Synthesis of 13C-labeled iodoacetanilide and application to quantitative peptide analysis by isotope differential mass spectrometry

Article abstract of DOI:10.1016/S0960-894X(03)00503-1

¹³C-Labeled and unlabeled iodoacetanilides have been synthesized for covalent modification of the sulfhydryl groups of cysteine residues in proteins or peptides. A combination of these reagents, coupled with mass spectrometry, is a powerful tool for quantitative analysis of peptides and hence proteins.

Full text of DOI:10.1016/S0960-894X(03)00503-1

Bioorganic & Medicinal Chemistry Letters 13 (2003) 2913–2916
Synthesis of 13C-Labeled Iodoacetanilide and Application to
Quantitative Peptide Analysis by Isotope Differential Mass
Spectrometry
Satomi Niwayama,a,* Sadamu Kuronob and Hiroyuki Matsumotob,*
aDepartment of Chemistry, Oklahoma State University, Stillwater, OK 74078-3071, USA
bDepartment of Biochemistry and Molecular Biology, NSF EPSCoR Oklahoma Biotechnology Networtk Laser
Mass Spectrometry Facility, University of Oklahoma Health Sciences Center,
Oklahoma City, OK 73190, USA
Received 26 February 2003; accepted 28 April 2003
Abstract—13C-Labeled and unlabeled iodoacetanilides have been synthesized for covalent modification of the sulfhydryl groups of
cysteine residues in proteins or peptides. A combination of these reagents, coupled with mass spectrometry, is a powerful tool for
quantitative analysis of peptides and hence proteins.
# 2003 Elsevier Ltd. All rights reserved.
Quantitative analysis of proteins is an essential part of
proteomics, which studies proteomes, a set of proteins
expressed under certain physiological conditions.
Examples of conventional methods for quantification
include densitometry of the gel and counting radio-
activities that are incorporated by metabolic labeling.1À3
Recently, stable isotope labeling followed by mass
spectrometry analysis has been emerging as a powerful
technology for more accurate quantification of proteins.
This technique is based on the assumption that the iso-
topically labeled molecule and its parent species behave
similarly and their ionization efficiencies are the same.4
Most common and classical approaches have relied on
incorporation of specific isotope atoms such as 15N,
18O, or D into growing cells in the isotope-enriched
media or into the digested peptides during the proteo-
lytic cleavage of proteins. However, such methods
require a long processing time and are laborious, and
may not be applicable to more complex systems such as
whole animals. In contrast, chemical modifications by
covalent labeling on specific amino acid residues using
isotope-labeled reagents followed by mass spectrometry
analysis is versatile and convenient, as it can handle any
protein extracts. The pioneering work by Aebersold et
al. utilized deuterium-labeled isotope-coded affinity tags
(ICATs).5 Their method applies a biotinylated con-
jugate of iodoacetamide, which is known to be a specific
sulfhydryl group modifier in cysteine residues in pep-
tides, and its deuterated derivative. In this method, the
biotinyl moiety is used to affinity-purify the resulting
peptides that are carrying the modified cysteine residues,
making the analysis of whole proteins feasible. If, how-
ever, each protein is separated and displayed on a two-
dimensional (2-D) gel electrophoresis, the affinity puri-
fication of the cysteine-modified peptides will be unne-
cessary, making the entire protocol significantly simpler
compared to the ICAT method. We have developed a
prototype of such a method using isotope-labeled
alkylmaleimides.6 The combination of isotope-labeled
and unlabeled chemical modification of specific amino
acid residues followed by 2-D gel electropheresis and
matrix-assisted laser desorption/ionization time-of-flight
mass spectrometry (MALDI-TOF MS) makes the
whole process handier and more economical compared
to the ICAT method.
Here, we report synthesis of another sulfhydryl-group-
specific modifier, 13C6-labeled and unlabeled iodoaceta-
nilides (IAA), and the application of this modifier to
quantitative analysis of peptides in the context of our
proteomics research. We believe that the success of this
work signifies success in quantification of tryptic
peptides, hence proteins (Scheme 1).
*Corresponding author. Tel.: +1-405-744-6594; fax: +1-405-744-
6007; e-mail: niwayama@biochem.okstate.edu (S. Niwayama); tel.:
+1-405-271-2227; fax: +1-405-271-3139; e-mail: hiro-matsumoto@
ouhsc.edu (H. Matsumoto).
0960-894X/03/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/S0960-894X(03)00503-1

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S. Niwayama et al. / Bioorg. Med. Chem. Lett. 13 (2003) 2913–2916
SDTCSSQKTEVSTVSSTQK, 2001.92 Da and 6.2 for
MAT 13, and ALVCEQEAR, 1017.49 and 4.4 for MAT
60. Aqueous solutions of these peptides with pH values
9.0 were treated with these labeled or unlabeled iodo-
acetanilides, and the ion peaks of these solutions were
analyzed by MALDI-TOF MS after adding a matrix.
The following charts (Fig. 1) show MAT 31 by itself
and MAT 31 reacted with unlabeled iodoacetanilide or
labeled iodoacetanilide.11 The ion peaks show the
monoisotopic mass for MAT 31 (1746.77 Da) and a
series of isotope peaks that are several Da higher than
the monoisotopic peak because of the existence of nat-
ural isotopes. The labeled and unlabeled iodoacetani-
lides completely reacted with this peptide within 10 min.
After reacting with the unlabeled or labeled iodoaceta-
nilide, the molecular weight of the IAA-modified or
13C6-IAA-modified MAT 31 increased by 133 or 139
Da, respectively, showing that a combination of these
modified peptides displays 6 Da difference.
Scheme 1.
Iodoacetanilide possesses the same reaction site toward
–SH as does iodoacetamide, the well-known cysteine-
specific modifier. These reagents are expected to be
more reactive than N-alkylmaleimide, they are expected
not to interfere with phosphine-based reducing
reagents,7 and the difference in the molecular mass is 6
Da rather than 5 Da; therefore, the combination of
13C6-labeled and unlabeled reagents is expected to pro-
vide sharper separation than that by N-alkylmaleimide
modification and hence more accurate quantification.
In the next step, in order to examine the applicability of
these reagents to quantification of peptides, several
aqueous MAT 31 solutions were prepared with pH 8.5
and these solutions were individually treated with
labeled or unlabeled iodoacetanilide. Then the differen-
tially labeled MAT 31 solutions were mixed. The ratios
of IAA-MAT 31 to 13C6-IAA-MAT 31 examined were
9:1, 6:1, 3:1, 1:1, and 1:3.12 The relative quantities of
MAT 31 in each mixed solution were measured from the
relative signal intensities for pairs of peptide ions
modified with labeled or unlabeled iodoacetanilide.
Synthesis of iodoacetanilide from aniline and iodoacetic
acid has been reported.8 Accordingly, 13C6-labeled
iodoacetanilide was synthesized from 13C6-labeled
aniline9 and iodoacetic acid (Scheme 2).10
The following charts show the MALDI MS spectrum
for each mixture (Fig. 2). The areas of the monoisotopic
peak and the following four isotopic peaks were sum-
med for each modified peptide. The areas of the small
7–10th isotopic peaks of IAA-MAT 31 were subtracted
from the monoisotopic peaks and the following high
isotopic peaks of 13C6-IAA-MAT 31. The relative ratios
of MAT 31 in the five sets of the two solutions obtained
in these experiments were plotted against their theoreti-
cal ratios in the following graph (Fig. 3).
Scheme 2.
We tested the reactivity of these reagents with three
synthetic peptides that possess different amino acid
sequences and molecular weights. We chose peptides
that have approximately 2000 Da or less, as the mole-
cular weights for most tryptic peptides associated with
proteomics are in this range. The three peptides are
MAT 31, MAT 13, and MAT 60, and the sequences,
monoisotopic masses, and estimated pIs are KEEPPH
HEVPESETC, 1746.75 Da, 4.5 for MAT 31,
The graph obtained in this way indicates that the
observed ratios and the theoretical ratios for 13C-labeled
Figure 1. MALDI-TOF MS spectra of MAT 31 andiodoacetanilide-modified MAT 31.

S. Niwayama et al. / Bioorg. Med. Chem. Lett. 13 (2003) 2913–2916
2915
Figure 2. MALDI-TOF MS spectra of IAA-modified/13C6-IAA-modified MAT 31 solutions with varied concentrations.
Figure 3. Quantitative analysis of MAT 31.
Figure 4. Quantitative analysis of MAT 13.
and unlabeled IAA-modified MAT 31 in the two solu-
tions show excellent correlation (r2=0.9995, inclina-
tion=1.0223). Therefore, as had been reported for d-
labeled and unlabeled N-alkylmaleimide modified pep-
tides earlier,6a we conclude that the ionization effi-
ciencies of the 13C-labeled IAA-MAT 31 and those for
unlabeled IAA-MAT 31 are the same within the experi-
mental error.
IAA-modified MAT 13 and MAT 60 are also well cor-
related (r2=0.9980 and 0.9995, inclination=0.9615 and
0.9789 respectively). These results also indicate that the
ionization efficiencies of the 13C-labeled and unlabeled
IAA-modified MAT 13 and MAT 60 are the same
within the experimental error. We safely conclude that
the relative molar ratio of the peptide in two different
sample solutions can be measured at a high accuracy by
this method.
We next applied this method to other peptides, MAT 13
and MAT 60, in order to demonstrate the general-
izability of this method. The results are shown in
Figures 4 and 5.
In summary, we developed a new method for quantita-
tive analysis of peptides for the purpose of quantification
of proteins. We synthesized 13C6-labeled iodoacetanilide
and have shown, as a model system for quantification of
proteins, that a combination of 13C6-labeled and
unlabeled iodoacetanilide enables quantitative analysis
As can be seen from these figures, the theoretical and
observed relative ratios for 13C-labeled and unlabeled

2916
S. Niwayama et al. / Bioorg. Med. Chem. Lett. 13 (2003) 2913–2916
2002, 2, 513. (j) Zhang, R.; Sioma, C. S.; Wang, S.; Regnier,
F. E. Anal. Chem. 2001, 73, 5142. (k) Yao, X.; Freas, A.;
Ramirez, J.; Demirev, P. A.; Fenseleau, C. Anal. Chem. 2001,
73, 2836. (l) Gu, S.; Pan, S.; Bradbury, E. M.; Chen, X. J. Am.
Soc. Mass Spectrom. 2003, 14, 1.
5. Gygi, S. P.; Rist, B.; Gerber, S. A.; Turecek, F.; Gelb,
M. H.; Aebersold, R. Nat. Biotechnol. 1999, 17, 994.
6. (a) Niwayama, S.; Kurono, S.; Matsumoto, H. Bioorg.
Med. Chem. Lett. 2001, 11, 2257. (b) Kurono, S.; Niwayama,
S.; Matsumoro, H. In American Society for Mass Spectro-
metry Conference, Mass Spectrometry and Allied Topics,
Orlando, FL, 2002. (c) Kurono, S.; Niwayama, S.; Matsu-
moro, H., 91st Annual Technical Meeting of the Oklahoma
Academy of Science, Oklahoma City, OK, 2002.
7. Getz, E. B.; Xiao, M.; Chakrabarty, T.; Cooke, R.; Selvin,
P. R. Anal. Biochem. 1999, 273, 73.
8. Benesch, R.; Edalji, R.; Benesch, R. E. J. Biochem. Biophys.
Methods 1979, 1, 129.
9. The 13C6-labeled aniline was purchased from Aldrich, Mil-
waukee, WI, USA.
10. The procedure for synthesis of 1 is as follows. Iodoacetic
acid (470 mg, 2.53 mmol) was dissolved in ethyl acetate and
the mixture was cooled to 0 ꢀC. To this solution, 13C6-labeled
aniline (250 mg, 2.53 mmol) was added, and subsequently a
solution of dicyclohexylcarbodiimide (520 mg, 2.53 mmol) was
added slowly with stirring. A white precipitate formed imme-
diately after addition of the dicyclohexylcarbodiimide. The
mixture was stirred at 0 ꢀC for 30 min and at rt for an hour.
The dicyclohexylurea was removed by Celite filtration and the
filtrate was evaporated to dryness and purified by silica gel
column chromatography (CH2Cl2–CHCl3=1/1 then CH2Cl2)
to afford the product (638 mg, 95% yield). The product was
recrystallized from CHCl3 (a yellowish solid, mp140–141 ꢀC).
1H NMR (300MHz, acetone-d6) d 9.51 (1H, br. s), 7.9–6.7
(5H, m), 3.88 (2H, s), 13C NMR (75 MHz, acetone-d6) 167.0,
140.0, 129.6, 124.5, 119.9, 0.71, HRMS m/z calcd for
12C213C6H8INO (M+H)+: 267.9933, found: 267.9933.
11. The MAT31 aqueous solution (0.6 mM, 2 mL), 2 mL of 50
mM TRIZMA1 Pre-set crystals pH 9.0 (Sigma Chemical Co.,
St Louis, MO, USA) and 2 mL of 10 mM IAA or 13C6-IAA
DMSO solution were mixed to approximately pH 8.5. This
mixture was incubated for 10 min at room temperature. Then,
1 mL of a-cyano-4-hydroxycinnamic acid (CHCA) matrix
solution (prepared by dissolving 10 mg of CHCA in 1 mL of
50% acetonitrile/0.1% trifluoroacetic acid) was added to 1 mL
of the sample solution, and 1 mL of this mixture was analyzed
by MALDI-TOF MS after evaporation of the solvent in
vacuo. The MALDI spectra were obtained from a Voyager
Elite BioSpectrometry Research Station, equipped with a
delayed extraction option (Applied Biosystems, Foster City,
CA, USA) operated at accelerating voltage, 20 kV; grid voltage,
74–75%; guide wire voltage, 0.05%; pulse delay time, 100–225
ns; vertical scale, 3000 mV; and vertical offset, 3%. A pulsed
nitrogen laser operating at 337 nm was used as a desorption/
ionization source. Mass spectrometry was performed in a reflec-
tor with positive ion detection. The ion signal was recorded using
a 500 MHz transient digitizer. The data were analyzed using
GRAMS/386 (Galactic Industries Corp., Salem, NH, USA).
12. The aqueous solution of MAT 31 (0.6 mM) was used for
quantitative analysis. The solution was incubated with an
excess amount of unlabeled or 13C6-labeled IAA DMSO solu-
tion for 10 min at room temperature, and the two solutions
were mixed with peptide molar ratios of 9:1, 6:1, 3:1, 1:1 or
1:3, and were subjected to MALDI-TOF MS analysis as
described in ref 11. Five data points were collected for analysis
of each ratio, and these values were averaged to be plotted on
the graph. Aqueous solutions that have the same concen-
tration (0.6 mM) were used for quantitative analysis of two
other peptides, MAT 13 and 30, as well.
Figure 5. Quantitative analysis of MAT 60.
of a variety of peptides with the molecular weight
1.0–2.0 kDa.
Acknowledgements
This work was supported by grants from the Elsa U.
Pardee Foundation and the National Institute of Health
(EY13877, EY06595, EY12190, and RR17703). All the
synthetic peptides described here were prepared by Dr.
Ken Jackson at the Molecular Biology Resource Facility,
University of Oklahoma Health Sciences Center. The
synthetic peptide, MAT 31, was a gift from Dr. Naoka
Komori, University of Oklahoma Health Sciences Center.
References and Notes
1. Matsumoto, H.; Kurien, B.; Takagi, Y.; Kahn, E. S.;
Kinumi, T.; Komori, N.; Yamada, T.; Hayashi, F.; Isono, K.;
Pak, W. L.; Jackson, K. W.; Tobin, S. L. Neuron 1994, 12,
997.
2. Komori, N.; Usukura, J.; Matsumoto, H. J. Cell Sci. 1992,
102, 191.
3. Gygi, S. P.; Rochon, Y.; Franza, B. R.; Aebersold, R. Mol.
Cell. Biol. 1999, 19, 1720.
4. (a) For example Oda, Y.; Huang, K.; Cross, F. R.; Cow-
burn, D.; Chait, B. T. Proc. Natl. Acad. Sci, U.S.A. 1999, 96,
6591. (b) Veenstra, T. V.; Martinovic, S.; Anderson, G.; Pasa-
Tolic, L.; Smith, R. J. Am. Soc. Mass Spectrom. 2000, 11, 78.
(c) Pasa-Tolic, L.; Jensen, P. K.; Anderson, G. A.; Lipton,
M. S.; Peden, K. K.; Martinovic, S.; Tolic, N.; Bruce, J. E.;
Smith, R. D. J. Am. Chem. Soc. 1999, 121, 7949. (d) Mirgor-
odskaya, O. A.; Kozmin, Y. P.; Titov, M. I.; Korner, R.;
Sonksen, C. P.; Roepstorff, P. Rapid Commun. Mass Spec-
trom. 2000, 14, 1226. (e) Chen, X.; Smith, L. M.; Bradbury,
E. M. Anal. Chem. 2000, 72, 1134. (f) Geng, M.; Ji, J.; Regnier,
F. E. J. Chromatgr. A 2000, 870, 295. (g) Wang, S.; Regnier,
F. E. J. Chromatgr. A 2001, 924, 345. (h) Sechi, S. Rapid
Commun. Mass Spectrom. 2002, 16, 1416. (i) Smith, R. D.;
Anderson, G. A.; Lipton, M. S.; Pasa-Tolic, L.; Shen, Y.;
Conrads, T. P.; Veenstra, T. D.; Udseth, H. R. Proteomics