Synthesis of d-labeled and unlabeled ethyl succinic anhydrides and application to quantitative analysis of peptides by isotope differential mass spectrometry

Article abstract of DOI:10.1016/j.bmcl.2016.08.079

Ethyl succinic anhydride and its d₅-labeled version have been synthesized and applied to quantitative analysis of peptides in combination with MALDI or ESI mass spectrometry. These modifiers react with amino groups in the N-termini and lysine side chains in proteins, and therefore the combination of these modifiers was shown to be a useful tool for quantification of peptides and hence for proteomics research.

Full text of DOI:10.1016/j.bmcl.2016.08.079

Accepted Manuscript
Synthesis of d-labeled and unlabeled ethyl succinic anhydrides and application
to quantitative analysis of peptides by isotope differential mass spectrometry
Satomi Niwayama, Masoud Zabet-Moghaddam, Sadamu Kurono, Pullaiah
Kattanguru, Aarif L. Shaikh
PII:
S0960-894X(16)30911-8
DOI:
http://dx.doi.org/10.1016/j.bmcl.2016.08.079
BMCL 24205
Reference:
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date:
Revised Date:
Accepted Date:
1 August 2016
19 August 2016
24 August 2016
Please cite this article as: Niwayama, S., Zabet-Moghaddam, M., Kurono, S., Kattanguru, P., Shaikh, A.L., Synthesis
of d-labeled and unlabeled ethyl succinic anhydrides and application to quantitative analysis of peptides by isotope
differential mass spectrometry, Bioorganic & Medicinal Chemistry Letters (2016), doi: http://dx.doi.org/10.1016/
j.bmcl.2016.08.079
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Synthesis of d-labeled and unlabeled ethyl succinic anhydrides and application to
quantitative analysis of peptides by isotope differential mass spectrometry
a,b,c
a, †
d,e
Satomi Niwayama, * Masoud Zabet-Moghaddam, Sadamu Kurono, Pullaiah
c
a
Kattanguru, and Aarif L. Shaikh
a: Department of Chemistry and Biochemistry, Texas Tech University, Lubbock, TX 79409-
061, USA
1
b: Department of Ophthalmology and Visual Sciences, Texas Tech University Health
Sciences Center, Lubbock, TX 79430, USA
c: Graduate School of Engineering, Muroran Institute of Technology, Muroran, Hokkaido,
0
50-8585, Japan
:
d Joint Research Laboratory of Molecular Signature Analysis, Division of Health Sciences,
Osaka University Graduate School of Medicine, 1-7 Yamadaoka, Suita, Osaka 565-0871,
Japan
e: Laboratory and Specialty Chemicals Division, Wako Pure Chemical Industries, Ltd., 3-1-2
Doshomachi, Chuo-ku, Osaka, Osaka 540-8605, Japan
Abstract:
Ethyl succinic anhydride and its d -labeled version have been synthesized and applied
5
to quantitative analysis of peptides in combination with MALDI or ESI mass spectrometry.
These modifiers react with amino groups in the N-termini and lysine side chains in proteins,
and therefore the combination of these modifiers was shown to be a useful tool for
quantification of peptides and hence for proteomics research.
Keywords: Proteomics, Quantitative analysis, Stable isotope labeling, Amino-group
modifiers, Soft ionization mass spectrometry
Proteomics comprehensively studies a set of proteins expressed under certain external
stimuli, and is increasingly becoming an important research area for study of biological
samples especially for comparison of protein expression patterns in different physiological
conditions. For this purpose, quantitative analysis of proteins and hence peptides derived
from enzymatic digestion of proteins, in particular in combination with mass spectrometry,
constitutes one of the essential parts of proteomics studies. Many studies for quantitative
analysis of proteins by various approaches have therefore been reported. One of the earliest
1
approaches is metabolic labeling. In this approach, cells are cultured in isotope-enriched or
in normal media and the relative abundance of specific proteins is quantitatively analyzed
from the mass spectra of each species. However, these approaches typically require a long
time for cell culturing, and they are not applicable to human proteins. Instead, chemical
modifications by covalent tagging of isotope-labeled and unlabeled modifiers on specific
functional groups of amino acid residues are expected to be applicable to any protein sample.
Therefore, many studies have been developed for quantitative analysis of proteins by
2
covalent labeling of specific amino acid residues in protein samples since the pioneering
3
work by the isotope-coded affinity tag (ICAT) method was reported.
In this context, we have also been developing our methodology with the use of
combinations of isotope-labeled and unlabeled small organic compounds that modify specific
functional groups of amino acid residues followed by mass spectrometric analysis. The
4
,5,6,7,8 13
13
combinations we reported are d -labeled and unlabeled N-ethyl maleimides,
C or C -
5
6
7
5
,6,7,8,9,10
labeled and unlabeled iodoacetanilides,
d7-labeled and unlabeled N--
1

1
1
12
naphthyliodoacetamides, and d -labeled and unlabeled benzoyloxysuccinimides. We have
5
demonstrated that these modifiers enable quantitative analysis of peptides and proteins, either
by electrophoresis or liquid chromatography followed by MALDI or ESI mass spectrometry.
However, most of these modifiers react specifically with the sulfhydryl group of the cysteine
residues, despite the fact that there are some proteins that do not contain cysteine residues.
1
2
Although the benzoyloxysuccinimides react with amino groups of N-termini and lysine, they
have rather limited solubility in aqueous media, and sometimes undesirable side reactions
occur, including reactions with other amino acid residues and methionine oxidations.
Therefore, we have been developing additional kinds of modifiers that react with amino
groups.
Here we report synthesis of ethyl succinic anhydride (ESA), 1, and its d -labeled
5
version, 2, and their application to quantitative analysis of peptides in combination with
MALDI or ESI mass spectrometry. Succinic anhydride is known to react with amino groups
of N-termini of peptides and lysine, and the combination of succinic anhydride and its d - or
4
1
3
13
C -labeled version has been applied to proteomics research. These combinations introduce
4
4
Da difference between the isotope-labeled and unlabeled succinic anhydride, however, we
had previously found that in our proteomics studies, having at least 5 Da difference facilitates
4
clear separation of the modified peptides. Therefore, we synthesized ethyl-substituted
succinic anhydride and its d -labeled version, allowing introduction of five deuterium atoms
5
(
Scheme 1).
Synthesis of ethyl succinic anhydride (ESA), 1, and its d -labeled version, 2, was
5
1
4
performed as in the following scheme (Scheme 2). We initially intended to synthesize them
by ethylation of monomethyl succinate prepared by selective monohydrolysis of dimethyl
1
5
succinate followed by dehydration, but the ethylation yielded greater percentages of the
corresponding diacid than the alkylated product, probably because of the small ester
functional group. Therefore, we introduced a bulkier and hence more hydrophobic tert-butyl
group. The preparation of tert-butyl succinate, 4, was performed by the reaction of succinic
1
6
anhydride, 3, and t-BuOH in the presence of N-hydroxysuccinimide (NHS). This mono
tert-butyl succinate was ethylated with 2 equivalents of LDA and ethyl iodide, followed by
the removal of the tert-butyl group, and subsequent cyclization to produce ethyl succinic
2

anhydride, 1, in a high yield. The corresponding d -labeled ethyl succinic anhydide, 2, was
5
obtained by substituting the ethyl iodide with C D I.
2
5
We tested the applicability of this combination to quantitative analysis of three
peptides, substance P, angiotensin, and neurotensin, utilizing both MALDI and ESI mass
17
spectrometry. The amino acid sequences and molecular weights of these peptides are
RPKPQQFFGLM-NH , DRVYIHPF, and pELYENKPRRPYIL; 1347.8, 1046.6, and 1672.9
2
+
Da ([M+H] ), respectively.
The following are ESI MS spectrum charts showing one of the above peptides,
angiotensin itself, and angiotensin reacted with d-unlabeled or d -labeled ethyl succinic
5
anhydride at pH 8.5 (Figure 1). As this peptide was identified from the doubly charged ion
2
+
[
M+2H] , the monoisotopic peak appeared as half of the molecular ion (523.7 Da). Because
of the existence of natural isotopes, the ion peaks show the monoisotopic and many isotopic
peaks that are several Dalton greater than it. Since this peptide does not contain a lysine
residue, the modification of this peptide with ethyl succinic anhydride (ESA) having the
molecular weight of 128 Da added 64 Da (=128/2) by the tagging of the terminal amino
group at this pH. The d -labeled ethyl succinic anhydride added 66.5 Da (=133/2) due to its
5
reactions with this amino group. Although modification of the amino group may be thought
to lead to a decrease in ionization efficiencies, we did not observe such a decrease of
ionization efficiencies after the modification, even though the ethyl succinic anhydride (ESA)
does not contain a nitrogen atom. Essentially the same MS spectra were obtained by MALDI
mass spectrometry as a result of the reaction with these modifiers (data not shown).
3

We next applied these modifiers to quantitative analysis of the three peptides for
assessment of general applicability to quantitative analysis. Solutions of the peptides were
prepared at pH 8.5 and were reacted with d-unlabeled or d -labeled ethyl succinic anhydride
5
(
ESA) solution at room temperature. The ESA-reacted peptide solution and d -ESA-reacted
5
peptide solution were mixed at several molar ratios, such as 0.25, 0.5, 0.75, 1, 2, 3, and 4
d /d ), and the relative ratios of the peptides in each mixed solution were measured from the
(
0
5
relative intensities of the monoisotopic peaks of the ESA-modified and d -ESA-modified
5
peptides by MALDI TOF mass spectrometer. In this way, the observed relative ratios of
ESA-modified and d -modified peptides were plotted against the theoretical ratios for all
5
18
three peptides. Figure 2 shows the results obtained by MALDI mass spectrometry.
4

6
5
4
3
2
1
0
"
"
"
"
"
"
"
2
R =0.9992
y=1.2147x -0.1846
0
"
0.5"
1"
1.5"
2"
2.5"
3"
3.5"
4"
4.5"
Theoretical Molar Ratio of ESA-peptide/d5-ESA-peptide
(a) quantitative analysis of substance P
5
"
2
R =0.9994
y"="1.1697x"*"0.1537"
R²"="0.99777"
4
3
2
1
0
.5"
y=1.0553x -0.0718
4
"
.5"
3
"
.5"
2
"
.5"
1
"
.5"
0
"
0"
0.5"
1"
1.5"
2"
2.5"
3"
3.5"
4"
4.5"
Theoretical Molar Ratio of ESA-peptide/d -ESA-peptide
5
(b) quantitative analysis of angiotensin
6
5
4
3
2
1
0
"
"
"
"
"
"
"
2
R =0.9978
y=1.2379x -0.2526
0"
0.5"
1"
1.5"
2"
2.5"
3"
3.5"
4"
4.5"
Theoretical Molar Ratio of ESA-peptide/d -ESA-peptide
5
(c) quantitative analysis of neurotensin
Figure 2. Quantitative analysis of 3 peptides with MALDI
As shown in the graphs, a good correlation between the theoretical ratios and
observed ratios was found for all the peptides. Two peptides, substance P and neurotensin,
contain one and two lysine residues respectively, in which the amino group also reacts with
5

these ESA or d -ESA, but significant differences among these three peptides were not
5
observed. Therefore, we conclude that ionization efficiencies of the ESA-modified peptides
and those of the d -ESA-modified peptides are the same within experimental errors, and
5
relative quantitative analysis of two peptide solutions is possible with the combination of
ESA and d -ESA.
5
However, at higher ratios of ESA-peptide/d -ESA-peptide, occasionally the observed
5
molar ratios for some of these peptides were notably higher than the theoretical molar ratios,
perhaps indicating “the suppression effect” known in the MALDI-ionization process due to
1
9
high concentration of salts. Therefore, we compared the quantitative analysis with ESI mass
20
spectrometry. The following are the results of quantitative analysis of the same peptides
with ESI mass spectrometry (Figure 3).
6

1
0
9
8
7
6
5
4
3
2
1
0
R² = 0.9841
y = 0.9462x + 0.3457
0
1
2
3
4
5
6
7
8
9
10
Theoretical Molar Ratio of ESA-peptide/d -ESA-peptide
5
(a) quantitative analysis of substance P
1
0
9
8
7
6
5
4
3
2
1
0
R² = 0.9923
y = 0.8579x + 0.0109
0
1
2
3
4
5
6
7
8
9
10
Theoretical Molar Ratio of ESA-peptide/d -ESA-peptide
5
(b) quantitative analysis of angiotensin
1
0
9
8
7
6
5
4
3
2
1
0
R² = 0.9976
y = 1.0429x + 0.075
0
1
2
3
4
5
6
7
8
9
10
Theoretical Molar Ratio of ESA-peptide/d -ESA-peptide
5
(c) quantitative analysis of neurotensin
Figure 3. Quantitative analysis of 3 peptides with ESI
7

As can be seen from the graphs, the observed ratios and theoretical ratios show
excellent correlation within experimental errors. Although the errors tend to become larger
with high ratios as in the previously reported quantitative analysis in our shotgun approach,
2
the correlation is still reasonably good, because the R is close to 1 in quantitative analysis of
all the peptides. This somewhat better correlation may be attributed to the homogeneous
nature of the analyte solutions in measuring ESI mass spectra and to reduced “ion
suppression effect” in ESI compared to MALDI because of the non-existence of excessive
salts in the analytes.
In summary, we synthesized d -labeled and unlabeled ethyl succinic anhydrides
5
(
ESAs), which specifically react with the amino group of lysine residues and N-termini of
peptides, and demonstrated that the combination of these modifiers and mass spectrometry
enables quantitative analysis of peptides with high accuracy. The reactions of these modifiers
with the amino group requires a longer time than those of cysteine modifiers, sometimes
necessitating overnight because of the reduced nucleophilicities of the amino group compared
to the sulfhydryl group, but quantitative analysis is possible with either MALDI or ESI mass
spectrometry, which are commonly applied soft-ionization mass spectrometry. Although a
side reaction of succinic anhydride with the hydroxyl group of tyrosine residue is sometimes
21
reported, no such side reaction was observed in our experiments. Therefore, the addition of
hydroxylamine for removal of the undesirable side reaction was unnecessary for modification
with these d -labeled and unlabeled ethyl succinic anhydrides (ESA), unlike the
5
1
2
benzoyloxysuccinimides, which induced some side reactions as we reported previously.
The solubility of ESA and d5-ESA also appear to have improved over
benzoyloxysuccinimides compared to the benzoyloxysuccinimides, as the reaction mixtures
were more homogeneous, allowing a reasonable quantification with ESI mass spectrometry.
In addition, while isotope effects are commonly observed during quantitative analysis with
the use of d-labeled modifiers, as in other modifiers we reported for our shotgun
4
,5,6,7,9,10,11
approach,
we did not observe an isotope effect in this quantitative analysis either with
MALDI or ESI. These modifiers can also be synthesized from readily available reagents
relatively inexpensively. Therefore, it is anticipated that the combination of d -labeled and
5
unlabeled ethyl succinic anhydrides (ESA) is a useful tool for quantification of peptides for
proteomics research without requiring existence of specific amino acid residues.
Acknowledgements
This work is supported by the Texas Tech University-Texas Tech University Health
Sciences Center Joint Initiative Grant and the Akiyama Life Science Foundation Grant. We
thank the Center for Biotechnology and Genomics, Texas Tech University for allowing us to
use the research facilities. We also thank Professor Ted W. Reid, in the Department of
Ophthalmology and Visual Sciences, Texas Tech University Health Sciences Center, for
providing us with a peptide.
References and Notes
*
Corresponding author; E-mail: sniwayama@hotmail.com
†
Current address: Center for Biotechnology and Genomics, Texas Tech University, Lubbock,
Texas 79409-3132, U.S.A.
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142.
1
0

1
4.
Synthetic procedures for ESA is as follows:
Synthesis of 4
Succinic anhydride, 3 (5.0 g, 49.96 mmol) was suspended in 30 mL of toluene under
nitrogen atmosphere. N-Hydroxysuccinimide (1.72 g, 14.98 mmol), 4-
(
dimethylamino)pyridine (0.610 g, 4.9 mmol), dry tert-butyl alcohol (15 mL, 150
mmol), and Et N (2.10 mL, 14.98 mmol) were added to the solution. Dissolution
3
occurred upon refluxing it for one day. After cooling, ethyl acetate (20 mL) was
added, and the organic layer was washed three times with 10 % citric acid and once
with brine, and dried over Na SO , filtered, and evaporated to afford 5.40 g (64%
2
4
yield) of brown solid. Recrystallization with ether gave white crystals of 4. The
1
22
1
structure was confirmed by H NMR. mp: 49-50 C (lit. 49-51 C) H NMR (500
1
3
MHz, CDCl )  2.61 (t, J = 6.7 Hz, 2H), 2.56–2.49 (m, 2H), 1.43 (s, 9H) ); C NMR
3
(
125 MHz, CDCl
3
) 178.7, 171.6, 81.2, 30.2, 29.3, 28.2.
Synthesis of 5
o
A THF (10 mL) solution of LDA (6.3 mL, 12.64 mmol) was stirred at -78 C for 15
minutes, and a solution of tert-butylsuccinate, 4, (1.0 g. 5.74 mmol) in THF (5 mL)
o
was added slowly. The reaction mixture was warmed to 0 C, stirred for 2h, cooled
o
again to -78 C. To this mixture was added ethyl iodide (0.64 mL, 8.04 mmol)
dropwise. The reaction mixture was allowed to warm to room temperature and stirred
for 24h. The reaction mixture was quenched with H O (3 mL) and concentrated. To
2
this residue was added ethyl acetate (30mL) and cold 1 M HCl (20 mL), and extracted
with ethyl acetate. The organic layer was dried (Na SO ), filtered, concentrated, and
2
4
purified by column chromatography (25% ethyl acetate in hexanes) to afford pure
1
product 5 (1.02 g, 88%) as an oil. The structure was confirmed by H NMR spectrum
1as follows.
H NMR (500 MHz, CDCl )  2.74–2.58 (m, 2H), 2.47–2.34 (m, 1H), 1.72–1.50 (m,
3
1
3
2
1
H), 1.42 (s, 9H), 0.92 (t, J = 7.6 Hz, 3H); C NMR (125 MHz, CDCl ): δ = 178.6,
3
74.1, 80.9, 43.4, 35.7, 28.1, 25.2, 11.4.
Synthesis of 6
Trifluoroacetic acid (12.34 mL, 160.25 mmol) was added to a solution of 2-ethyl-tert-
butylsuccinate (1.029 g, 5.09 mmol) in dry CH Cl (40 mL). The reaction mixture was
2
2
stirred at r.t. for 24 h, and was then concentrated and purified by column
chromatography (50% ethyl acetate in hexane) to produce diacid 6 (0.609 g, 82 %) as
1
white crystals. The structure was confirmed by the melting point and H NMR. mp:
1
6
o
1
9
6-97 C (lit. 96-97 C) H NMR (300 MHz, DMSO-d )  12.15 (brs, 2H), 2.48–2.34
6
(
m, 2H), 2.28 (dd, J = 16.5, 4.7 Hz, 1H), 1.59–1.36 (m, 2H), 0.81 (t, J = 7.6 Hz, 3H);
1
3
C NMR (125 MHz, DMSO-d ): δ = 176.0, 173.4, 42.1, 35.2, 24.5, 11.3.
6
Synthesis of ethyl succinic anhydride, 1
A mixture of 2-ethyl succinic acid, 6, (0.100 g, 0.684 mmol) and acetyl chloride (0.29
mL, 4.21 mmol) was refluxed for 3 h. The solution was concentrated to give the
desired anhydride 1 (0.080 g, 92 %) as an oil. The structure of ethyl succinic
1anhydride, 1, was confirmed by spectral data as follows.
H NMR (500 MHz, CDCl )  3.24–2.94 (m, 2H), 2.83–2.55 (m, 1H), 2.09–1.78 (m,
3
1
3
1
1
H), 1.80–1.60 (m, 1H), 0.92 (t, J = 7.6 Hz, 3H); C NMR (125 MHz, CDCl
73.8, 170.4, 42.0, 33.7, 24.3, 11.1.; HRMS m/z calcd for C
3
): δ =
(M+H) : 129.0551,
+
6
H
8
O
3
found: 129.0554.
The corresponding d -ethyl succinic anhydride, 2, was synthesized by substituting
5
1
C H I with C D I in the above procedure. H NMR (500 MHz, CDCl )  3.16–2.99 (m,
2
5
2
5
3
1
1

1
3
2
2
H), 2.74–2.60 (m, 1H); C NMR (125 MHz, CDCl ): δ = 173.5, 170.2, 41.7, 33.5,
3
+
3.23, 9.87; HRMS m/z calcd for C
6
H
3
D
5
3
O (M+H) : 134.0865, found: 134.0870.
1
1
1
1
5.
6.
7.
8.
Niwayama, S. J. Org. Chem. 2000, 65, 5834
Bergmeier, S. C.; Ismail, K. A. Synthesis, 2000, 10, 1369.
These peptides were purchased from Sigma-Aldrich and Akaal Organics.
The typical procedure with a MALDI mass spectrometer is as follows: The stock
solution of each peptide was prepared at the concentration of 0.19 mM in
triethylammonium bicarbonate (TEAB) buffer (pH=8.5, 50 mM). The stock solution
of ethyl succinic anhydride (ESA) and that of d -ESA were prepared at the
5
concentration of 20 mM in the solution containing 60% acetonitrile and 40% TEAB
buffer (pH=8.5, 50 mM). The reaction started by mixing 2 μL of the peptide stock
solution with 2 μL of ESA or d -ESA stock solution, which was left at room
5
temperature to an elevated temperature for one hour to overnight. In order to stop the
reaction, 2 μL of lysine solution (20 mM in TEAB buffer) was added to the mixture
and left at room temperature for an additional half hour. For the quantitative analysis,
the ESA-peptide solution and the d -ESA-peptide solution were mixed together at the
5
molar ratios of 0.25, 0.5, 0.75, 1, 2, 3, and 4 (ESA-peptide/d -ESA-peptide), and
5
TM
subjected to MALDI MS analysis by MALDI TOF/TOF 4800 plus (Applied
Biosystems). The sample at each ratio was spotted 5 times to overcome the problem
of sample inhomogeniety in MALDI analysis.
The MS spectra were acquired automatically in the positive mode as previously
12
reported. Five data points were collected for each ratio, and the S/N values of the
monoisotopic peaks of ESA or d -ESA-modified peptides were used for the
5
calculation of the relative ratios. The averages of these data were plotted on the
graph.
1
2
9.
0.
Knochenmuss, R. and Zenobi, R. Chem. Rev. 2003, 103. 441.
The typical procedure with an ESI mass spectrometer is as follows: Each peptide was
dissolved in 50 mM ammonium bicarbonate at around pH 9 for modification with
ESA (light, 1) or d -ESA (heavy, 2). Each peptide solution was divided into the
5
designated ratios (8:1, 4:1, 2:1, 1:1, and 1:2 for light/heavy modification) and then
incubated with an excess amount of 100 mM ESA (1) or d -ESA (2) acetonitrile
5
solution overnight at 60 ℃. Next, the peptide solutions were mixed in the designated
ratios above as sample solutions. Subsequently, 10% trifluoroacetic acid
corresponding to 5% of the solution was added to make the pH acidic. The amount of
5
L of sample solution was injected into a nano LC (UltiMate 3000, Thermo
Scientific Dionex, San Jose, CA, USA) with a L-column 2 C18 (150 x 0.075 mm id
analytical column, containing 3 m particles; Chemicals Evaluation and Research
Institute (CERI), Kitakatsushika, Saitama, Japan), equipped with a C18 guard column
(
5 x 0.3 mm id, containing 5 m particles; CERI). The measurement time and the
flow rate were 45 min and 300 nL/min, respectively; mobile phases A and B consisted
of 0.1% formic acid (pH 2.0) and 0.1% formic acid in acetonitrile, respectively.
Sample separation was accomplished with the following linear gradient: 2-8% B over
1
9
min; 8-20% B over 19 min; 20-35% B over 5 min; 35-95% B over 0.01 min; held at
5% B over 5 min; 95-2% B over 0.01 min; and held at 2% B until the end of the
measurement. The LC was connected directly to a nanoelectrospray ionization ion
1
2

trap (nano-ESI-IT)-MS (HCTultra, Bruker Daltonik GmbH, Bremen, Germany)
equipped with a nano-ESI ion source. A PicoTip emitter (10 m id, New Objective,
Woburn, MA, USA) was used to generate nano-ES(Electrospray). The heated
nitrogen gas temperature, gas flow rate, and spray voltage were 160 °C, 4.0 L/min,
and 1.2 kV, respectively. The LC/MS measurements collected from m/z 300 to 1500
were obtained three times for each sample. The LC/MS/MS measurements were
conducted for data-dependent ion trap MS/MS acquisition of the four most abundant
precursor ions, and an active exclusion time of 30 s was used to differentiate these
precursor ions from previously analyzed ions. For the quantitative analysis of the
peptides, the relative ratio of the EIC peak areas corresponding to the monoisotopic
ions of a peptide modified with ESA (1) or d -ESA (2) was calculated and taken as the
5
average of three measurements.
2
2
1.
2.
Gounaris, A. D.; Perlmann, G. E. J. Biol. Chem. 1967, 242, 2739.
Guzzo, P. R.; Miller, M. J. J. Org. Chem. 1994, 50, 4862.
1
3

Graphical abstract
1
4