Synthesis of d-labeled and unlabeled benzoyloxysuccinimides and application to quantitative analysis of peptides and a protein by isotope differential mass spectrometry

Article abstract of DOI:10.1016/j.bmcl.2011.05.090

Benzoyloxysuccinimide and its d₅-labeled version, which react with amino groups in the N-termini and lysine side chains in proteins, were synthesized and applied to quantitative analysis of peptides and a commercially available protein in combi

Full text of DOI:10.1016/j.bmcl.2011.05.090

Bioorganic & Medicinal Chemistry Letters 21 (2011) 4629–4632
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis of d-labeled and unlabeled benzoyloxysuccinimides and application
to quantitative analysis of peptides and a protein by isotope differential
mass spectrometry
⇑
Masoud Zabet-Moghaddam , Aarif L. Shaikh, Lauren B. Jones, Satomi Niwayama
Department of Chemistry and Biochemistry, Texas Tech University, Lubbock, TX 79409-1061, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Benzoyloxysuccinimide and its d5-labeled version, which react with amino groups in the N-termini and
lysine side chains in proteins, were synthesized and applied to quantitative analysis of peptides and a
commercially available protein in combination with a MALDI mass spectrometer.
Ó 2011 Elsevier Ltd. All rights reserved.
Received 10 February 2011
Revised 21 May 2011
Accepted 23 May 2011
Available online 30 May 2011
Keywords:
Amino group modifier
Stable isotope-labeling
Proteomics
Mass spectrometry
Proteomics is becoming an important research area in the
study of proteomes, a set of proteins expressed under specific
physiological conditions, for example, the examination of
comprehensive protein expression patterns expressed under
different external stimuli. In particular, quantitative analysis of
relative amounts of expressed proteins and identification of
the proteins in combination with mass spectrometry are central
in proteomics studies. Many research groups have reported
methodologies for quantitative analysis of proteins or peptides
in this context, in particular, in the incorporation of stable
isotopes. These methods include metabolic labeling,1 enzyme
labeling,2 and chemical modifications.3 Among these methods,
chemical modification is expected to be applicable to any protein
samples, including mammalian proteins. Most of these methods
of chemical modification rely on liquid chromatography (LC) for
separation and purification of the proteins and on subsequent
mass spectrometry analysis for identification and quantitative
analysis of the peptides, exemplified by the ICAT method and
its derivatives.
versions. Small organic molecules are expected to have greater
solubility in sample solutions. Furthermore, we have thus far not
observed isotope effects in our methods. We have reported four
kinds of modifiers and their d- or 13C-labeled versions that
specifically react with the sulfhydryl group of cysteine residues,
and have demonstrated that all these reagents can successfully
measure the relative amounts of peptides or proteins in
combination with electrophoresis and soft ionization mass
spectrometry such as MALDI or ESI.4–9 However, these methods
rely on the existence of cysteine residues. Since some proteins do
not contain cystein residues, there is a need to develop a method
for noncysteine-containing proteins. Here we report synthesis of
a new modifier that reacts with the amino group and its d5-labeled
version and their application to quantitative analysis of peptides
and a protein in combination with MALDI TOF MS.
N-hydroxysucciniminyl esters are among the most commonly
used compounds for modification of amino groups in peptides
(Scheme 1). Münchbach et al. also reported 1-(nicotinoyloxy)
succinimide and its deuterated versions for quantitative analysis
of proteins in combination with electrophoresis,10 and later other
groups also reported derivatives of (nicotinoyloxy)succinimide.11
Although these nicotinoylating reagents are also small organic
molecules, the difference between the isotope-labeled and
unlabeled versions are 4 Da or less. In our case, we can introduce
However, due to the problems associated with LC separation,
such as isotope effects, at our laboratory we have been developing
a practical methodology that makes use of electrophoresis rather
than LC, as well as small organic molecules that specifically react
with certain amino acid residues and their stable isotope-labeled
a
5 Da difference, which potentially allows more accurate
quantitative analysis. In addition, benzoic acid and deuterated
benzoic acid are far less expensive than nicotinic acid and
deuterated nicotinic acid, thus making our reagents more
accessible.
⇑
Corresponding author. Tel.: +1 806 742 3118; fax: +1 806 742 1289.
E-mail address: satomi.niwayama@ttu.edu (S. Niwayama).
Present address: Center for Biotechnology and Genomics, Texas Tech University,
Lubbock, TX 79409-1061, USA
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.05.090

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M. Zabet-Moghaddam et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4629–4632
O
1296.730
O
O
D
D
O
D
O N
O N
angiotensin I
D
D
O
O
d -benzoyloxysuccinimide
benzoyloxysuccinimide
(BSI)
5
(d -BSI)
5
1
2
angiotensin I
+ BSI
1400.766
O
O
- NHS
104 Da
O N
O
O
D
5
peptide
NH
peptide
NH
2
1405.787
angiotensin I
D
5
+
+ d -BSI
109 Da
O
5
- NHS
HO N
O
Scheme 1. D5-benzoyloxysuccinimide, benzoyloxysuccinimide, and their reactions
with an amino group.
Figure 1. MALDI MS spectra of angiotensin I and benzoyloxysuccinimide(BSI)-
modified angiotensin I.
D5-labeled benzoyloxysuccinimide (BSI), 1, and unlabeled
benzoyloxysuccinimide (BSI), 2, were synthesized by a coupling
reaction of benzoic acid and N-hydroxysuccinimide (NHS) by
modification of a known procedure (Scheme 2).12,13
We next examined the general applicability of these reagents to
quantitative analysis of peptides.15 Several aqueous solutions of
the peptide, angiotensin I, were prepared with a pH of 8.5, and
these aqueous solutions were treated with d5-labeled or unlabeled
benzoyloxysuccinimide, 1 or 2, and an aqueous hydroxylamine
solution was added to this mixture. The differentially labeled
angiotensin I solutions were then mixed in the ratio of 0.5, 1, 2,
4, 6, and 9 (d0/d5). The relative quantities of this peptide in each
mixed solution were measured from the relative intensities of
the signals corresponding to monoisotopic peaks for the peptide
ions modified with d5-labeled or unlabeled benzoyloxysuccinimide.
The relative ratios of BSI-modified and d5-BSI-modified peptides
observed in this way were plotted against the theoretical ratios
as in the graph. An excellent correlation between the theoretical
ratios and the observed ratios is shown here (Fig. 2a).
O
O
O
O
DCC
THF
r.t.
OH
+ HO N
O N
O
O
D
D
5
5
1 or 2
Scheme 2. Synthesis of d5-benzoyloxysuccinimide and benzoyloxysuccinimide.
In order to test the applicability of the combination of these
reagents to quantitative analysis of peptides, solutions of two model
peptides, angiotensin I (human) and ACTH 18-39, were prepared,
and the amino modifiers, 1 and 2, were reacted with these peptides.
The amino acid sequences and molecular weights of these peptides
are DRVYIHPFHL, 1296.7 Da, and RPVKVYPNGAEDESAEAFPLEF,
2465.6 Da, respectively.14 Various aqueous solutions of these model
peptides with pH 8.5 were reacted with d5-labeled or unlabeled
reagents and the relative molar ratios were quantitatively analyzed
by MALDI TOF MS.
In order to further examine the general applicability of these
reagents, we applied this method to quantitative analysis of
another peptide, ACTH 18–39, in the same way, and plotted the
observed relative ratios against the theoretical relative ratios. This
peptide has one lysine residue which has an e-amino group, and
therefore the reactions with these modifiers increased 208 and
218 Da, respectively, but the results were similar to those of angio-
tensin I. As can be seen in the following graphs, there is excellent
correlation between the theoretical ratios and the observed ratios
(Fig. 2b).
The following charts show a peptide, angiotensin I itself, and
angiotensin I reacted with the d-unlabeled or d5-labeled modifier
( Fig. 1). Due to the natural isotopes, the ion peaks show the
monoisotopic mass as well as a series of isotopic peaks several
Daltons greater than it. As this peptide does not contain a
lysine residue, this reaction increased 104 Da as a result of the
modification of the terminal amino group, although the reagents
Next we applied the combination of these reagents to quantitative
analysis of a commercially available protein, bovine serum albumin
(BSA), after the cysteine residues were blocked with a cysteine
modifier, iodoacetamide.16 Several solutions with differing amounts
of BSA were prepared and loaded in each well of 1D
electrophoresis. The proteins were excised and in-gel digested, and
the extracted peptide solutions were modified with the d5-labeled
or unlabeled modifier. Six peptides were identified from the mixtures
in this way. The d5-labeled and unlabeled peptides were mixed in
various ratios and quantitatively analyzed in the same manner as
the peptides above, and the averages of the observed ratios were
plotted against theoretical ratios. As can be seen from the graph
below, good correlation between the theoretical ratios and observed
ratios was obtained in accordance with the results obtained from the
two model peptides (Fig. 3).
can react with both the N-terminal amino group and the e-amino
group of the lysine at this pH. The d5-labeled version added
109 Da. Although reactions with other amino acid residues such
as tyrosin are commonly reported for other N-hydroxysuccinimide,
these esters were hydrolyzed by addition of hydroxylamine. In
addition, reactions with these reagents did not diminish the
ionization efficiencies of the peptide, even though the benzoyl
group from these modifiers does not contain a nitrogen atom.

M. Zabet-Moghaddam et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4629–4632
4631
quantitative analysis of peptides and proteins with high accuracy.
As with other reagents we reported earlier that specifically modify
cysteine residues, we did not observe any isotope effects. Although
a decrease in the ionization efficiencies as a result of blocking the
amino groups may be anticipated, we did not observe a decrease in
ionization efficiencies in the model peptides or the commercial
protein reported here. While addition of hydroxylamine for
removal of excessive reactions with hydroxyl groups is required
for these modifiers, they do not require the existence of particular
amino acid residues and are thus expected to be applicable to
quantitative analysis of a wider variety of proteins. These modifiers
are prepared relatively inexpensively from readily available
sources. We are developing additional kinds of amino-group
modifiers and the results will be reported in due course.
Acknowledgments
This work is supported by the Texas Tech University-Texas Tech
University Health Sciences Center Joint Initiative Grant. We also
thank the Center for Biotechnology and Genomics, Texas Tech
University for allowing us to use the research facilities.
References and notes
1. For example (a) Gouw, J. W.; Tops, B. B. J.; Mortensen, P.; Heck, A. J. R.;
Krijgsveld, J. Anal. Chem. 2008, 80, 7796; (b) Colzani, M.; Schutz, F.; Potts, A.;
Waridel, P.; Quadroni, M. Mol. Cell. Proteomics 2008, 7, 927; (c) Hebeler, R.;
Oeljeklaus, S.; Reidegeld, K. A.; Eisenacher, M.; Stephan, C.; Sitek, B.; Stuehler,
K.; Meyer, H. E.; Sturre, M. J. G.; Dijkwel, P. P.; Warscheid, B. Mol. Cell.
Proteomics 2008, 7, 108; (d) Hathout, Y.; Flippin, J.; Fan, C.; Liu, P.; Csaky, K. J.
Proteome Res. 2005, 4, 620; (e) Ong, S.-E.; Blagoev, B.; Kratchmarova, I.;
Kristensen, D. B.; Steen, H.; Pandey, A.; Mann, M. Mol. Cell. Proteomics 2002, 1,
376; (f) Myint, K. T.; Aoshima, K.; Tanaka, S.; Nakamura, T.; Oda, Y. Anal. Chem.
2009, 81, 1121; (g) Gouw, J. W.; Krijgsveld, J.; Heck, A. J. R. Mol. Cell. Proteomics
2010, 9, 11; (h) Collier, T. S.; Sarkar, P.; Franck, W. L.; Rao, B. M.; Dean, R. A.;
Muddiman, D. C. Anal. Chem. 2010, 82, 8696; (i) Jehmlich, N.; Fetzer, I.; Seifert,
J.; Mattow, J.; Vogt, C.; Harms, H.; Thiede, B.; Richnow, H.-H.; von Bergen, M.;
Schmidt, F. Mol. Cell. Proteomics 2010, 9, 1221.
2. (a) Yao, X.; Freas, A.; Ramirez, J.; Demirev, P. A.; Fenselau, C. Anal. Chem. 2001,
73, 2836; (b) Mirgorodskaya, O. A.; Kozmin, Y. P.; Titov, M. I.; Körner, R.;
Sönksen, C. P.; Roepstorff, P. Rapid Commun. Mass Spectrom. 2000, 14, 1226; (c)
Sakai, J.; Kojima, S.; Yanagi, K.; Kanaoka, M. Proteomics 2005, 5, 16.
3. For example, (a) Gygi, S. P.; Rist, B.; Gerber, S. A.; Turecek, F.; Gelb, M. H.;
Aebersold, R. Nat. Biotechnol. 1999, 17, 994; (b) Ross, P. L.; Huang, Y. N.;
Marchese, J. N.; Williamson, B.; Parker, K.; Hattan, S.; Khainovski, N.; Pillai, S.;
Dey, S.; Daniel, S.; Purkayastha, S.; Juhasz, P.; Martin, S.; Bartlet-Jones, M.; He,
F.; Jacobson, A.; Pappin, D. J. Mol. Cell. Proteomics 2004, 3, 1154; (c) Xia, B.;
Feasley, C. L.; Sachdev, G. P.; Smith, D. F.; Cummings, R. D. Anal. Biochem. 2009,
387, 162; (d) Xiang, F.; Ye, H.; Chen, R.; Fu, Q.; Li, L. Anal. Chem. 2010, 82, 2817;
(e) Thompson, A.; Schafer, J.; Kuhn, K.; Kienle, S.; Schwarz, J.; Schmidt, G.;
Neumann, T.; Hamon, C. Anal. Chem. 2003, 75, 1895; (f) Storme, M. L.; van
Bocxlaer, J. F. Handbook on Mass Spectrometry 2009, 113; (g) Zhou, H.; Ranish, J.
A.; Watts, J. D.; Aebersold, R. Nat. Biotechnol. 2002, 19, 512; (h) Goshe, M. B.;
Conrads, T. P.; Panisko, E. A.; Angell, N. H.; Veenstra, T. D.; Smith, R. D. Anal.
Chem. 2001, 73, 2578; (i) Qiu, Y.; Sousa, E. A.; Hewick, R. M.; Wang, J. H. Anal.
Chem. 2002, 74, 4969.
Figure 2. Quantitative analysis of two model peptides. (a) Quantitative analysis of
angiotensin I. (b) Quantitative analysis of ACTH 18-39.
4. Niwayama, S.; Kurono, S.; Matsumoto, H. Bioorg. Med. Chem. Lett. 2001, 11,
2257.
5. Niwayama, S.; Kurono, S.; Matsumoto, H. Bioorg. Med. Chem. Lett. 2003, 13,
2913.
6. Niwayama, S.; Kurono, S.; Cho, H.; Matsumoto, H. Bioorg. Med. Chem. Lett. 2006,
16, 5054.
7. Kurono, S.; Kurono, T.; Komori, N.; Niwayama, S.; Matsumoto, H. Bioorg. Med.
Chem. 2006, 14, 8197.
8. Zabet-Moghaddam, M.; Kawamura, T.; Yatagai, E.; Niwayama, S. Bioorg. Med.
Chem. Lett. 2008, 18, 4891.
9. Niwayama, S.; Zabet-Moghaddam, M.; Kurono, S.; Cho, H. Bioorg. Med. Chem.
Lett. 2009, 19, 5698.
10. Münchbach, M.; Quadroni, M.; Miotto, G.; James, P. Anal. Chem. 2000, 72, 4047.
11. (a) Tsumoto, H.; Murata, C.; Miyata, N.; Taguchi, R.; Kohda, K. Chem. Pharm.
Bull. 2003, 51, 1399; (b) Tsumoto, H.; Ra, M.; Samejma, K.; Taguchi, R.; Kohda,
K. Rapid Commun. Mass Spectrom. 2008, 22, 965; (c) Tsumoto, H.; Murata, C.;
Miyata, N.; Kohda, K.; Taguchi, R. Rapid Commun. Mass Spectrom. 2007, 21,
3815; (d) Schmidt, A.; Kellermann, J.; Lottspeich, F. Proteomics 2005, 5, 4.
12. Zhao, Y.-Y.; Cai, L.-S.; Jing, Z.-Z.; Wang, H.; Yu, J.-X.; Zhang, H.-S. J. Chromatogr. A
2003, 1021, 175.
Figure 3. Quantitative analysis of bovine serum albumin (BSA).
In summary, we synthesized benzoyloxysuccinimide and its
deuterated version, which react with amino groups in peptides,
and demonstrated that the combination of these reagents enables
13. Synthesis of the benzoyloxysuccinimide (BSI), 2, is as follows: benzoic acid
(0.353 g, 2.89 mmol) was dissolved in 50 mL of dry THF, and N-

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M. Zabet-Moghaddam et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4629–4632
hydroxysuccinimide (0.40 g, 3.47 mmol) and DCC (0.776 g, 3.76 mmol) were
d5-BSI-modified peptides were used for calculation of the relative ratios. The
averages of these data were plotted on the graph.
16. The procedure for quantitative analysis of bovine serum albumin (BSA) is as
follows: the stock solutions of 1 and 2 were prepared at a concentration of
100 mM in 75% acetonitrile/water. The stock solutions of BSA were prepared at
added. The mixture was stirred at room temperature for 2 days. The soluble
portion was separated from the viscous residue and the solvent was removed
under reduced pressure. The residue was dissolved in CHCl3 (50 mL) and
washed with brine (30 mL). After the CHCl3 layer was separated and dried over
anhydrous Na2SO4, the solvent was removed by evaporation to dryness. The
crude product was purified by column chromatography with 40% ethyl acetate
a
concentration of 1.25 mg/mL in
bicarbonate (TEAB), 50 mM, pH 8.5). This stock solution (50
with 20 L of DTT (10 mM) solution, heated to 100 °C, and cooled to room
temperature. To this mixture was added 20 L of iodoacetamide (IA) solution
a
sample buffer (triethylammonium
lL) was mixed
in hexane to afford
a
white solid (0.586 g, 93%). The white solid was
l
recrystallized from 2-propanol.
l
mp 132–133 °C (lit. 134–136 °C).17 1H NMR (300 MHz, CDCl3) d 2.88 (4H, s),
7.49–8.13 (4H, m), Anal. Calcd for C11H9NO4: C, 60.29; H, 4.14; N, 6.39. Found:
C, 60.52; H,4.00; N, 6.40. MS (MALDI TOF) m/z calcd for C11H9NO4Na (M+Na)+
242.0, found: 242.0.
(250 mM). The mixture was left for two additional hours in darkness for
completion of the alkylation by IA, and was then subjected to 1D
electrophoresis. Different amounts (1.5, 3, 6, 12, and 27 pmol) of this BSA
solution were loaded in each well, and the purification of the protein in this 1D
electrophoresis was performed for 1 h at 120 V. The gel was stained with
Coomassie staining solution, and the protein bands were excised. After the
decoloration of the excised bands, protein digestion was performed overnight
by addition of 50 ng of Trypsin in TEAB buffer (50 mM, pH 8.5). After extraction
The corresponding d5-labeled version, 1, was synthesized in the same manner
from d5-benzoic acid (purchased from Cambridge Isotope Laboratories, Inc.) in
91% yield. mp 132–133 °C. 1H NMR (300 MHz, CDCl3) d 2.88 (4H, s), MS (MALDI
TOF) m/z calcd for C11H4D5NO4Na (M+Na)+ 247.1, found: 247.1.
14. These peptides were purchased from Sigma–Aldrich.
15. The typical procedure is as follows: the stock solutions of the two peptides were
prepared at a concentration of 0.19 mM in triethylammonium bicarbonate
(TEAB) buffer (50 mM, pH 8.5). The stock solutions of the d5-labeled or
unlabeled BSI were prepared at a concentration of 20 mM in 50% acetonitrile/
of the peptides, the samples were dried and mixed with 20
The samples prepared this way (1.5, 3, 6, 12, and 27 pmol) were modified with
10 L of d-unlabeled benzoyloxysuccinimide, 2, and another 3 pmol sample
was separately modified with d5-benzoyloxysuccinimide, 1, and 10 of
acetonitrile was added to each sample in order to avoid potential precipitation.
After 1 h, an additional 5 L of the solution of 1 or 2 was added, and the
mixture was left for two additional hours at room temperature. For removal of
the esters derived from esterification reactions, 10 L of N-hydroxylamine
lL of TEAB buffer.
l
l
L
water solution. The reaction was started by mixing 2
solution with 2 L of BSI or d5-BSI stock solution, and left at room temperature
for 1 h. Another 1 of BSI was added to this mixture and left for one
additional hour to ensure completion of the reaction. To this mixture was
added of hydroxylamine (1.4 M, pH 10–12) and left for 10 min to
lL of the peptide stock
l
l
lL
l
(1.4 M, pH 11–12) was added to each mixture. The samples were dried, re-
suspended in 0.5% TFA and 5% acetonitrile for purification with the use of
3 lL
hydrolyze additional esters potentially formed on other amino acids. The
sample mixture was analyzed by MALDI mass spectrometer every 30 min in
order to examine the completion of the reaction. The BSI-peptide and d5-BSI-
peptide solutions prepared in this way were mixed in the molar ratios of BSI/
d5-BSI = 0.5, 1, 2, 4, 6, and 9. These mixtures were diluted with 50% acetonitrile,
0.1% TFA and subjected to MALDI MS analysis by MALDI TOF/TOF 4800 plusTM
(Applied Biosystems). The MS spectra were acquired automatically in the
positive mode and a total of 1000 shots were accumulated per spectrum. The
mass range was selected between 600 and 4000 m/z. Five data points were
collected for each ratio, and the S/N values of the monoisotopic peaks of BSI or
PepClean C-18 Spin Colums, again dried, and dissolved in 20
acetonitrile and 0.1% TFA for further analysis by MALDI TOF. For quantitative
analysis, 2 L of each sample solution modified with 2 was mixed with 2 L of
L of this mixture was spotted on
L of a matrix solution consisting
lL of 50%
l
l
the sample solution modified with 1, and 0.5
the MALDI-plate followed by mixing with 0.5
l
l
of 5 mg/mL of a-cyano-4-hydroxycinnamic acid (CHCA) in 50% acetonitrile and
0.1% trifluoroacetic acid (TFA).
17. Bailén, M. A.; Chinchilla, R.; Dodsworth, D. J.; Nájera, C. Tetrahedron Lett. 2002,
43, 1661.