SR13_004E

S h i m a d z u

R e v i e w

Development of Iodoacetyl-Based Ionic Mass Tags for Improved Sensitivity in the Detection of
Cysteine-Containing Peptides by MALDI-TOF MS
by Takashi SHIMADA1, Hiroki KUYAMA2, Takaaki SATO3, and Koichi TANAKA4

Abstract
We developed and characterized six new ionic mass tags that bond to cysteine residues and enable high-sensitivity analysis of peptides. Each mass
tag molecule was designed with the following structural characteristics: 1) an iodoacetyl group that binds to thiols; 2) hydrophilic properties that
reduce losses due to physical adsorption of the sample; 3) a tertiary amine, quaternary ammonium, or guanidino group to retain strong proton
affinity; and 4) no amide bonding to minimize fragmentation caused by collision-induced dissociation. We analyzed peptides for their MS sensitivity
using the new mass tags and achieved improvements in sensitivity of approx. 2 to 200 times the sensitivity obtained with a carbamide-methylated
peptide control sample. The new ionic mass tags are ideal for analytical mass spectrometry of hydrophobic peptides, low-abundance biomarkers, andcirculating hormones.

1. Introduction

2. Experiments

Living systems contain a wide dynamic range of proteins, and theaccompanying molecular complexity contributes to maintaining themiraculous balance observed in biological phenomena. Overcomingthat complexity is an extremely large challenge for proteomics, which
involves the exhaustive analysis of the proteome1-2). With current MS
technology available, it is difficult to detect proteins present at low
levels amongst highly abundant proteins. Many different techniques
have therefore been developed for the pretreatment of samples
before introduction to the mass spectrometer, including multi-stage
separations and affinity purification3-8). Derivatizations that target
cysteine are the most common form of irreversible modification used
for biochemical binding due to the universal distribution of cysteine
residues in diverse proteomes, the low abundance of the cysteineamino acid, and high chemical reactivity of the thiol group9). To date,a diverse range of reagents that typically contain a maleimide group,iodoacetyl group, or vinyl group have been used to introduce afunctional group with chemical affinity, as crosslinking reagents, influorescent probes as activity markers, and in quantitative analysis. An
in silico analysis that we performed using the human proteins
database registered in the International Protein Index (IPI) found 97 %of human proteins contain at least one cysteine residue, and that 17% of predicted tryptic fragments also contain at least one cysteine
residue. This means that predicting a proteome based only on thefraction of its peptides that contain cysteine could result in awell-described proteome. The results also suggest that samples can be
reduced to about one-fifth of their original complexity without loss ofproteome coverage. The selection of cysteine-containing proteins as
the subject of proteomic analysis is likely to contribute to the field ofproteomics through a bottom-up approach.

2.1 Reagents
All commercially available reagents were used without further
purification. Bovine insulin (MW = 5734), P14R synthetic peptide (MW= 1533), ammonium bicarbonate, and ammonium acetate werepurchased from Sigma-Aldrich (St. Louis, MO, US). NC4 CLAC-P
peptide (collagen-like Alzheimer amyloid plaque component precursor)
641-654 (LGPDGLP-MPG CWQK, MW = 1499) and S26C amyloid-beta
17-40 peptide (LVFFAEDVGC NKGAIIGLMV GGVV, MW = 2409) werepurchased from Anaspec (Fremont, CA, US). Alpha-fetoprotein waspurchased from SCIPAC (London, UK). High-purity
alpha-cyano-4-hydroxycinnamic acid (CHCA) and 2,5-dihydroxybenzoic
acid (DHB) were purchased from Shimadzu GLC (Tokyo, Japan). Other
solvents and reagents were purchased from Wako Pure Chemical
Industries (Osaka, Japan).

(Received December 17, 2012)1

Doctor of Philosophy, Life Science Research Center, Technology Research Laboratory

2

Doctor of Agriculture, Life Science Research Center, Technology Research Laboratory

3

Doctor of Medicine, Life Science Research Center, Technology Research Laboratory

4

Koichi Tanaka Laboratory of Advanced Science and Technology

2.2 Mass Tag Synthesis
For details on the six new cysteine mass tags, see the "References"section 10).
2.3 Labeling Peptides with Mass Tags
Peptides were dissolved in an aqueous solution of 50 % acetonitrile
containing 0.05 % trifluoroacetic acid to a final concentration of 100
pmol/µL. An amount of this solution that contained 200 pmol of
peptide was then added to 10 µL of an aqueous solution of 100 mM
ammonium bicarbonate and 10 mM dithiothreitol, and the resulting
mixture was heated at 56 °C for 60 minutes to reduce the disulfide
bonds. After the reduction reaction ended, 15 µL of a 100 mM
ammonium bicarbonate and 50 mM mass tag reaction solution wasadded to the mixture and stirred gently at room temperature for 45
minutes to allow s-alkylation. Trifluoroacetic acid was then added to a
final concentration of 1 % to end the peptide labeling reaction.
2.4 Mass Spectrometry and Data Analysis
2 pmol of mass tag-labeled peptides was diluted in 0.1 %trifluoroacetic acid, purified using ZipTip C18 (Millipore, Billerica, MA,US) according to a standard protocol, spotted directly onto a stainlesssteel MALDI target, and then air-dried. CHCA was added to an
aqueous solution of 50 % acetonitrile and 0.1 % trifluoroacetic acidto make a CHCA concentration of 5 mg/mL, then P14R was added tothis mixture to a final concentration of 300 fmol/µL.

This matrix solution was then placed over the spotted samples on the
MALDI target. MALDI-TOF MS was performed using the AXIMA
Performance (Shimadzu Corporation, Kyoto, Japan). Data was
acquired in linear positive ion mode using a UV nitrogen laser (337nm) and in automatic measurement mode at an accelerating voltage
of 20 kV and with 300 profiles/run. The intensity of target peptide
peaks was normalized using the peak intensity of P14R, which wasadded as an internal standard. Outliers were discarded by determining
the 35 % trimmed mean of the normalized peak intensity (n = 12).
The resulting spectra were then evaluated statistically.
2.5 Serum Peptidome Analysis
15 µL of blood serum from a seven-year-old C57BL/6N mouse (Charles
River Laboratories, Yokohama, Japan) was denatured in 70 % acetic
acid at 100 °C for 30 minutes. After centrifugation (20,000 g for 60
minutes), the supernatant was filtered through a 0.45 µm
Ultrafree-MC (Millipore), and the resulting sample was separated bygel filtration chromatography on a Superdex Peptide PC 10/300 GL(GE Healthcare, Waukesha, WI, US). Eluent fractions were analyzed
using an aqueous solution of 50 mM ammonium acetate (pH 7) and
20 % acetonitrile at a flowrate of 0.35 mL/min. The peptide fraction
below 6 kDa was collected using the external standards ribonucleaseA (13.7 kDa) and aprotinin (6.5 kDa) and concentrated using acentrifugal evaporator. 5 pmol of each NC4 CLAC-P peptide(carbamide-methylated or mass tag (e)-labeled) was added to the
prepared serum peptidome. Each sample was purified using MonoTip
C18 (GL Sciences, Tokyo, Japan). The serum peptidome was separated
and analyzed using the Prominence nanoLC system (Shimadzu),
MU701 capillary UV-VIS detector (GL Sciences), and AccuSpot MALDI
spotter (Shimadzu). The following HPLC conditions were used:
Eluent A: 0.5 % acetonitrile containing 0.1 % heptafluorobutyric acidEluent B: 80 % acetonitrile containing 0.1 % heptafluorobutyric acid
Gradient program: 1 % B (35 min), 1-12 % B gradient (10 min),12-52 % B gradient (135 min), and 52-100 % B gradient
Column: Presto FF-C18 (non-porous 2 µm silica, 0.5 × 150 mm, Imtakt
Corporation, Kyoto, Japan)
Flowrate: 1.6 µL/min
Interval between MALDI target spots: 20 sec/well
MALDI-TOF MS was performed using the AXIMA Performance. Data
was acquired in linear positive ion mode and in automatic
measurement mode with 600 profiles/run. All MS spectra werenormalized using the peak intensity of the P14R internal standard.

3. Results and Discussion
To date, during our research into the development of functional masstags, we have used mass tags to improve MS sensitivity, efficiently
analyze variability in gene expression, and analyze the amino acidsequence of proteins11-13). This report describes newly developed
cysteine mass tags that can be used as sensitivity enhancers. The
molecular design of these cysteine mass tags has four features, as
illustrated in Fig. 1: (1) an iodoacetyl structure for binding to thiolgroups with high sensitivity and minimizing side reactions with otheramino acid residues; (2) a hydrophilic structure to avoid peptide
recovery losses caused by physical adsorption; (3) tertiary amine,
quaternary ammonium, or guanidine groups to confer strong proton
affinity. (There are already a number of reports on the technique ofincreasing the detection efficiency of MS by introducing a fixed positive
charge to the molecule14-16).); and (4) an ester bond between theiodoacetyl group and positive ion receptor to minimize mass tag
fragmentation due to collision-induced dissociation17). Six cysteine
mass tags were synthesized incorporating these four features in theirmolecular structures (Fig. 2).
Four model peptides (insulin alpha-chain and beta-chain, NC4 CLAC-P,
and S26C beta-amyloid 17-40) were used to optimize the labelingreaction conditions of the synthesized mass tags and verify theirenhancement of the MS signal. The optimal reaction conditions were
45 minutes at room temperature in the dark, during which almost all
reactions with cysteine were completed. MS signals were undetectablefor unreacted peptides and side products derivatized to non-cysteineamino acid residues (Fig. 3).
The sample area of a MALDI target is well known to have "sweet
spots" that generate extremely strong ion signals and "non-sweet
spots" that generate almost no signal—a phenomenon that is reported
to be affected by the state of co-crystallization between the analyte
and the matrix18). Consequently, it must always be bore in mind that
abnormal values will occur when MALDI-TOF MS is used forquantitative analysis. Ionization efficiency also varies substantially withdifferent peptide sequences. We therefore devised a new method forquantitating peptides using MALDI-TOF MS that is composed of threesteps: (1) obtaining multipoint data (n = 12) by normalizing individual
sets of data with an internal standard; (2) discarding outlier peakintensities by using a 35 % trimmed mean; and (3) statistical analysis toevaluate the significance of sensitivity improvements. This analyticaltechnique makes highly accurate quantitative analysis possible using
MALDI-TOF MS. Typical MS spectra are shown in Fig. 4, where P14R isused as the internal standard.

fig 1. Mass tag structure with multi-functional property
R1; NH (amide), O (ester)R2; hydrocarbon (linker), ethyleneglycol (hydrophilic linker)
R3; ter tiar y amine, quater nar y amine, guanidine, triazine (proton acceptor)

(a)

(d)

(b)

(c)

(e)

(f)

Fig 2. Structure of cysteine mass tags with CLogP value of derivatized form(a) DE-IAA, 1-(N,N-diethylamino)-3-iodoacetamidopropane hydroiodide (0.849)(b) TE-IAA, 3-iodoacetamidopropyltriethylammonium iodide (-2.890)(c) DE-IA, 1-(N,N-diethylamino)-3-iodoacetoxypropane hydroiodide (1.857)(d) TE-IA, 3-iodoacetoxypropyltriethylammonium iodide (-1.882)(e) TM-DEG-IA, 8-iodoacetoxy-3,6-dioxaoctyltrimethylammonium iodide (-3.538)(f) Gu-DEG-IA, 8-guanidino-1-iodoacetoxy-3,6-dioxaoctane hydroiodide (-1.091)

(a)

(b)

(c)

(d)

(e)

(f)

(g)

Fig 3. MS spectra of tag-derivatized NC4 CLAC-P peptide
The tags used were : (a) DE-IAA : m/z 1668.81 as [M+H]+ , (b) TE-IAA : 1696.85 as [M]+, (c) DE-IA : 1669.86 as [M+H]+, (d) TE-IA :1697.83 as [M]+, (e) TM-DEG-IA : 1729.70 as [M]+, (f) Gu-DEG-IA : 1729.75 as [M+H]+, and (g) IAA-derivatized NC4 CLAC-P control:
1555.71 as [M+H]+

(a)

(b)

(c)

(d)

(e)

(f)

(g)

fig.4 MS spectra of tag-derivatized NC4 CLAC-P peptide along with P14 R as an internal standard for quantitative peptide
analysis
The tags used were : (a) DE-IAA: m/z 1669.59, (b) TE-IAA: 1697.62, (c) DE-IA: 1670.66, (d) TE-IA: 1698.68, (e)TM-DEG-IA: 1730.66, (f) Gu-DEG-IA: 1730.54, and (g) IAA-derivatized NC4 CLAC-P as a control: 1556.80. Mass
numbers are shown as averaged in this figure.

In terms of improving the sensitivity of MS, the quaternary ammonium
tags (b and d) were more effective than the tertiary amine tags (a andc). Comparing the quaternary ammonium tag (e) and guanidine tag (f)shows the quaternary ammonium tag was more effective. Verifying
these effects using peptides of various amino acid sequences revealed
that the quaternary ammonium tag (e) improves sensitivity mostremarkably among the six mass tags. Table 1 summarizes the results of
the improvements. By using the mass tags developed in this researchfor MS analysis of peptides, we successfully improved sensitivity by astatistically significant 2 to 200 times over the carbamide-methylated
peptide control sample.
Next, we evaluated the sensitivity improvements from using the masstags with fragments from in-gel tryptic digestion of alpha-fetoprotein(AFP). AFP is a known protein biomarker of liver cancer19). Particularattention has been focused on the lectin 3-fraction of AFP (AFP-L3
fraction) as a specific biomarker that distinguishes between liver cancer
and cirrhosis. Table 2 reveals that, even given the fact that the tryptic

digest is a peptide fragment with high ionization efficiency, thesensitivity improvements depend significantly on the amino acidsequence of the peptide. This is probably because the rate of recoveryof the peptide fragment from the gel also depends significantly on its
physicochemical properties, which may substantially affect the absoluteamounts to be used during analysis. The hydrophilic regions of thesynthesized mass tags probably also help improve the rate of recoveryof the peptide from the gel. We also discovered peptide fragments only
detectable when labeled with the new mass tags. In addition, somedata, not shown here, indicated that the new mass tags are also usefulwhen used with peptides having an amino acid sequence that isdifficult to detect with MS due to an absence of a basic amino acid atthe c-terminal, contributing to a dramatic improvement in detention
efficiency. The new mass tags show great potential for thedevelopment of new biomarkers through mass spectrometry.

Table 1 Enhanced sensitivity ratio of mass tag-derivatized peptides
Ratios (enhanced ratio) of tag-derivatized peptides are shown compared to carbamydomethylation (CAM) control withrespective p-values.
Insulin alpha and beta, derived from reductive alkylation of insulin
NC4, NC4 CLAC-P 641-654 (collagen-like Alzheimer amyloid plaque component precursor)
S26C, S26C beta-amyloid 17-40
Tag IDs are described in Figure 2.

Mass Tag ID
Peptide

(a)

(b)

(c)

(d)

(e)

(f)

Ratio

44.2

58.0

18 .5

121 .1

222 .5167.7

p-value

1.40E-09

1.13E-12

3 .59E-11

5.65E-12

3 .22E-06

6 .02E-10

Ratio

6.4

28.2

4.4

58 .1

58 .3

30 .8

p-value

2.92E-09

2 .38E-12

6 .47E-16

2 .54E-12

1.34E-14

4.22E-10

Ratio

1.2

3.0

1.3

25.8

33 .5

12 .4

p-value

9.89E-03

3.42E-09

4 .51E-03

1.08E-08

1.57E-08

2 .17E-07

Ratio

1.4

24.2

1.4

16.6

16 .5

11.1

p-value

0.027

1 .49E-05

0.010

5 .08E-06

7.54E-06

1.94E-05

Insulin alphaInsulin beta

NC4

S26C

Table2 Result of creating calibration curve of VP standard samples calculated from derivative spectrum chromatogram
Each peptide intensity was normalized by highest peak of m/z 1582.9 signal (aa. 348-360 NIFLASFVHE YSR) in the same
MS spectra.
# Cys; Number of cysteine in each peptide sequence
ND*; Not detected
P**;
Detection of probe-coupled peptides, but not detected from control

Position

Cys-peptide sequence of AFP

#Cys

(a)

(b)

(c)

(d)

(e)

(f)

5-42

NEYGIASILD SYQCTAEISLADLATIFFAQ FVQEATYK

1

ND*

ND*

ND*

ND*

ND*

ND*

50-83

DALTAIEKPT GDEQSSGCLENQLPAFLEEL CHEK

2

0.5

0.2

ND*

ND*

1.7

ND*

89-102YGHSDCCSQS EEGR

2

1.4

0.2

ND*

0.0

0.0

ND*

103-110HNCFLAHK

1

2.5

8.9

0.0

1.5

2.5

0.0

111-136

KPTPASIPLF QVPEPVTSCE AYEEDR

1

0.6

0.0

0.2

0.0

0.01

ND*

169-175

IIPSCCK

2

14.6

11.0

0.6

0.0

0.7

ND*

176-186

AENAVECFQT K

1

3.3

20.7

0.0

38.7

29.8

3.1

196-209

ESSLLNQHAC AVMK

1

26.7

97.5

5.3

103.4

44.5

0.8

239-252

LVLDVAHVHE HCCR

2

1.1

0.1

0.0

0.0

0.1

ND*

253-264

GDVLDCLQDG EK

1

4.3

13.9

ND*

31.4

24,8

4.6

265-279IMSYICSQQD TLSNK

1

P**

P**

P**

P**

P**

P**

280-285

ITECCK

2

916.6

962.9

0.8

6.7

538.5

192.3

292-313

GQCIIHAEND EKPEGLSPNL NR

1

0.7

0.05

ND*

0.5

0.8

0.2

365-377

CFQTENPLEC QDK

2

243.7

23.0

44.3

7.9

10.4

31.9

396-402

SCGLFQK

1

2.5

13.8

0.1

3.7

2.3

0.1

435-449

MAATAATCCQ LSEDK

2

P**

P**

P**

P**

P**

P**

450-468

LLACGEGAAD IIIGHLCIR

2

1.0

0.2

0.0

0.0

0.1

0.0

469-489

HEMTPVNPGV GQCCTSSYAN R

2

0.3

0.0

ND*

0.0

0.0

0.0

490-511RPCFSSLVVD ETYVPPAFSD DK

1

0.8

0.2

0.3

ND*

ND*

ND*

517-530

DLCQAQGVAL QTMK

1

2.6

15.1

ND*

18.4

18.8

1.4

563-579

CCQGQEQEVC FAEEGQK

3

1.5

0.7

ND*

0.0

0.1

0.2

We also analyzed serum peptidome to verify how much sensitivity isimproved by using the mass tags for proteomics. Diagnostic techniques
employing multiple biomarkers in blood serum and blood plasma holdgreat potential as clinical applications of mass spectrometry, and of
those techniques, peptidome analysis is being increasingly recognized
as most effective in searching for biomarkers20). The results of thepeptidome analysis are shown in Fig. 5. The improvements in sensitivity
were verified by comprehensively analyzing about 700 serum peptides,
using an internal standard to obtain definite quantitative results, andadding a carbamide-methylated peptide control sample and peptides
labeled with mass tag (e). The results demonstrated that sensitivity isimproved by labeling peptides with the mass tags even in an extremelycomplex sample such as serum peptidome.
The mass tag (e) likely achieves high sensitivity in the mass spectrometric analysis of peptides by improving peptide recovery rates due to its
hydrophilic linker and by improving sensitivity due to the attachment of
positive ions to peptide molecules. The peptides NC4 and S26C play arole in Alzheimer's disease, which is caused partly by tissue fibrosis andplaque formation21). Tissue fibrosis, in particular, is an important

histopathological indication associated with progression of thedisease22). The new mass tags can be used in the high-sensitivity
analysis of clinically valuable molecules with a hydrophobic domain
susceptible to fibrosis. Also, peptides of biological or clinicalsignificance are often hormones that circulate in the bloodstream, suchas insulin, natriuretic hormone, and growth factors, or peptide
fragments that have undergone disease-specific cleavage23). These
peptides are present in very small quantities and often contain astrongly hydrophobic surface as a result of their unique steric structure.
Some of them also have no c-terminal basic amino acid, which makes ahigh-sensitivity analysis difficult to perform. The further development ofmethods that improve the sensitivity of peptide analysis with mass
spectrometry will lead to the development of valuable biomarkers andthe discovery of unknown biological systems. We consider cysteinecontaining peptides, in particular, to be important candidates for
detection, and anticipate that they will find application in quantitative
analysis by means of stable isotope labeling or specific-fragment
indices24).

(b)

(a)

Serum peptidome fraction
RT［min］(d)(c)

NC4 / IAA

Relative intensity (NC4/tag(e))

NC4 / tag(e)Relative intensity (NC4/IAA)

Fig 5. Enhanced sensitivity by tag-derivatized peptide in serum peptidome(a) UV (220 nm)-absorption spectra of serum protein by size exclusion chromatography (SEC). Solid line shows the profile of C57B6-7w
mice serum, and dotted is the molecular weight standard. The peak of ribonuclease A (Ribo) and aprotinin (Aprot) are indicated by arrows. Serum peptidome fraction is shown as side arrows.
(b) UV (220 nm)-absorption spectra of serum peptide fraction by reverse-phase chromatography. Left axis shows the UV-absorption andright is the percentage of solvent B concentration. Solid and dotted lines are the profile of serum peptidome spiked with NC4/IAA peptide and NC4/tag (e) peptide, respectively. The spotting point on MALDI target is from 65 min to 195 min for 20 sec/well interval.
(c) Scatter plot of peptide intensity from serum peptidome analysis. Horizontal and vertical axes indicate peptide intensity from peptidome
spiked with NC4/IAA peptide and NC4/Tag-e peptide, respectively.
(d) Representative MS spectra of IAA-derivatized NC4 peptide and tag (e)-derivatized NC4. Enhanced sensitivity ratio by tag (e) derivatization is about 6.2-fold.

4. Conclusion
We carried out the molecular design and synthesis of six new
cysteine-binding ionic mass tags to improve the sensitivity of peptide
analysis by mass spectrometry. Of these, mass tag (e), with aquaternary ammonium and hydrophilic linker, caused the greatest
increase in sensitivity. The six novel mass tags are expected to be
applicable in MS analysis of hydrophobic peptides, low-abundancebiomarkers, and circulating hormones.
This research is supported by the Japan Society for the Promotion of
Science through its "Funding Program for World-Leading Innovative
R&D on Science and Technology" (FIRST Program). Cysteine mass tag
synthesis was performed with the support of Dr. Katsuya Noguchi(Dojindo Molecular Technologies).

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