Peptide peak intensities enhanced by cysteine modifiers and MALDI TOF MS

Article abstract of DOI:10.1002/jms.3093

Two cysteine-specific modifiers we reported previously, N-ethyl maleimide (NEM) and iodoacetanilide (IAA), have been applied to the labeling of cysteine residues of peptides for the purpose of examining the enhancement of ionization efficiencies in combination with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF MS). The peak intensities of the peptides as a result of modification with these modifiers were compared with the peak intensities of peptides modified with a commercially available cysteine-specific modifier, iodoacetamide (IA). Our experiments show significant enhancement in the peak intensities of three cysteine-containing synthetic peptides modified with IAA compared to those modified with IA. The results showed a 4.5-6-fold increase as a result of modification with IAA compared to modification with IA. Furthermore, it was found that IAA modification also significantly enhanced the peak intensities of many peptides of a commercially available proteins, bovine serum albumin (BSA), compared to those modified with IA. This significant enhancement helped identify a greater number of peptides of these proteins, leading to a higher sequence coverage with greater confidence scores in identification of proteins with the use of IAA.

Full text of DOI:10.1002/jms.3093

Research article
Received: 29 June 2012
Revised: 14 August 2012
Accepted: 17 August 2012
Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI 10.1002/jms.3093
Peptide peak intensities enhanced by cysteine
modifiers and MALDI TOF MS
Masoud Zabet-Moghaddam,a,b Aarif L. Shaikha and Satomi Niwayamaa*
Two cysteine-specific modifiers we reported previously, N-ethyl maleimide (NEM) and iodoacetanilide (IAA), have been applied
to the labeling of cysteine residues of peptides for the purpose of examining the enhancement of ionization efficiencies in
combination with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF MS). The peak
intensities of the peptides as a result of modification with these modifiers were compared with the peak intensities of peptides
modified with a commercially available cysteine-specific modifier, iodoacetamide (IA). Our experiments show significant
enhancement in the peak intensities of three cysteine-containing synthetic peptides modified with IAA compared to those
modified with IA. The results showed a 4.5–6-fold increase as a result of modification with IAA compared to modification with
IA. Furthermore, it was found that IAA modification also significantly enhanced the peak intensities of many peptides of a
commercially available proteins, bovine serum albumin (BSA), compared to those modified with IA. This significant enhancement
helped identify a greater number of peptides of these proteins, leading to a higher sequence coverage with greater confidence
scores in identification of proteins with the use of IAA. Copyright © 2012 John Wiley & Sons, Ltd.
Keywords: ionization efficiencies; peak enhancement; cysteine modifier; MALDI TOF; alkylation
Here, we examined the effects on enhancement of ionization
efficiencies as a result of modification with two cysteine-specific
Introduction
Structural modification of peptides by tagging reagents plays an
important role in combination with mass spectrometry in various
biochemical and biological applications. Some examples include
selective separation of the tagged peptides by selective adsorption
through affinity tag technology[1] and identification of spatial
relationships between amino acids in proteins or protein complexes
by cross-linking.[2] One of the most beneficial aspects of chemical
modification of peptides for observation of the mass spectrum is
improvement of the ionization efficiencies, allowing detection of
the peptides at a lower concentration.[3] This improvement also
assists in identification of proteins. More recently, various tagging
reagents have been invented for the purpose of identification and
quantitative analysis of proteins or peptides in proteomics research.
The combination of stable isotope-labeled and unlabeled tagging of
peptides and proteins allows quantitative analysis of peptide or
protein samples obtained from two different external stimuli. Some
of the most well-known examples include the ICAT[4] and iTRAQ
reagents,[5] and many other researchers reported other tagging
reagents.[6] In this context, earlier we reported several sets of
isotope-labeled and unlabeled small organic molecules that specif-
ically react with cysteine residues for the purpose of identification
and quantitative analysis of peptides and proteins in combination
with 1D and 2D electrophoresis and soft ionization mass spectrom-
etry, such as electrospray ionization (ESI) and matrix-assisted laser
desorption/ionization (MALDI).[7–11] We demonstrated that these
sets of reagents enable quantitative analysis of proteins and
peptides as well as identification of proteins, and hence they are
expected to be a useful tool for proteomics research. In particular,
we reported that N-ethyl maleimide (NEM), 1, and iodoacetanilide
(IAA), 2, exhibited significant enhancement of ionization efficiencies
of peptides in combination with ESI. However, since MALDI is as
commonly applied as ESI, it is also important to assess the effects
by these modifiers in combination with MALDI.
modifiers, NEM, 1, and IAA, 2, in MALDI mass spectrometry
(Scheme 1).[12,13] In particular, we compared the effects of these
modifiers with the effects of commercially available iodoaceta-
mide (IA), 3, which is commonly applied.
Results and discussion
Peak enhancement of peptides
We first examined the effects of enhancement of peak intensities of
three model peptides with different amino acid sequences and
molecular weights after the modification of cysteine residues. The
model peptides were PEP 60, PEP 13 and PEP 31 with the amino
acid sequences, molecular weight and pI values of ALVCEQEAR,
1017.49 Da, 4.4; SDTCSSQKTEVSTVSSTQK, 2001.92 Da, 6.2 and
KEEPPHHEVPESETC, 1746.75 Da, 4.5, respectively, and each model
peptide contained a cysteine residue. Each peptide was modified
with IAA, NEM or IA, and the signal-to-noise (S/N) ratios were
compared. As each peptide was reacted with an excess of each
modifier, no unmodified peptide was observed in each case. The
relative S/N ratios which were calculated by the S/N ratios of
IAA- or NEM-modified peptides divided by those of IA-modified
*
Correspondence to: Satomi Niwayama, Department of Chemistry and
Biochemistry, Texas Tech University, Lubbock, Texas 79409-1061, USA.
E-mail: satomi.niwayama@ttu.edu
a Department of Chemistry and Biochemistry, Texas Tech University, Lubbock,
Texas 79409-1061, USA
b Current address: Center for Biotechnology and Genomics, Texas Tech University,
Lubbock, Texas 79409-3132, USA
J. Mass Spectrom. 2012, 47, 1546–1553
Copyright © 2012 John Wiley & Sons, Ltd.

Peak enhancement by cysteine modification
measurement of the S/N ratios was performed to find out the
LOD of the peptide modified with each modifier. The results
obtained are summarized in Table 1. As can be seen, the LOD
was reduced fourfold particularly as a result of IAA modification
over IA modification for PEP 60 and PEP 13, and twofold for PEP
31, while NEM modification improved the S/N ratio only for PEP 60.
Figure 2 shows the spectra of PEP 60 modified by IAA, IA and NEM
in four successive dilutions. The starting amount of each modified
peptide on the MALDI-plate was 1.6 fmol followed by dilution to
0.8, 0.4 and 0.2 fmol. In accordance with the above result, the S/N
ratio of the IAA-peptide (252) was far higher than the two other
modified peptides, IA (46) or NEM-peptide (37), at the same amount
of 1.6 fmol on the plate. As the sample was diluted by half, 0.8 fmol
for each, the S/N ratios of all three peptides were reduced but still
identifiable. At the concentration of 0.4 fmol, the signals of IA- and
NEM-peptides were no longer identifiable, whereas a clear signal
with the S/N ratio of 49 was observed for the IAA-peptide. The signal
corresponding to the IAA-modified peptide was identified even
after dilution to 0.2 fmol on the plate with the S/N ratio of 18,
demonstrating a fourfold improvement of LOD for PEP 60 in
comparison with the other two modifiers, IA and NEM.
Protein identification
Scheme 1. N-ethylmaleimide, iodoacetanilide, iodoacetamide and their
reactions with cysteine.
Next, we examined the effects of these modifiers in comparison to
IA on alkylation of a commercial protein, bovine serum albumin
(BSA). BSA was alkylated with an excess of IA, IAA or NEM, and each
sample was spotted six times to overcome the problem caused by
non-homogeneity of samples in matrix crystals on the MALDI plate.
All the spectra were checked carefully, to ensure in each sample
that the masses of alkylated peptides (m/z) were truly derived from
the peptides where all the cysteine residues were alkylated rather
than non-alkylated peptides with the same m/z or those where
another amino acid residue was alkylated, as each alkylation
reagent adds a different mass to the respective peptides. The
masses of 57, 133 and 125 Da for IA, IAA and NEM, respectively,
were added to the masses of peptides for calculation of the
alkylated peptide masses in the spectra. These extra masses were
subjected to a MASCOT database search. Figure 3 shows examples
of the spectra obtained as a result of modification by each
alkylation reagent investigated in this study.
The alkylated peptides with their masses for each alkylation
reagent are listed in Table 2. The alkylated peptides were taken into
account only if they were identified in at least four out of six identically
measured spots. Therefore, although the IA-peptide No. 12 was
identified in three spots, it was not counted in Table 2. As can be seen,
a greater number of alkylated peptides and alkylated cysteine
residues was identified as a result of IAA modification than IA modi-
fication. The NEM modification indicated the smallest number of
identified alkylated peptides. A total of 23 alkylated peptides were
identified using IA, whereas this number was 28 for IAA-peptides,
which indicates an 8% improvement in identification of cysteine-
containing peptides, as the total theoretical number of cysteine-
containing peptides is 71. The total number of identified peptides
alkylated with NEM was only 14. In addition, the number of alkylated
cysteine residues was greater with the use of IAA (30) compared to IA
(28). Only 17 alkylated cysteines were identified with NEM.
Figure 1. The average relative S/N ratios of NEM-peptides/IA-peptides and
IAA-peptides/IA-peptides. The results show a nearly sixfold enhancement by
IAA over IA for PEP 60 and PEP 13 and a nearly twofold enhancement for PEP
31. Error bars show the error ranges in five independent measurements.
peptides (IAA or NEM-modified peptide/IA-modified peptide) are
summarized in Fig. 1.
As can be seen from Fig. 1, significant enhancement was
observed as a result of IAA modification compared to IA modification.
The S/N ratios were increased about six times for PEP 60 and PEP 13
and more than two times for PEP 31 as a result of IAA modification
over IA modification. The modification with NEM also enhanced
the S/N ratios for all these peptides over the modification with IA,
but the effect was not as remarkable. Only the smallest peptide,
PEP 60, showed a threefold enhancement with NEM over IA.
The enhancement observed above also resulted in improvement
of the limit of detection (LOD). Each peptide modified with IAA,
NEM or IA was mixed together at the same concentration, and
The influence on ionization efficiencies of alkylated peptides
by IA, IAA and NEM was more closely examined on the identical
peptides modified with IA, IAA or NEM. The comparison was
made between IAA-peptides/IA-peptides and NEM-peptides/IA-
J. Mass Spectrom. 2012, 47, 1546–1553
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M. Zabet-Moghaddam, A. L. Shaikh and S. Niwayama
Table 1. Limit of detections (LOD) and the S/N ratios for three model peptides modified by IA, IAA and NEM. The amounts on the plate and
corresponding S/N value are given for each peptide. The LOD was calculated based on the ratios of amounts of peptides in which the signal was
observed to the amount of peptide detected only by IAA for all three model peptides modified by IA, IAA and NEM
Peptides
IA-Peptide amount on
NEM-Peptide amount
IAA-Peptide amount
Improved LOD by IAA
the plate/S/N value
on the plate/S/N value
on the plate/S/N value
PEP 60
800 amol/19
not detected
800 amol/33
not detected
800 amol/117
200 amol/18
fourfold
PEP 31
PEP13
800 amol/21
not detected
800 amol/26
not detected
800 amol/45
300 amol/18
2.6-fold
fourfold
10 fmol/22
10 fmol/23
10 fmol/143
2.5 fmol/34
not detected
not detected
identified either by IAA modification or IA modification, as shown
also in Table 2. NEM not only delivered the lowest number of
peptides, but also showed a negative influence of ionization
efficiencies on most of the peptides in comparison to IAA or IA.
Previously, we found that NEM is less reactive than IAA or IA under
certain conditions,[10] but the tendency of the enhanced ionization
efficiencies by IAA is consonant with the synthetic peptides
described above. Depending on the sequence of amino acids of
the peptides and the length of the peptides, the effects of IAA, IA
and NEM on ionization of the peptides were different. In a certain
case, a nearly 50-fold enhancement was observed with the use of
IAA over IA (peptide with m/z of 1024.521). In some cases, the S/N
values of IA-peptides were greater than those of IAA-peptides. For
example, the two peptides with the highest enhancements with the
use of IA over IAA were observed in the peptide with m/z 2441.251
with a fivefold enhancement and the peptide with m/z 2472.286 with
a 3.5-fold enhancement. However, this enhancement is far lower
compared to those achieved by IAA (up to 50-fold). Overall, the alky-
lated peptides showed far higher S/N values when IAA was applied
in comparison to IA. Interestingly, the influence of IAA on ionization
efficiencies of the peptides was more pronounced for peptides with
smaller molecular weights (m/z up to about 2000 Da), whereas IA
showed greater enhancement of ionization efficiencies for peptides
with greater molecular weights (m/z > about 2000 Da) (Fig. 4) for BSA.
Due to the enhancement of peak intensities by use of IAA, some
of the peptides were identified uniquely by IAA. Although some
peptides were uniquely identified by IA modification, the number
uniquely identified by IAA modification (9) increased by more than
50% in comparison to those uniquely identified by IA modification
(4). In accordance with the earlier results mentioned above, all
the peptides uniquely identified by IA are peptides with greater
molecular weights, with m/z greater than 2000 Da, and those
uniquely identified by IAA modification are peptides with smaller
molecular weights, with m/z less than 2000 Da.
We further confirmed that the S/N values of the non-alkylated
peptides are in a similar error bar range in all the samples we
attempted to react with IA, IAA or NEM, indicating that the changes
in peak intensities of cysteine-containing peptides are mainly the
result of modification by IA, IAA or NEM (data not shown).
Figure 2. The spectra of PEP 60 modified with IAA, NEM or IA. The
amounts of each peptide on the plate for each spectrum are A) 1.6 fmol,
B) 0.8 fmol, C) 0.4 fmol and D) 0.2 fmol. M: matrix cluster.
peptides. Figure 4 shows the logarithmic scale of the ratio of
S/N values of IAA/IA-peptides and NEM/IA-peptides.
Protein identification at the LOD
Ionization efficiencies of the peptides with respect to the alkylation
reagent used were observed to be different. The S/N values of most
of the peptides modified by IAA were enhanced compared to those
modified by IA and NEM. Some of the peptides were uniquely
Identification of proteins in a small amount is still challenging in
proteomics. Therefore, peak enhancement of the peptides obtained
from digestion of a protein is expected to make a significant
contribution to detection of small quantities of proteins. In order to
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Peak enhancement by cysteine modification
Figure 3. The spectra of BSA digest alkylated with a) IAA b) IA and c) NEM. Some S/N values are shown in parentheses. The peptides assigned letters are
non-alkylated. The inserted spectra show enlarged views of the m/z range of 1050–1350 Da. The numbered peptides shown in the inserted spectra are
referred to in Table 2.
take advantage of peak enhancement using IAA, we compared the
number of identified peptides from a small amount of the BSA digest
solutions (1.5 fmol) modified with IA or IAA on the MALDI plate. NEM
was not tried since it did not show any significant improvement in
the ionization efficiencies in comparison to the use of IA.
modification were those with m/z less than 2000 Da. These results
are consistent with the earlier results shown above, wherein detec-
tion was more effective by IAA for smaller peptides (m/z< 2000 Da).
As a result of peak enhancement with the use of IAA, the
sequence coverage of the protein improved from 24% with the
use of IA to 33% with the use of IAA (Table 3). This improvement
in the sequence coverage was entirely due to the uniquely
alkylated peptides identified by IAA. Similarly, the score of the
identified protein was also increased using IAA (255) compared
to IA (203), indicating higher confidence for identification of
protein as a result of IAA modification over IA modification.
Table 3 lists all the peptides obtained from the in-gel digestion of
gel bands containing BSA. As can be seen, the total number of
peptides identified (alkylated or non-alkylated) was 14 with the
use of IA, and 19 with the use of IAA. The five additional peptides
identified with the use of IAA were all alkylated peptides. It should
be mentioned here that the number of non-alkylated peptides
identified was the same with the use of IA and IAA, and hence the
alkylated peptides constitute the main difference in this comparison.
The identified number of cysteine residues alkylated with IAA was
12, and therefore far greater than that with IA (3), again demonstrat-
ing enhanced ionization efficiencies by IAA. Six peptides were
uniquely identified with the use of IAA, while only one peptide
was identified with the use of IA. The only peptide uniquely identi-
fied by IA modification was peptide No. 17 (Table 3) with a high
m/z of 2529.03 Da. All the peptides uniquely identified by IAA
Conclusions
Both the NEM and IAA we reported before showed enhancement
of MALDI ionization efficiencies over IA in model peptides and a
commercial protein. In particular, IAA modification showed an up
to 50-fold enhancement in ionization efficiencies compared to IA
modification. This enhancement with the use of IAA led to
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M. Zabet-Moghaddam, A. L. Shaikh and S. Niwayama
Table 2. The identified peptides from BSA modified with the three different alkylation reagents IA, IAA or NEM. The numbers represent the
obtained masses of the respective alkylated peptide. MC: missed cleavage, C: no. of cysteines
No
Position
Peptide
MC
C
IA
IAA
NEM
1
45 – 65
K.GLVLIAFSQYLQQCPFDEHVK.L
K.TCVADESHAGCEK.S
K.SLHTLFGDELCK.V
0
0
0
1
0
1
0
1
0
1
0
0
1
0
1
0
1
0
0
1
1
0
1
0
0
0
0
0
0
1
0
1
1
2
1
2
2
1
1
1
2
3
1
1
3
1
2
1
2
2
1
1
1
1
1
2
1
1
2
2
1
1
1
3
2492.314(1)
1463.633(2)
1419.717(1)
–
–
–
2
76 – 88
1615.663(2)
1495.736(1)
2172.918(2)
1646.586(2)
1977.920(1)
1652.819(1)
2096.025(1)
1899.793(2)
–
–
3
89 – 100
1487.718(1)
4
101 – 117
106 – 117
123 – 138
139 – 151
139 – 155
184 – 197
184 – 204
198 – 204
223 – 228
267 – 285
286 – 297
298 – 309
310 – 318
372 – 386
375 – 386
387 – 399
387 – 401
402 – 420
413 – 420
413 – 433
460 – 468
469 – 482
483 – 489
499 – 507
499 – 507
508 – 523
524 – 544
529 – 544
581 – 597
K.VASLRETYGDMADCCEK.Q*
R.ETYGDMADCCEK.Q*
R.NECFLSHKDDSPDLPK.L
K.LKPDPNTLCDEFK.A
K.LKPDPNTLCDEFKADEK.K
K.YNGVFQECCQAEDK.G
K.YNGVFQECCQAEDKGACLLPK.I
K.GACLLPK.I
–
5
–
–
6
–
–
7
1576.798(1)
2020.012(1)
1747.745(2)
2430.202(2)
758.441(1)
–
1644.790(1)
8
2087.997(1)
9
–
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
–
834.464(1)
782.391(1)
–
–
R.CASIQK.F
774.375(1)
K.ECCHGDLLECADDRADLAK.Y
K.YICDNQDTISSK.L
2247.996(3)
1443.725(1)
1532.813(2)
–
–
1519.692(1)
1684.863(2)
1148.566(1)
1966.937(2)
1654.731(2)
1630.677(1)
1871.897(1)
–
1511.671(1)
K.LKECCDKPLLEK.S
–
K.SHCIAEVEK.D
–
R.LAKEYEATLEECCAK.D
K.EYEATLEECCAK.D
1814.867(2)
–
–
1638.857(2)
–
K.DDPHACYSTVFDK.L
K.DDPHACYSTVFDKLK.H
K.HLVDEPQNLIKQNCDQFEK.L
K.QNCDQFEK.L
–
1795.858(1)
2355.167(1)
1068.456 (1)
2529.286(1)
–
1863.870(1)
–
1144.477(1)
2605.262(1)
1318.569(2)
1816.878(1)
974.516(1)
1157.555(1)
1290.570(2)
1956.971(1)
2574.219(1)
1983.977(1)
2155.926(3)
30 (85.7%)
28 (39.5%)
9
1136.463(1)
–
K.QNCDQFEKLGEYGFQNALIVR.Y
R.CCTKPESER.M
1302.552(2)
1808.859(1)
–
R.MPCTEDYLSLILNR.L*
R.LCVLHEK.T
1740.864(1)
898.504(1)
–
K.CCTESLVNR.R
–
K.CCTESLVNR.R
1138.521(2)
1880.958(1)
2498.254 (1)
1907.963(1)
1927.848 (3)
28 (80%)
23 (32%)
4
1274.542(2)
1948.955(1)
2566.248(1)
1975.950(1)
2131.899(3)
17 (48.5%)
14 (20%)
R.RPCFSALTPDETYVPK.A
K.AFDEKLFTFHADICTLPDTEK.Q
K.LFTFHADICTLPDTEK.Q
K.CCAADDKEACFAVEGPK.L
No. of alkylated Cysteines identified (theoretical 35)
No. of alkylated peptides identified (theoretical 71)
No. of uniquely alkylated peptides identified
improvement of identification of proteins in a smaller amount
over IA, and a higher sequence coverage and a higher score in
the database search. Therefore, we found that IAA has considerable
advantages over IA in combination with MALDI.
at a concentration of 0.6 mM (Tris-buffer, 50 mM, pH = 8.5).
The stock solutions of IAA, IA and NEM were, respectively,
prepared in DMSO at a concentration of 20 mM, in a Tris-
buffer at a concentration of 50 mM at pH = 8.5 and in DMSO
at a concentration of 10 mM. Each peptide stock solution
(2 mL) was mixed with 2 mL of the stock solution of IA, IAA
or NEM and left for 0.5–1 h in a dark place. A large excess
of b-mercaptoethanol was added to each solution to termi-
nate the alkylation reaction. Samples were diluted with 50%
acetonitrile containing 0.1% TFA prior to MS measurement
to obtain a sufficient amount on the MALDI-plate. The
comparison of the peak intensities was performed against
IA-modified peptides. The alkylated peptides (IAA or NEM)
were mixed with IA-peptides at the same amount of 8 fmol
each (IAA-peptides with IA-peptides or NEM-peptides with
IA-peptides) for PEP 61 and PEP 31, and 50 fmol for PEP 13
on the plate. For experiments of the LOD, peptides alkylated
with IA, IAA or NEM were mixed together in an equal mole,
and the MALDI measurement of successive dilution of the
mixture was performed to determine the LOD for each alkylating
reagent used in this study.
Earlier, we observed that NEM modification showed ionization
efficiencies more enhanced than IAA modification in combination
with ESI, which significantly contrasts with the present results with
MALDI. While the reason is not clear, it may be speculated that the
existence of the phenyl ring might increase absorption at the laser
wavelength applied in MALDI, contributing to more desorption of a
greater amount of peptides modified with IAA. These observations
are anticipated to be useful in designing new modifiers according
to the types of mass spectrometer to be applied, and in more
confident identification of proteins in the future.
Experimental
Alkylation of peptides
The alkylation of model peptides was performed as described
earlier.[1] Briefly, the stock solutions of peptides were prepared
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Peak enhancement by cysteine modification
Alkylation of a protein
The procedure for cysteine alkylation of a model protein, BSA, was
performed before the sample was subjected to SDS-PAGE as
described previously.[10] The stock solution of protein was prepared
in a buffer containing Tris-HCl (100 mM), 3% SDS, 20 mM
tributylphosphine at a concentration of 0.1mg/mL and pH= 8.5,
and left for 1 h at room temperature for completion of the
reduction. The protein solution (10 mL) was incubated with 1 mL
of the solution of alkylation reagent, IAA (200 mM in DMSO), IA
(200 mM in the above Tris-buffer) or NEM (100 mM in DMSO). The
mixture was shaken and kept in a dark place for 2 additional hours.
1D SDS-PAGE
To the protein reaction mixture prepared above was added 2 mL of a
solution containing 20% (v/v) glycerol and 0.1% (w/v) bromophenol
blue. The amount of protein loaded on the gel was 1 mg. The mixture
was directly subjected to SDS-PAGE, and separation was performed
at 120 V for 90 min. The gel was stained by Coomassie Brilliant Blue
for overnight.
In-gel digestion
The protein bands from the 1D electrophoresis above were
washed with 100 mL H2O for 5 min and with 100 mL of a solution
H2O/acetonitrile (50/50) at 37ꢀC and 600 rpm on a Thermomixer
comfort (Eppendorf). The latter step was repeated until the
Figure 4. The logarithmic scale of the S/N values of IAA-peptides/IA-
peptides (top) and NEM-peptides/IA-peptides (bottom). The m/z values
are the masses of peptides without modification by alkylation reagent
IA, IAA or NEM.
Table 3. The identified peptides (alkylated and non-alkylated) obtained from the digestion of BSA alkylated either by IAA or IA. The numbers
represent the obtained masses of the respective alkylated peptide. The numbers given in parentheses are the S/N values of the respective peptides.
MC: missed cleavage, C: no. of cysteines
No.
Position
Peptide
MC
C
IA
IAA
1
25–34
R.DTHKSEIAHR.F
1
1
0
0
0
0
1
1
0
1
0
1
0
1
0
1
0
0
0
0
0
0
0
2
1
0
0
0
1
2
0
0
0
1
0
0
2
1
2
1
1193.520 (30)
1249.517 (38)
1163.534 (17)
---------
1193.505 (20)
1249.522 (13)
1163.541 (13)
1615.549 (26)
1495.606 (67)
927.416 (137)
1083.506 (18)
1001.500 (48)
1519.550 (29)
1684.716 (28)
1567.626 (93)
1439.693 (192)
1305.600 (24)
----------
2
35–44
R.FKDLGEEHFK.G
2
66–75
K.LVNELTEFAK.T
3
76–88
K.TCVADESHAGCEK.S
K.SLHTLFGDELCK.V
K.YLYEIAR.R
4
89–100
1419.581 (45)
927.418 (237)
1083.513 (15)
1001.506 (36)
---------
5
161–167
161–168
233–241
286–297
298–309
347–359
360–371
402–412
413–433
421–433
437–451
460–468
469–482
499–507
508–523
6
K.YLYEIARR.H
9
R.ALKAWSVAR.L
11
12
13
14
16
17
18
19
20
21
22
23
K.YICDNQDTISSK.L
K.LKECCDKPLLEK.S
K.DAFLGSFLYEYSR.R
R.RHPEYAVSVLLR.L
K.HLVDEPQNLIK.Q
K.QNCDQFEKLGEYGFQNALIVR.Y
K.LGEYGFQNALIVR.Y
R.KVPQVSTPTLVEVSR.S
R.CCTKPESER.M
---------
1567.632 (131)
1439.704 (219)
1305.607 (16)
2529.030 (15)
1479.684 (48)
1639.821 (111)
----------
1479.673 (46)
1639.808 (128)
1318.450 (29)
1816.718 (11)
1290.467 (18)
1956.901 (11)
19
R.MPCTEDYLSLILNR.L
K.CCTESLVNR.R
---------
----------
R.RPCFSALTPDETYVPK.A
1880.785 (15)
14
No. of peptides identified
No. of unique peptides identified
1
6
No. of Cysteine containing peptides identified
No. of cysteine alkylated
Sequence coverage
3
8
3
12
24%
33%
Score
203
255
J. Mass Spectrom. 2012, 47, 1546–1553
Copyright © 2012 John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/jms

M. Zabet-Moghaddam, A. L. Shaikh and S. Niwayama
Kinumi, Y. Shimomae, R. Arakawa, Y. Tatsu, Y. Shigeri, N. Yumoto, E. Niki.
Effective detection of peptides containing cysteine sulfonic acid using
matrix-assisted laser desorption/ionization and laser desorption/
ionization on porous silicon mass spectrometry. J. Mass Spectrom.
2006, 41, 103; (f) C. Hagman, M. Ramstrom, P. Hakansson, J. Bergquist.
Quantitative analysis of tryptic protein mixtures using electrospray
ionization Fourier transform ion cyclotron resonance mass spectrometry.
J. Proteome Res. 2004, 3, 587; (g) V. A. Gorshkov, K. A. Artemenko, T. Y.
Samgina, A. T. Lebedev. Signal enhancement for protonated peptides
in matrix-assisted laser desorption/ionization by doping with organic
molecules. Mass-Spektrometriya 2007, 4, 5; (h) J. L. Frahm, I. D. Bori, D.
L. Comins, A. M. Hawkridge, D. C. Muddiman, Anal. Chem. 2007, 79,
3989; (i) D. K. William, C. W. Meadows, I. D. Bori, A. M. Hawkridge, D. L.
Comins, D. C. Muddiman. J. Am. Chem. Soc. 2008, 130, 2122.
Coomassie Brilliant Blue dye was completely removed, and the
protein bands were incubated for 1 min in 100 mL of 100%
acetonitrile. After removal of the acetonitrile, the protein bands
were dried for 15 min. Digestion was performed by the addition
of 10 mL of an aqueous ammonium hydrogen carbonate
(30 mM) containing 300 ng of trypsin (Promega Sequencing
Grade Modified Trypsin, Promega Corporation, Madison, USA) at
37ꢀC overnight, and peptide extraction was performed as
described previously.[10] The extracted peptide solution was dried
by Speed Vacuum and kept at À20ꢀC for further analysis by the
MALDI mass spectrometer.
[4] S. P. Gygi, B. Rist, S. A. Gerber, F. Turecek, M. H. Gelb, R. Aebersold.
Quantitative analysis of complex protein mixtures using isotope-coded
affinity tags. Nat. Biotechnol. 1999, 17, 994.
Mass spectrometric analysis
[5] P. L. Ross, Y. N. Huang, J. N. Marchese, B. Williamson, K. Parker, S. Hattan,
N. Khainovski, S. Pillai, S. Dey, S. Daniel, S. Purkayastha, P. Juhasz,
S. Martin, M. Bartlet-Jones, F. He, A. Jacobson, D. J. Pappin. Multiplexed
protein quantitation in Saccharomyces cerevisiae using amine-reactive
isobaric tagging reagents. Mol. Cell. Proteomics 2004, 3, 1154.
[6] For example, (a) M. B. Goshe, R. D. Smith. Stable isotope-coded
proteomics mass spectrometry. Curr. Opin. Biotechnol. 2003, 14, 101;
(b) W. A. Tao, R. Aebersold. Advances in quantitative proteomics via
stable isotope tagging and mass spectrometry. Curr. Opin. Biotechnol.
2003, 14, 110; (c) M. R. Flory, T. J. Griffin, D. Martin, R. Aebersold,
Advances in quantitative proteomics using stable isotope tags.
Trends Biotechnol. 2002, 20, S23; (d) S. D. Patterson. Proteomics: The
industrialization of protein chemistry. Curr. Opin. Biotechnol. 2000,
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2001, 12, 607; (f) R. Aebersold, D. R. Goodlett. Mass spectrometry in
proteomics. Chem. Rev. 2001, 101, 269; (g) M. Hamdan, P. G. Righetti.
Modern strategies for protein quantification in proteome analysis:
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carboxylic groups. Mol. Cell. Proteomics 2008, 7, 800; (m) J. Zhang, L.
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All the mass spectrometric analysis described here with peptides and
proteins was performed with a MALDI- time-of-flight (TOF)/TOF 4800
mass spectrometer (Applied Biosystems). The dried protein samples
were mixed with 100 mL of the solution consisting of 50% CH3CN,
0.1% trifluoroacetic acid (TFA). The protein digest sample or peptide
sample solution (0.5 mL) was spotted on the MALDI plate followed
by the addition of 0.5 mL of a matrix consisting of 5 mg/mL
of a-cyano-4-hydroxycinnamic acid in 50% acetonitrile and 0.1%
TFA. The mass spectra were acquired automatically in the positive
mode, and a total of 1000 shots were accumulated per spectrum.
The mass range was selected to be between 600 and 4000m/z.
Identification of proteins
For identification of the protein, the MASCOT search engine
(Matrix Science, V2.1) was utilized to perform the search for the
obtained MS data. The following parameters were used for the
search: enzyme, trypsin; allowed missed cleavages, 1; variable
modification, oxidation of methionine. The mass tolerance for
precursors was set to Æ50 ppm for MS. The fixed modification
was introduced for IAA-modified peptides (+133 Da) and
NEM-modified peptides (+125 Da).
Acknowledgements
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.
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Copyright © 2012 John Wiley & Sons, Ltd.
J. Mass Spectrom. 2012, 47, 1546–1553

Peak enhancement by cysteine modification
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J. Mass Spectrom. 2012, 47, 1546–1553
Copyright © 2012 John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/jms