSI) is a
powerful technique to visualize the distributions of biomolecules without any labeling. In MALDI-MSI
experiments, the choice of matrix deposition method is important for acquiring favorable MSI data with highsensitivity and high spatial resolution. Generally, manual or automated spray-coating and automated sublimation
methods are recognized as established matrix coating techniques for MSI. Manual or automated spray-coating
methods are relatively convenient, but they show low reproducibility of metabolite detection. The automatedsublimation is useful to obtain high spatial resolution and high reproducibility, but its signal intensity is often
less than the spray-coating method capable of sensitive detection of such metabolites due to a solvent extraction
effect. A recrystallization of sublimated matrix in an organic solvent atmosphere is alternative method to thespray-coating method, and effective to improve sensitivity by solvent extraction effect. To date, there have been
no other reports of 9-aminoacridine (9-AA)-based sublimation combined with recrystallization, whereas thematrix is useful to ionize endogenous metabolites. Herein, we present a novel matrix deposition method of
sublimation coupled with recrystallization using 9-AA.
Methods/Results: We attempted to improve the matrix sublimation method by coupling it with arecrystallization reaction with solvent treatment, using 9-AA as a matrix for visualizing the distributions ofendogenous metabolites. The matrix vapor deposition system iMLayer (Shimadzu), which can automaticallymonitor the temperature and thickness of a matrix deposited during sublimation, was used. Our procedure formatrix sublimation and recrystallization on a tissue sample (mouse brain: C57BL/6J, 6 weeks, male) consisted oftwo steps. The first step was formation of the matrix layer on mouse brain sections by sublimation using
iMLayer. The next step was co-crystallization of the matrix and analyte on the tissue, with exposure to solvent
vapor to promote recrystallization of the matrix, using a hand-made reaction chamber. We evaluated our
proposed procedure for its ability to provide MSI data with high sensitivity, good spatial resolution, andexcellent reproducibility, using a MALDI-TOF-MS instrument (AXIMA Performance, Shimadzu). Both asolvent concentration and reaction temperature were optimized to perform a recrystallization of sublimated 9-AA.
This optimized method showed excellent reproducibility and spatial resolution compared to the spray-coating
method. Furthermore, the recrystallization step after sublimation remarkably improved the detectability of
metabolites, including amino acids, nucleotide derivatives, and lipids, compared with the conventional
sublimation method. The present method provides an easy, sensitive, and reproducible matrix deposition method
for MALDI-MSI of endogenous metabolites.
Conclusion/Future aspects: We established a 9-AA sublimation method with coupling to a recrystallization
step, which was optimized for the solvent concentration and reaction temperature. This methodology is very
simple but provides excellent detection sensitivity for endogenous metabolites and yields highly reproducible
data. The present sample preparation method and its optimization strategy could be applied to tissue sections
from different types of organisms and other matrices. This method could be used to obtain precise information
about the distributions of endogenous molecules, which could be applied to biological discovery and biomarker
development.
References
1) D. Miura et al., Biochem. Biophys. Res. Commun., 496, 140-146 (2018).
2) D. Miura et al., Anal. Bioanal. Chem., 409, 1697-1706 (2017).

2P-65 (LB)
LC-MS-Compatible Salt-free High-Resolution Microfractionation of Tryptic Peptides at
Nanogram Levels(Okinawa Institute of Science and Technology1)
A.Villar-Briones1
Keywords: sample preparation, proteomics, salt-free, microfractionation, peptides
The intrinsic complexity of enzymatically digested samples is greatly reduced by employing off-line
fractionation. The use of C18 columns with high-pH solvents addresses the problem of buffers that areincompatible with mass spectrometry (MS) and provides a semi-orthogonal separation of the sample compared
to traditional low-pH separation with the same column. However, this technique still relies on the use of anammonium salt as a buffer to achieve the desired chromatographic resolution. Electrostatic
Repulsion-Hydrophilic Interaction Chromatography (ERLIC) 1) presents an attractive solution to this problem byusing a salt-free solvent, fully compatible with tandem MS analysis. This technique has most often been
implemented using a Weak Anion Exchange (WAX) column with a 4.6-mm i.d., requiring hundred(s) of
micrograms of sample. In addition, it uses solvents supplemented with up to 2% formic acid (FA) for elution,
which adversely affects ionization efficiency.
Here we present an optimized method for microfractionation of tryptic digests using ERLIC separation,compatible with sample loads ranging from micrograms to nanograms, using alkaline solvents similar to
those for reverse phase separations in tandem with MS.
To this end, we used a capillary-size WAX column (1-mm i.d.), at a flow rate of 40 µL/min, withwater/acetonitrile containing 0.1% FA and 0.05% ammonium hydroxide, delivered by a binary HPLC pump.
Sample elution was monitored at 214 nm and fractions were collected in 96-well plates using an autosampler
with a MALDI-Spot accessory. Peptide resolution and recovery were evaluated using 10 µg of a tryptic digest ofproteins extracted from mouse brain, separated on a 60-min gradient. Fraction volume was ~120 µL. This systemachieved a 2x increase in the number of proteins identified, compared to traditional shotgun analysis using thesame amount of sample. More low-concentration proteins were revealed due to the reduced complexity of thefractionated sample. Peptide elution profiles were highly reproducible, making this technique suitable fortargeted proteomics. In addition, salts from the digestion buffer elute in the chromatographic void volume; hence,
they do not influence mass spectrometry.
This method allows us routinely to microfractionate peptides from small biological samples (several
micrograms), considerably increasing the number and coverage of identified proteins, with special emphasis on
low-abundance components. Separation of peptides in a reduced volume improves their re-suspension prior to
LC-MS injection. Although the method presented here was employed off-line, it can easily be adapted foron-line separation (2D LC) with tandem MS, reducing sample handling and machine time.
This optimized method facilitates peptide fractionation using sample amounts similar to those of traditional
shotgun proteomics. Therefore, it is especially useful in cases in which other methods cannot be employed due
to limited sample and risk of sample loss due to sample dilution or downstream desalting.
References
1) Hao P. et al., J Proteome Res. 2010 Jul 2;9(7):3520-6.

3P-51 (LB)
Improved iST workflows for the streamlined analysis of tissues and high-throughput preparation ofsamples using isobaric labeling1

Garwin Pichler1; Fabian Hosp1; Nils Kulak1
PreOmics GmbH, Planegg/Martinsried, Germany

Introduction
Recently, the straightforward and robust in-StageTip (iST) method for streamlined sample processing ofvarious sample types including cell lines and body fluids (e.g. plasma, serum, CSF, urine) was described(Kulak et al., Nat Meth, 2014). Here, we present further developments based on the iST technology.
Methods
The iST method is a 3-step procedure performed in a single, enclosed volume, which thereby
circumvents the likelihood of contamination and sample loss. Moreover, it allows rapid sample
preparation from lysing the sample to ready-to-measure peptides in less than 3 hours while keeping
excellent reproducibility and sensitivity. Due to its straightforward nature, the method can readily be
performed in a 96-well format on liquid handling robotic systems. The iST workflow is highly compatible
with established and novel StageTip based pre-fractionation methods and thereby allows in-depth
analysis of complex proteomic samples.
Preliminary DataFirst, we present the iST-NHS adaptation that allows chemical labeling of peptides in the very same
reaction device used for cell lysis, protein denaturation, reduction, alkylation and digestion, thus
minimizing sample loss, overall hands-on time and the amount of required chemical labels. Using yeast
cells, we employed this iST-NHS approach and compared different ratios of tandem mass tag (TMT)
reagents per µg of peptides input material. While most labeling workflows utilize a TMT to peptide ratioof 8:1, we found that a ratio of 4:1 results in the highest number of identified peptides while stillachieving labeling efficiencies of more than 99.5% using the iST-NHS method. In addition, the iST-NHS
method is fully compatible with 96well plate formats and automated robotic systems enabling highthroughput isobaric labeling experiments.
Second, we present an improved iST workflow for the lysis of tissue (fresh brain, heart and liver samples,
as well as FFPE samples) directly in the reaction device used for the downstream sample preparation,
thus avoiding lysate transferring steps and minimizing sample loss. We present identification of 6,00010,000 proteins for the aforementioned tissue types demonstrating in-depth proteome analysis withminimal sample preparation efforts. With this improved iST workflow, up to 24 tissue samples can beprepared in parallel and in less than 3 hours from wet tissue to ready-to-measure peptides.
Novel Aspect
The new iST developments enable streamlined analysis of tissues and high-throughput preparation ofchemically labeled samples.

3P-52 (LB)
A Oncoproteogenomics Strategy for Multiplexed Screening of EGFR Mutationsin Non-small-cell Lung Cancer
Chi-Ting Lai1,2, Wai-Kok Choong3, Yi-Ju Chen1, Hui-Yin Chang3, Ting-Yi Sung3, Hsuan-Yu Chen2,4,
Yu-Ju Chen1,2*, Chia-Li Han5*
1

Institute of Chemistry, Academia Sinica, Taipei, Taiwan; 2Genome and Systems Biology Degree
Program, National Taiwan University, Taipei, Taiwan; 3Institute of Information Science, Academia
Sinica, Taipei, Taiwan; 4Institute of Statistical Science, Academia Sinica, Taipei, Taiwan; 5Master
Program in Clinical Pharmacogenomics and Pharmacoproteomics, College of Pharmacy, Taipei
Medical University, Taipei, Taiwan

Oncogenes with somatic mutations contribute to the development and progression ofcancers. However, the expression levels of mutation bearing oncogenes and theirtranslated protein variants are largely unexplored. Thus, we aimed to develop a mass
spectrometry (MS)-based oncoproteogenomics strategy for multiplexed screening oftumor-specific mutations on oncoproteins. The non-small-cell lung cancer (NSCLC)
which harbor different EGFR genotypes were used as study model. The
oncoproteogenomics strategy integrated bioinformatics analysis of mutant protein
sequences for protease prediction, affinity purification of EGFR protein complex,
parallel enzymatic gel-assisted digestions, LC-MS/MS analysis, and customized
database searching using multiple engines, for unambiguous identification of 34
mutated and 33 wild-type EGFR proteins. The abundances of cellular wild-type andmutant EGFR proteins were measured using LC-PRM-MS in a series of NSCLC celllines. The results revealed concomitant and heterogeneous expressions of mutant andwild-type EGFR proteins in NSCLC. We expected that our developed
oncoproteogenomics strategy can precisely determine the multiple somatic mutationsat protein level and provide a bettering understanding towards the molecular impact of
EGFR genetic mutations during tumor progression and adaption to tyrosine kinase
inhibitor treatments.
Keywords
EGFR, lung cancer, oncoproteogenomics

3P-53 (LB)High Resolution - Mass Spectrometry Cellular Thermal Shift Assay (HR-MS-CETSA)post-translational modifications impact on thermal
protein stability
School of Biological Sciences, Nanyang Technological University, Singapore1. Institute of Molecular and Cell
Biology (IMCB), Agency for Science, Technology and Research (A-STAR), Singapore2, Department of
Oncology-Pathology , Karolinska Institutet, Stockholm, Sweden3
Lingyun Dai1 , Tianyun Zhao1 , Liyan Chen1 , Chris Soon Heng Tan2, Yan Ting Lim1 , Par Nordlund1,2,3,○Radoslaw M. Sobota

2

Keywords: PTM, Thermal Shift Assay, MS-CETSA, drug target deconvolution
Observation of the proteins thermal stability towards increasing temperature forms the foundation for methods that
explore thermally induced protein unfolding. Heat treatment induced protein unfolding and aggregation can begraphically presented as a sigmoidal melting curve. It was shown that ligand engagement changes the functional
state of the protein and induces thermal shift. MS- based Cellular Thermal Shift Assay (CETSA) monitors
thermal induced unfolding of individual proteins in lysates/intact cells/tissue samples using quantitative mass
spectrometry. To further extend CETSA application, here we proposed high resolution MS-CETSA to increase atarget specificity. We investigated further impact of post-translational modifications on the protein stability. We
were able to detect in the sample with limited input more than 5000 distinct phosphorylation residues, representing
over 5000 unique melting profiles. We demonstrated that phosphorylated proteins exhibit different stability
towards increasing temperature. Results point destabilizing effect of the phosphorylation, although detailed
mechanism has to be further determined. Furthermore, presented method could help to identify functional
phosphorylation sites in the protein which occupancy would be reflected in protein stability. The Cellular PTM
resolved MS-CETSA has potential to add a valuable dimension to pre-clinical drug development defining drug
target engagement and off-target effects in relevant tissue systems.

1.

2.

PTM

PTM

Fraction folded protein

PTM

3.

Temperature

Fraction folded protein

5.
drug

Temperature

Temperature

PTM

Fraction folded protein

Fraction folded protein

Fraction folded protein

4.

PTM

1 2 3

4

Temperature

Temperature

Figure1. Influence of PTM on protein stability - High resolution PTM resolved MS-CETSA principle. (1)Baseline- unmodified protein with distinct melting properties. (2). Modified protein with PTM affecting
melting behaviour. Additional PTM could affect melting properties directly (3) or indirectly (4) by triggering
new complex formation or cellular localization context. (5) Drug candidate distinguishing specific PTM stateand preventing protein further activity.
1.

2.

Thermal proximity coaggregation for system - wide profiling of protein complex dynamics in cells. Tan
Soon Heng Ch. Go Ka Diam, Bisteau X., Dai Lingyun, Prabhu N., Ozturk B., Lim YT. , Sreekumar L.,
Chern Han Yong , Lengqvist J.,Tergaaonkar V., Kaldis P., Sobota R.M., Nordlund P. Science 08 Feb
2018: eaan0346 DOI: 10.1126/science.aan0346
Dual Blockade of the Lipid Kinase PI5P4Ks and Mitotic Pathways Leads to Cancer-selective Lethality
Kitagawa, M., Liao, Pei-Ju , Wong J., Cheng Shang See , Minami, N., Sampetrean, O., Saya, H., Dai
Lingyun, Prabhu, N., Go Ka Diam, Sobota, R. , Larsson, A., Nordlund P., McCormick, F., Ghosh S.,
Epstein,D., Dymock, B., Sang Hyun Lee. Nature Communications, 2017 Dec 19;8(1):2200. doi:
10.1038/s41467-017-02287-5

3P-54 (LB)
Urine proteome profiling predicts lung cancer from control cases and other tumors
Chunchao Zhang3, Wenchuan Leng1,2, Changqing Sun2, Tianyuan Lu1, Zhengang Chen2, Xuebo
Men2, Yi Wang1,3, Guangshun Wang2, Bei Zhen1,2, Jun Qin1,2,3
1.

State Key Laboratory of Proteomics, National Center for Protein Sciences (The PHOENIX Center, Beijing),
Beijing Proteome Research Center, Beijing, 102206, China
2. Joint Center for Translational Medicine, Tianjin Baodi Hospital, Tianjin, 301800, China
3. Alkek Center for Molecular Discovery, Verna and Marrs McLean Department of Biochemistry and Molecular
Biology, Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas 77030,
USA

Development of noninvasive, reliable biomarkers for lung cancer diagnosis has many clinicalbenefits knowing that most of lung cancer patients are diagnosed at the late stage. For this purpose,
we conducted proteomic analyses of 231 human urine samples in healthy individuals (n = 33),
benign pulmonary diseases (n = 40), lung cancer (n = 33), bladder cancer (n = 17), cervical cancer(n = 25), colorectal cancer (n = 22), esophageal cancer (n = 14), and gastric cancer (n = 47) patientscollected from multiple medical centers. By random forest modeling, we nominated a list of urine
proteins that could separate lung cancers from other cases. With a feature selection algorithm, weselected a panel of five urinary biomarkers (FTL: Ferritin light chain; MAPK1IP1L: MitogenActivated Protein Kinase 1 Interacting Protein 1 Like; FGB: Fibrinogen Beta Chain; RAB33B:
RAB33B, Member RAS Oncogene Family; RAB15: RAB15, Member RAS Oncogene Family)
and established a combinatorial model that can correctly classify the majority of lung cancer cases
both in the training set (n = 46) and the test sets (n = 14 – 47 per set) with an AUC ranging from
0.8747 to 0.9853. A combination of five urinary biomarkers not only discriminates lung cancer
patients from control groups but also differentiates lung cancer from other common tumors. The
biomarker panel and the predictive model may be used as an auxiliary diagnostic tool along withimaging technology for lung cancer detection.

3P-55 (LB)
Novel interaction of RSK3 and IκBα induced cell growth and survival in breast cancer cells
Hee-Sub Yun

1, 3

1

, Sung Ho Yun , Eun-Hee Han

1, 2

1

4

, Sung hoon Choi , Ju hee Park Young-Ho Chung

1,

4

1

Division of Bioconvergence Analysis, Drug & Disease Target Group, Korea Basic Science
2

Institute(KBSI), Ochang, Republic of Korea; Department of Functional Genomics, University of3

Science and Technology (UST), Daejeon, Republic of Korea; Korea University, Seoul, Republic of
4

Korea; GRAST, Chungnam National University, Daejeon, Republic of Korea
Keywords: Protein-Protein Interaction (PPI), Breast Cancer cells, RSK3, IκBα, Inhibitor
A number of cancer-related biological processes are mediated by protein-protein interaction (PPIs).
Therefore, targeting PPIs are emerging as a promising new class of drug targets. But, although PPIs
have essential role in cancer diseases, there are few PPIs target as a therapeutic targets in breast
cancer. The RSK (90 kDa ribosomal S6 kinase) family comprises a group of highly related
serine/threonine kinases that regulate diverse cellular processes, including cell survival, proliferation,
growth. Although RSK3 was initially suggested to play positive roles in cell proliferation, a detailed
functions and mechanisms are currently unknown. Our group has over the 500 fluorescence tagging
kinase vector libraries for screen of kinase PPI as a drug target. Several candidates have been
identified as RSK3 binding partner through CUPID analysis. IκBα is one of the RSK3 novel binding
partner and is phosphorylated by active RSK3. Besides, A screened PPI inhibitor targeting RSK3/IκBαcomplex which is playing anti-cancer activity in breast cancer cells such as cell proliferation andsurvival. Taken together, these observations suggest that RSK3 phosphorylation and RSK3/IκBαcomplex formation are may be functionally important in breast tumorigenesis. Indeed, the validation of
RSK3/IκBα specific inhibitor can provides a lead to development new anti-cancer drugs.

Fig. 1. RSK3/IkBa Direct Interaction (myc/His-RSK3 and GFP-IkBa co-transfected)
References
1) SERRA, Violeta, et al. RSK3/4 mediate resistance to PI3K pathway inhibitors in breast cancer. The
Journal of clinical investigation, 2013, 123.6: 2551-2563.
2) EISINGER-MATHASON, TS Karin; ANDRADE, Josefa; LANNIGAN, Deborah A. RSK intumorigenesis: connections to steroid signaling. Steroids, 2010, 75.3: 191-202.

4P-41 (LB)
Quantitative analysis of ERBB protein complex assembly as a diagnostic tool for abnormal
signaling in cancer cells(State Key Laboratory of Proteomics, Beijing Proteome Research Center, National Center for Protein Sciences(Beijing), Beijing Institute of Lifeomics, Beijing, China1, Lunenfeld Tanenbaum Research Institute, Mount Sinai
Hospital, Toronto, Canada2, Novartis Institutes for Biomedical Research, Cambridge, USA3, McGill University
Health Centre, Montreal, Canada4, Department of Molecular Genetics, University of Toronto, Toronto, Canada5)
Shujuan Wang1#, Cunjie Zhang2#, Adrian Pasculescu2, Pan Zhang1, Sihui Li1, Xiaojing Wu1, Xiao Li3, Rita Das3,
Qing Sheng3, Lorne Taylor2,4, Vivian Nguyen1, Tony Pawson2,5, Carl Uli Bialucha3, Karen Colwill2*, Yong
Zheng1*#: equal contribution
Keywords: quantitative mass spectrometry, protein complex, drug resistanceProteins rarely act by their own but rather as part of large protein complexes formed through dynamic
non-covalent protein-protein interactions in response to specific environmental cues. Precisely controlledassembly of protein complexes is critical for the fidelity and magnitude of downstream signaling transduction
while its dysregulation leads to aberrant signaling and cancer. Identifying abnormal protein complex formation
is key to understand underlying mechanisms of oncogenesis.
We developed an affinity purification and quantitative mass spectrometry-based workflow (AP-MRM and
AP-PRM) to quantify 37 interacting proteins in ERBB-mediated protein complexes in 19 cancer cell lines bothin basal and ligand stimulated states. ERBB family members are “driver genes” in several cancers including
breast cancer. We show that the intrinsic differences between cell lines is the biggest differential as the cell lines
varied significantly in the type and number of proteins recruited to the complex. Temporal analysis of ERBB
complexes in three breast cancer cell lines suggested that ERBB complexes tend to assembled in a functionallydistinct modular fashion in individual cell line which may correlate with prejudicial activation of downstream
signaling. Thus, the quantitative characteristics of key protein complexes may serve as a diagnostic tool toidentify rewired signaling in cancer.

4P-42 (LB)
Urinary metabolomics study of the therapeutic mechanisms of Ding-Zhi-Xiao-Wan
against Alzheimer's disease rat model using UPLC-Q-TOF-MS(Department of Chemistry and Pharmacy, Zhuhai College of Jilin University, Zhuhai 519041, China1
National Center for Mass Spectrometry in Changchun, Changchun Institute of Applied Chemistry, Chinese
Academy of Sciences, Changchun 130022, China2)
1
1
Yimin Wang , ○Hui Zhou , Shu Liu2, Zifeng Pi2, Yufei Sun2, Zhiqiang Liu2
Keywords: Metabolomics, Ding-Zhi-Xiao-Wan, Alzheimer’s disease
Background: As a well-known traditional Chinese medicine (TCM) formula, Ding-Zhi-Xiao-Wan (DZXW) is
commonly used for the treatment of mental disorders, especially amnesia and depression1). The aim of this study
was to investigate the potential curative mechanisms of DZXW on learning and memory in Aβ25-35
peptide-induced Alzheimer's disease (AD) rat model.
Methods: Rats were injected with Aβ25-35 peptide in bilateral hippocampus CA1 area2). After injection 7 days,
DZXW were administered once daily for 30 days. Learning and memory abilities tests were then performed byMorris water maze. Furthermore, a urinary metabolomics study was carried out based on ultra high-performanceliquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS), andfollowed with multivariate analysis to screen potential biomarkers.
Results: DZXW administration significantly improved dysmnesia in Morris water maze (P＜0.05) compared tothe model group. A total of 28 metabolites were identified as potential biomarkers of AD. The main metabolism
pathway in which DZXW affected on AD rats were taurine and hypotaurine metabolism, citrate cycle metabolism,
purine metabolism, vitamin B6 metabolism and so on. These findings begin to illuminate therapeutic mechanisms
of DZXW upon anti-oxidant protection, energy metabolism regulation, neurons protection and intestinal
microbes improvement against AD.

Fig.1. Correlation networks of main potential biomarkers in response to the therapeutic effects of DZXW on AD
rats

References
1) Lu Xu et al., Journal of Chromatography B., 1026, 36-42 (2016).
2) Min Liu et al., BMC Complementary and Alternative Medicine., 15, 382(2015).

This work is supported by the National Natural Science Foundation of China Key Program (no. 81530094),
National Natural Science Foundation of China (No. 81403077), Natural Science Foundation of Guangdong
Province (No. 2014A030310494).

3A-02-P-1555
Large-scale Identification and Visualization of N-glycome with Primary Structures
Using GlySeeker(School of Chemical Science & Engineering, Tongji University, Shanghai 200092, China)
Kaijie Xiao, Yuyin Han, Zhixin Tian*
Key words: N-glycome, primary structure, identification, visualization, N-glycan databasesearch engine GlySeeker
Aberrant glycosylation has been commonly observed in various types of cancers, and quite afew glycoproteins have been approved by US Food and Drug Administration (FDA) as markers
for early diagnosis; yet, effective markers with satisfactory sensitivity and specificity are stillwidely lacking. Mass spectrometry-based glycomics is the state-of-the-art instrumental
analytical pipeline for high-throughput analysis of N-glycomes.[1-4] With the combination of
CH3I permethylation, PGC enrichment, RPLC-MS/MS analysis and our recently developed Nglycan database search engine GlySeeker based on isotopic envelope fingerprinting [5, 6],large-scale identification and visualization of N-glycomes with primary structures (not onlymonosaccharide composition, but also sequence & linkage) have become possible. In thispresentation, we will present our development of GlySeeker as well as its application incharacterization of differentially expressed N-glycans in hepatocellular carcinoma HepG2 cells(vs. normal liver LO2 cells). The integrated platform can, in principle, be adopted for both
qualitative and quantitative characterization of any N-glycome system.

Fig. 1. Schematic diagram of N-glycome characterization using GlySeeker
References[1] K. Tanabe, et al. J Proteome Res, 15, 2935-2944 (2016).
[2] L. Cheng, et al. Oncotarget, 7, 61199-61214 (2016).
[3] Y. Qin, et al. Glycoconj J, doi 10.1007/s10719-017-9768-5 (2017).
[4] Y. Huang, et al. Electrophoresis, doi 10.1002/elps.201700025 (2017).
[5] K. Xiao, et al. Anal and Bioanal Chem, doi 10.1007/s00216-018-1070-2 (2018).
[6] K. Xiao, et al. Rapid Commun Mass Spectrom, 32, 142-148 (2018).