Development of a novel imidazolium-based aromatic quaternary ammonium tag: Synthesis and application to the efficient analysis of cysteinyl-peptides by mass spectrometry

Article abstract of DOI:10.1002/rcm.6785

RATIONALE Chemical derivatization is a very promising technique for improving analysis of peptides by mass spectrometry (MS). In this study, a novel kind of imidazolium-based aromatic quaternary ammonium tag, 1-[3-[(2-iodo-1-oxoethyl)amino]propyl]-3-butyl

Full text of DOI:10.1002/rcm.6785

Research Article
Received: 21 September 2013
Revised: 30 October 2013
Accepted: 12 November 2013
Published online in Wiley Online Library
Rapid Commun. Mass Spectrom. 2014, 28, 256–264
(wileyonlinelibrary.com) DOI: 10.1002/rcm.6785
Development of a novel imidazolium-based aromatic quaternary
ammonium tag: Synthesis and application to the efficient
analysis of cysteinyl-peptides by mass spectrometry
1
1,2
1
1
2
*
2**
Xiaoqiang Qiao , Rui Wang , Hongyuan Yan , Tao Wang , Qun Zhao , Lihua Zhang
and Yukui Zhang2
1Key Laboratory of Pharmaceutical Quality Control of Hebei Province & College of Pharmaceutical Sciences, Hebei University,
Baoding 071002, China
2Key Laboratory of Separation Science for Analytical Chemistry, National Chromatographic Research & Analysis Center, Dalian
Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China
RATIONALE: Chemical derivatization is a very promising technique for improving analysis of peptides by mass
spectrometry (MS). In this study, a novel kind of imidazolium-based aromatic quaternary ammonium tag, 1-[3-[(2-
iodo-1-oxoethyl)amino]propyl]-3-butylimidazolium bromide (IPBI), designed with strong gas-phase basicity and a
permanent positive charge, was firstly synthesized and further used for derivatization of cysteinyl-peptides with
improved ionization efficiency and higher charge states.
METHODS: Both the model peptides and tryptic digests of proteins were used to evaluate the effect of IPBI
derivatization on the MS performance of the derivatized peptides, and the results were further compared with
the commonly used iodoacetamide (IAA) tag. Matrix-assisted laser desorption/ionization time-of-flight
(MALDI-TOF)-MS and electrospray ionization (ESI)-MS were used to evaluate the ionization efficiency and charge
states of the derivatized peptides.
RESULTS: With model peptides as samples, a nearly 100% derivatization efficiency and superior stability were achieved
via IPBI derivatization. By further analysis of both standard peptides and tryptic protein digests, the ionization efficiency
and charge states of IPBI-derivatized peptides could be remarkably improved. For example, for protein bovine serum
albumin, compared with the commercial available IAA tag, the identification efficiency of cysteinyl-peptides was
increased about 67% by combining with IPBI derivatization.
CONCLUSIONS: The results indicated that the novel tag is an effective derivatization reagent for cysteinyl-peptide
identification. We hope it could be further used for high-efficiency cysteinyl-peptide identification in proteome
research, especially those with low abundance and poor ionization efficiency. Copyright © 2013 John Wiley &
Sons, Ltd.
In the post-genomic era, protein research has been paid much
attention. Because of the superior sensitivity, accuracy,
and ability to provide structural information, the mass
spectrometry (MS)-based technique has increasingly become
the analytical method of choice in the field of proteome research
in recent years.[1–4] However, for comprehensive peptide/
protein profiling, a major limitation of MS-based proteomics
is the inefficient and differential ionization of peptides
with different size, charge, hydrophobicity, and secondary
structure.[5] In particular, the high complexity and tremendous
dynamic range of real samples (i.e. human plasma) render the
detection of low-abundance peptides still a challenge.[6]
Therefore, the development of highly sensitive detection
technology for the analysis of peptides with low-abundance
or low-ionization efficiency is crucial in proteome research.
Chemical derivatization has long been employed in the field
of MS for various purposes, thereby dramatically expanding
the usefulness of the measurements.[7–9] More recently, various
tagging reagents have been developed and further used
for derivatization of the amino group,[10–15] the carboxyl
group,[16–21] the hydroxyl group[22,23] on peptides, as well as
those with post-translational modifications,[24,25] allowing the
analysis of peptides with improved ionization efficiency
thereby increased sensitivity. Aldehyde-based tags, butanal
and hexanal, were developed by Kulevich et al. for the
* Correspondence to: X. Q. Qiao, Key Laboratory of
Pharmaceutical Quality Control of Hebei Province &
College of Pharmaceutical Sciences, Hebei University,
Baoding 071002, China.
E-mail: xiaoqiao@hbu.edu.cn
** Correspondence to: L. H. Zhang, Key Laboratory of
Separation Science for Analytical Chemistry, National
Chromatographic Research & Analysis Center, Dalian
Institute of Chemical Physics, Chinese Academy of
Sciences, Dalian 116023, China.
E-mail: lihuazhang@dicp.ac.cn
Rapid Commun. Mass Spectrom. 2014, 28, 256–264
Copyright © 2013 John Wiley & Sons, Ltd.

Novel tag for high efficient analysis of cysteinyl-peptides
derivatization of primary amines to increase the
hydrophobicity of peptides. By analyzing both the unmodified
and butanal-modified tryptic digests, the combined sequence
coverage of bovine serum albumin (BSA) increased by 7%,
providing complementary peptide identification.[11] Ko
and Brodbelt reported the derivatization of the carboxyl
groups of peptides with benzylamine, 1-benzylpiperazine,
carboxymethyl trimethylammonium chloride hydrazide, and
(2-aminoethyl)trimethylammonium chloride hydrochloride
(AETMA). The improved electron transfer dissociation
(ETD) efficiencies and greater c- and z-ion populations were
obtained via derivatization, especially for the fixed charge
reagent, AETAM.[20]
Recently, cysteine has been paid much attention due to its
high reactivity, low abundance, and universal distribution
in a variety of proteomes.[26] Biological thiols, such as
glutathione and thiol proteins, are critical physiological
components of fluids and tissues, involved in a variety of
important biological events, such as cellular redox signaling
and antioxidant defenses, modulation of various cellular
activities, and so on.[27] To increase the ionization efficiency
of cysteinyl-peptides, a variety of hydrophobic alkyl tags,
such as 2-iodo-N-octylacetamide, 2-iodo-N-dodecylacetamide
and 2-iodo-N-benzylacetamide, were developed.[28,29] For
example, the limit of detection of B-type Natriuretic
Peptide was reduced ~3.5-fold via 2-iodo-N-octylacetamide
derivatization.[30] Zabet-Moghaddam et al. reported,
using hydrophobic alkyl tag iodoacetanilide (IDA) for
derivatization of cysteinyl-peptides, a 4.5-6-fold increment
in the peak intensities for model peptides.[31] Quaternary
ammonium tags[32] could introduce a permanent positive
charge to the modified peptides, benefiting for the
analysis of peptides difficult to protonate in MS. For example,
EXPERIMENTAL
Chemicals and reagents
1-Butylimidazole, iodoacetic acid, tris(2-carboxyethyl)phosphine
hydrochloride (TCEP), iodoacetamide (IAA), catalase from
bovine liver, BSA, and TPCK-treated trypsin (from bovine
pancreas) were purchased from Sigma-Aldrich (St. Louis, MO,
USA). 3-Bromopropylamine hydrobromide was obtained
from
Aladdin
reagent
(Shanghai,
China).
1-(3-
Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride
(EDC∙HCl) and peptides with sequences CDPGYIGSR,
ALVCEQEAR, MECFG, and KEEPPHHEVPESETC were
from GL Biochem (Shanghai, China). Trifluoroacetic acid
(TFA) was purchased from Acros Organics (Geel, Belgium).
α-Cyano-4-hydroxycinnamic acid (CHCA) was obtained
from Bruker Daltonics (Bremen, Germany). HPLC-grade
acetonitrile was from Merck (Darmstadt, Germany). All
inorganic reagents were analytical-reagent grade and other
reagents were of HPLC grade. Water was purified by a
Milli-Q system (Millipore, Molsheim, France).
Synthesis of 1-(3-aminopropyl)-3-butylimidazolium
bromide
The synthetic procedure for 1-(3-aminopropyl)-3-butylimidazolium
bromide was according to the previous report.[35] In brief,
1-butylimidazole (1.0 g, 8 mmol) and 3-bromopropylamine
hydrobromide (1.2 g, 5 mmol) were firstly dissolved in 10 mL
of anhydrous ethanol, followed by refluxing at 80 °C for 24 h
under nitrogen atmosphere. Subsequently, ethanol was
removed under vacuum and the crude residue was dissolved
in 2 mL of water that was brought to pH ~8 by the addition
of solid potassium hydroxide. Finally, the water was removed
under vacuum again, and the crude residue was redissolved
in 30 mL of tetrahydrofuran/methanol (1:1, v/v), filtered, dried
under vacuum, and thoroughly washed with dichloromethane
to yield the final product (yield, 75%).
a
quaternary ammonium tag, (3-acrylamidopropyl)-
trimethylammonium chloride (APTA), was developed by
Vasicek and coworkers for alkylation of cysteinyl-peptides
prior to ETD analysis. The Sequest score of tryptic digests
of BSA increased by 6-fold via APTA derivatization, allowing
highly credible identification.[33] Shimada et al. synthesized
several iodoacetic acid-based tags to improve the analysis
of cysteinyl-peptides in matrix-assisted laser desorption/
ionization time-of-flight (MALDI-TOF)-MS, and the synthetic
tag, 8-iodoacetoxy-3,6-dioxaoctyltrimethylammonium iodide,
incorporating quaternary and hydrophilic linker moieties,
showed an optimal sensitivity-enhancing effect for the
derivatized peptides.[34] Although a variety of tags have
been developed for thiol group derivatization in peptides,
the improvement in ionization efficiency is relatively limited
and often peptide-dependent. Therefore, the development of
novel tags is an imperative task for highly efficient cysteinyl-
peptide identification.
Herein, an imidazolium-based aromatic quaternary
ammonium tag, 1-[3-[(2-iodo-1-oxoethyl)amino]propyl]-3-
butylimidazolium bromide (IPBI), designed with strong
gas-phase basicity and a permanent positive charge, was
synthesized and further exploited for the derivatization
of thiol groups in peptides. Through the analysis of both
standard peptides and tryptic protein digests, our results
demonstrate that highly efficient cysteinyl-peptide
identification can be achieved by MALDI-TOF MS and
electrospray ionization (ESI)-MS.
Synthesis of IPBI
1-(3-Aminopropyl)-3-butylimidazolium bromide (125 mg,
0.48 mmol) was firstly dissolved in 2 mL of acetonitrile/water
(1:4, v/v) and the resulting mixture was stirred at 0 °C for
10 min. Then, iodoacetic acid (54 mg, 0.21 mmol) and
EDC∙HCl (68 mg, 0.35 mmol) were sequentially added, and
the resulting mixture was stirred at 0 °C for an additional
1 h. Finally, the crude product was purified by reversed-phase
HPLC with acetonitrile/water containing 0.1% (v/v) TFA as
the mobile phase to yield the final product (yield, 73%).
Fourier transform infrared (FT-IR) measurement was
performed using a Bruker Vertex 70 spectrometer (Bruker
Optik GmbH, Ettlingen, Germany) in the transmission mode.
NMR spectrum was obtained on a Bruker AVANCE III
600 MHz spectrometer (Bruker, Bremen, Germany), and
high-resolution (HR) MS spectrum was determined on a Bruker
apex ultra 7.0 T Fourier transform mass spectrometer (Bruker,
Bremen, Germany). 1H NMR (600 MHz, DMSO) δ = 9.15
(s, 1H), 7.17 (s, 1H), 7.09 (s, 1H), 7.00 (s, 1H), 4.19–4.16
(m, 4H), 3.63 (s, 2H), 1.98–1.94 (m, 2H), 1.79–1.76 (m, 2H),
1.27–1.23 (m, 4H), 0.91 (t, 3H, J = 7.4 Hz). HRMS, m/z:
Rapid Commun. Mass Spectrom. 2014, 28, 256–264
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X. Qiao et al.
350.0719. The HRMS, NMR, and FT-IR spectra are shown
in Supplementary Figs. S1, S2, and S3 (see Supporting
Information).
MS analysis
MALDI-TOF MS experiments were performed on a Bruker
Ultraflex III TOF/TOF mass spectrometer (Bruker, Bremen,
Germany) in positive ion reflectron mode. An aliquot of 1 μL
of native or derivatized peptides was directly deposited and
dried on the polished steel target, followed by 1 μL of matrix
solution (7 mg/mL CHCA in 0.1% TFA/60% acetonitrile)
deposition. The instrument was immediately calibrated using
standard peptides before experiments. MS spectra were
processed and analyzed with the software FlexAnalysis
(version 3.0) and BioTools (version 3.2) (Bruker, Bremen,
Germany). The protein was identified by peptide mass
fingerprinting (PMF), and database searching was performed
on the MASCOTserver (Matrix Science, London, UK) to search
the database of bovine, with parameters as following: (i)
enzyme: trypsin; (ii) fixed modification: carbamidomethyl (C)
or modifying cysteine residue with 222.16064 Da for IPBI
derivatization; (iii) variable modification: oxidation (M); (iv)
peptide mass tolerance: 200 ppm; (v) missed cleavages: 2.
A Finnigan surveyor MS pump coupled with a LTQ linear
ion trap mass spectrometer (both Thermo Finnigan, San Jose,
CA, USA) were used for ESI-MS determination.
Peptide derivatization
In a typical experiment, 5 μL of 1 mg/mL peptides ALVCEQEAR,
CDPGYIGSR, KEEPPHHEVPESETC or 0.5 mg/mL MECFG was
initially mixed with 20 μL of 10 mM TCEP in 50 mM Tris-HCl
buffer (pH ~8.4). After the peptides were reduced at 50 °C
for 1 h, an aliquot of 50 μL of 50 mM IPBI was added,
followed by incubation at 37 °C in the dark for 2 h with
end-over-end rotation. The derivatized peptides could be
directly applied for MALDI-TOF MS or ESI-MS analysis
without any additional cleanup step. The alkylation
procedure of the peptides with IAA or IDA was similar to
the peptide derivatization experiment, except that 50 mM
of IPBI was replaced by 50 mM of IAA or IDA.
Protein derivatization and digestion
In brief, 1.0 mg of protein catalase or BSA was firstly
dissolved in 70 μL of 8 M urea and then denatured at 56 °C
for 1 h. Subsequently, 70 μg of proteins were reduced by
10 mM TCEP at 50 °C for 1 h. After cooling to room
temperature, 50 μL of 50 mM IPBI were added, followed by
further incubation at 37 °C in the dark for 2 h with end-
over-end rotation. After the proteins were desalted and
further re-dissolved in 300 μL of 60 mM Tris-HCl buffer
(pH 8.3), trypsin was added with enzyme/protein ratio of
1:20 (w/w). The sample was incubated at 37 °C overnight,
ready for analysis. The alkylation procedure of the protein with
IAA was similar to the derivatization procedure, except that
50 mM of IPBI was replaced by 50 mM of IAA.
RESULTS AND DISCUSSION
Gas-phase basicity and hydrophobicity are crucial factors
affecting the ionization of analytes by MS.[5,36] In the present
study, a novel kind of imidazolium-based aromatic tag
was designed and synthesized by a quaternization reaction
(Fig. 1(a)). The nitrogen atoms in imidazole give the tag strong
gas-phase basicity, while the introduced n-butyl and n-propyl
groups could attenuate the hydrophilicity of the quaternized
cation tag. These structural characteristics are particularly
beneficial for subsequent MS analysis. By further activation
with iodoacetic acid, the tagging agent could readily react
with the thiol group of cysteine via alkylation derivatization
(Fig. 1(b)).
HPLC analysis
HPLC experiments were performed using a P230II system and
a Sinchrom ODS-AP column (5 μm, 300 Å, 4.6 × 250 mm i.d.,
Dalian Elite Analytical Instruments, Dalian, China). Eluent A
was 100% water with 0.1% (v/v) TFA; eluent B was 100%
acetonitrile with 0.1% (v/v) TFA. The gradient was set as
follows: 0 min, 5% B, 13 min, 31% B, with a flow rate of
1.0 mL/min. The UV wavelength was 214 nm.
Derivatization efficiency
The model peptide CDPGYIGSR with the cysteine residue
located in the N-terminal part was firstly used to evaluate the
derivatization efficiency. Figure 2(a) shows the representative
chromatograms of native peptide CDPGYIGSR and that
Figure 1. Scheme of IPBI synthesis (a) and peptide/protein derivatization (b).
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Novel tag for high efficient analysis of cysteinyl-peptides
Figure 2. Chromatograms of peptides CDPGYIGSR (a) and ALVCEQEAR (b) before
(1) and after (2) IPBI derivatization. a′ and b′ are the MALDI-TOF MS spectra of
IPBI-derivatized peptides. Peaks* are the peptide peaks.
derivatized by IPBI. It can be seen that, under the optimal
Effect of derivatization on ionization efficiency
derivatization conditions, the peak representing the native
peptide had fully disappeared and converted into the
corresponding derivatized product. The peptide derivative
was further verified via MALDI-TOF MS (Fig. 2(a′)).
The cysteine residues located in the interior domain of
the peptide possessing relative large steric hindrance are
more difficult to thoroughly label during the derivatization
procedure. Therefore, the model peptide with the sequence
ALVCEQEAR was further used to evaluate the derivatization
efficiency. Similarly, the native peptide peak completely
disappeared and converted into the corresponding derivative
(Fig. 2(b)), indicating a 100% derivatization efficiency. The above
results demonstrated that high derivatization efficiency could be
achieved by the developed tag.
The stability of peptide derivatives was further investigated
with the model peptide ALVCEQEAR as the sample. As shown
in Supplementary Fig. S4 (Supporting Information), even though
the IPBI-derivatized peptides were stored at room temperature
for up to 1 week, no noticeable change in the MALDI-TOF MS
profiling was observed, indicating its good stability. As extra
reagents would not induce a side-chain reaction, therefore, no
additional cleanup step is needed before MS analysis.
IAA is the most commonly used tag for thiol alkylation in
proteome research. Thus, the effect of derivatization on the
ionization efficiency of peptides via IPBI was firstly compared
with the IAA-modified counterpart. For the model peptides
CDPGYIGSR and ALVCEQEAR, an aliquot of IPBI-derivatized
peptide was respectively mixed with its IAA-modified
counterpart in equimolar quantities and subsequently subjected
to MALDI-TOF MS analysis. According to the representative
MALDI-TOF MS spectra shown in Figs. 3(a) and 3(b), the
signal-to-noise (S/N) ratios of IPBI-derivatized peptides were
respectively 62 and 97 times (n=5) higher than that of those
labeled with commercial IAA. For peptides MECFG and
KEEPPHHEVPESETC, most strikingly, even though the S/N
ratios of the peptides derivatized via IPBI reached 278 and
1250, respectively, the peptide peaks representing the IAA-
modified species were still not observable (Figs. 3(c) and 3(d)),
indicating that the ionization efficiency of the peptides
derivatized via IPBI was largely increased.
To further evaluate the effect of derivatization on ionization
efficiency, peptides derivatized by IPBI were also compared
with its native cognates. For peptides CDPGYIGSR and
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X. Qiao et al.
Figure 3. MALDI-TOF mass spectra of equimolar mixtures of IPBI- and IAA-derivatized peptides
CDPGYIGSR (a), ALVCEQEAR (b), MECFG (c), and KEEPPHHEVPESETC (d).
ALVCEQEAR, the ionization efficiency was respectively
is roughly equal to the IAA-modified counterpart, the
increased by 100- and 114-fold (n = 5) via IPBI derivatization
ionization efficiency of peptides CDPGYIGSR, MECFG,
and KEEPPHHEVPESETC were respectively increased by
20-, 627-, and 4-fold via IPBI derivatization, which further
demonstrated that IPBI is an efficient tag for highly sensitive
peptide detection in MS.
(Table
1
and Supplementary Fig. S5, Supporting
Information). Moreover, the developed tag is more efficient
than a recently reported tag, IDA,[31] which has been used
to increase peptide peak intensities in MALDI-TOF MS.
Compared with those labeled by IDA, the ionization
efficiency of peptides CDPGYIGSR, ALVCEQEAR, and
KEEPPHHEVPESETC were respectively increased by 43-, 4-
, and 60-fold (n = 5) via IPBI derivatization (Table 1 and
Supplementary Fig. S6, Supporting Information).
MS/MS analysis of model peptide
To further evaluate the effect of derivatization on collision-
induced dissociation (CID) fragmentation, the model
peptide CDPGYIGSR was respectively derivatized by IAA
and IPBI and further analyzed via MALDI-TOF MS/MS.
The MS/MS spectra and the peak map are shown in Fig. 4.
It can be seen that four b-type ions and three y-type ions
are found from the peptide derivatized via IAA (Fig. 4(a))
while respectively three b-type ions and three y-type ions
can be successfully recognized from the peptide derivatized
via IPBI (Fig. 4(b)). Thus, the product ions could be used for
the deduction of the peptide sequence.
Furthermore, in ESI-MS, peptides derivatized by IPBI also
showed improved ionization efficiency. For example, even
though the ionization efficiency of the peptide ALVCEQEAR
Table 1. S/N ratios of model peptides derivatized by IPBI
vs that modified by IAA, IDA or the native cognates
IPBI/IAA IPBI/Native IPBI/IDA
Effect of derivatization on charge states
CDPGYIGSR
ALVCEQEAR
MECFG
62
97
278*
1250*
100
114
472*
3602*
43
4
180*
60
Higher charge states are beneficial for the highly confident
identification of peptides and proteins by MS, because the
ion’s S/N ratio, mass resolving power, and mass accuracy
are all proportional to the charge states of analytes in the
highest resolution mass analyzer (such as ion cyclotron
resonance and orbitrap). Furthermore, for the relative novel
KEEPPHHEVPESETC
*The data represent the S/N values of the peptides
derivatized by IPBI.
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Novel tag for high efficient analysis of cysteinyl-peptides
Figure 4. CID product ion mass spectra of the peptide CDPGYIGSR derivatized
with IAA (a) and IPBI (b).
dissociation model, ETD and electron capture dissociation
(ECD), with the increment of the square of ion charge, the
MS/MS efficiency could be increased.[37] Therefore, the effect
of derivatization on the charge states of peptides was also
investigated. The average charge state was calculated using
the formula:
where n represents the number of ion-charge states while ci
and Ai respectively represent the net charge of the i-th charge
state and the signal intensity of the i-th charge state.
Figure 5 shows the representative ESI mass spectra of
the peptide CDPGYIGSR derivatized by IAA and IPBI,
respectively. When the peptide was modified with IAA,
both the doubly charged species and the singly charged
species were observed, giving an average charge state
of 1.89 (Fig. 5(a)). However, after the peptide had
n
n
caverage ¼ ∑ ciAi=∑ Ai
i
i
Figure 5. ESI mass spectra of peptide CDPGYIGSR respectively derivatized by
IAA (a) and IPBI (b).
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X. Qiao et al.
Analysis of tryptic digests of proteins
To evaluate the efficiency of IPBI derivatization on
complex samples, protein catalase containing four
a
cysteine residues was firstly analyzed. The protein was
respectively derivatized via IAA and IPBI, digested with
trypsin and further analyzed via MALDI-TOF MS. Figure 6
shows the representative MS spectrum of an equimolar
mixture of IPBI- and IAA-modified peptides. For IAA
derivatization, only one cysteinyl-peptide with the
sequence R.LGPNYLQIPVNCPYR.A was recognized.
However, after it was modified with IPBI, the two
strongest peaks were recognized as the cysteinyl-peptides
R.LGPNYLQIPVNCPYR.A and R.LCENIAGHLK.D. Most
strikingly, peptide R.LCENIAGHLK.D, which was not
even detected via IAA modification, gave the highest
signal intensity via IPBI derivatization.
Figure 6. MALDI-TOF mass spectrum of equimolar mixtures
of tryptic digests of protein catalase respectively derivatized
by IAA and IPBI. Peaks* represent the non-cysteinyl-peptides
recognized from the protein.
BSA is a more complex protein with 607 amino acid units,
of which 35 amino acids are cysteine residues. Thus, the
developed tag was further used for the analysis of tryptic
digests of BSA, and the results were further compared with
the commonly used IAA tag. By combining the three
consecutive results, in total 15 cysteinyl-peptides were
identified via IAA derivatization (Table 2). However, when
it was labeled by IPBI, a total of 21 cysteinyl-peptides were
confidently recognized, among which 10 were not even
detected via IAA derivatization. Thus, the identification
efficiency of cysteinyl-peptides was increased about 67%
(10/15) by combining with IPBI derivatization. It clearly
shows that highly efficient peptide detection could be
achieved by combining with IPBI derivatization in a
relatively complex sample.
beenderivatized by IPBI, the singly charged species
completely disappeared, and the triply charged species
could be observed, achieving an average charge state of
2.07 (Fig. 5(b)). A dramatic shift toward higher charge states
has also been observed for the peptides ALVCEQEAR,
MECFG and KEEPPHHEVPESETC. As shown in Sup-
plementary Fig. S7 (Supporting Information), the average
charge state of the peptides derivatized by IPBI could be
respectively increased to 2.67, 1.88, and 3.51 from 1.91,
1.00, and 2.94. Thus, the charge states of the derivatized
peptides could be simultaneously increased with the
developed tag.
Table 2. The identified cysteinyl-peptides from BSA modified with IAA and IPBI
No
Position
Peptide sequence
No of cysteines
IAA
IPBI
1
76-88
K.TCVADESHAGCEK.S
2
1
2
1
1
1
1
1
3
1
2
2
1
2
2
1
1
2
1
1
1
2
1
3
1
√
√
√
√
√
√
√
√
√
√
2
89-100
K.SLHTLFGDELCK.V
3
106-117
118-130
118-138
139-151
139-155
223-228
264-285
286-297
298-309
300-309
310-318
310-340
375-386
387-401
413-420
460-468
483-489
483-495
483-498
499-507
508-523
581-597
588-597
R.ETYGDMADCCEK.Q
K.QEPERNECFLSHK.D
K.QEPERNECFLSHKDDSPDLPK.L
K.LKPDPNTLCDEFK.A
K.LKPDPNTLCDEFKADEK.K
R.CASIQK.F
4
5
√
√
6
7
8
9
K.VHKECCHGDLLECADDRADLAK.Y
K.YICDNQDTISSK.L
√
√
√
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
√
√
√
√
K.LKECCDKPLLEK.S
K.ECCDKPLLEK.S
K.SHCIAEVEK.D
K.SHCIAEVEKDAIPENLPPLTADFAEDKDVCK.N
K.EYEATLEECCAK.D
K.DDPHACYSTVFDKLK.H
K.QNCDQFEK.L
√
√
√
√
√
√
√
√
√
R.CCTKPESER.M
R.LCVLHEK.T
R.LCVLHEKTPVSEK.V
R.LCVLHEKTPVSEKVTK.C
K.CCTESLVNR.R
R.RPCFSALTPDETYVPK.A
K.CCAADDKEACFAVEGPK.L
K.EACFAVEGPK.L
√
√
√
√
√
√
√
√
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Copyright © 2013 John Wiley & Sons, Ltd.
Rapid Commun. Mass Spectrom. 2014, 28, 256–264

Novel tag for high efficient analysis of cysteinyl-peptides
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CONCLUSIONS
An imidazolium-based aromatic quaternary ammonium
tag, IPBI, was designed, synthesized, and further exploited
for cysteinyl-peptide derivatization. The high derivatization
efficiency and superior stability of IPBI derivatives could
ensure good reproducibility of sample labeling. Once peptides
have been labeled with IPBI, the group with strong gas-phase
basicity and a permanent positive charge could be introduced.
Thus, both the ionization efficiency and charge states of
derivatized peptides could be remarkably improved, which
was proved by applying the developed tag to derivatize both
standard peptides and tryptic protein digests. We expect the
novel tag will be very promising in high-efficiency cysteinyl-
peptide identification, especially those with low abundance
and poor ionization efficiency.
Acknowledgements
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We are grateful for the financial support from the National
Natural Science Foundation of China (21205027), the
National Basic Research Program of China (2012CB910604)
and the Natural Science Foundation of Hebei Province
(B2012201095, B2012201052).
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Copyright © 2013 John Wiley & Sons, Ltd.
Rapid Commun. Mass Spectrom. 2014, 28, 256–264