N-terminal derivatization of peptides with isothiocyanate analogues promoting edman-type cleavage and enhancing sensitivity in electrospray ionization tandem mass spectrometry analysis

Article abstract of DOI:10.1021/ac8021136

N-terminal derivatization of peptides with Edman's reagent, phenyl isothiocyanate (PITC), promotes gas-phase Edman cleavage that yields abundant complementary b ₁ and y _n-1 ion pairs by tandem mass spectrometry (MS/MS). The formation

Full text of DOI:10.1021/ac8021136

Anal. Chem. 2009, 81, 1893–1900
N-Terminal Derivatization of Peptides with
Isothiocyanate Analogues Promoting Edman-Type
Cleavage and Enhancing Sensitivity in
Electrospray Ionization Tandem Mass
Spectrometry Analysis
Dongxia Wang,* Sunan Fang, and Robert M. Wohlhueter
Biotechnology Core Facility Branch, Division of Scientific Resources, National Center for Preparedness, Detection,
and Control of Infectious Disease, Centers for Disease Control and Prevention (CDC), Atlanta, Georgia 30333
N-terminal derivatization of peptides with Edman’s re-
agent, phenyl isothiocyanate (PITC), promotes gas-phase
Edman cleavage that yields abundant complementary b1
and yn-1 ion pairs by tandem mass spectrometry (MS/
MS). The formation of b1 ions can be utilized as a mass
tag to enhance the interpretation of MS/MS spectra and
increase the confidence of peptide identification during
mass spectrometry analysis. Derivatization of tryptic
peptides with another isothiocyanate analogue, 4-sul-
fophenyl isothiocyanate, also produces signature ions
resulting from Edman cleavage and facilitates peptide
sequencing on linear or branched peptides. The limita-
tion of these derivatizations, however, is reduced MS
signal intensities of modified peptides, due presumably
to the tags themselves. Here we have demonstrated
that several other isothiocyanate analogues bearing
basic moieties can derivatize peptides and significantly
improve the MS sensitivity of tagged analytes, while
promoting Edman fragmentation and maintaining other
sequence fragments as well.
Scheme 1. Preferential Gas-Phase Edman
Cleavage of the Peptide Derivatized by Phenyl
Isothiocyanate (PITC) or 4-Sulfophenyl
Isothiocyanate (SPITC)
Chemical derivatization of protein digests has been used to
facilitate peptide sequencing via tandem mass spectrometry (MS/
MS) in the “bottom-up” proteomic approach. MS/MS analysis of
derivatized peptides can elevate ionization efficiency, induce
favorable fragmentation patterns, or generate specific signature
fragments. As consequence, increased sensitivity of detection and
improved de novo sequencing of peptides can lead to highly
confident identification of proteins.1-4
of analytes can be determined by sequentially analyzing modified
N-terminal residues cleaved from a thiocarbamoyl derivative
labeled with Edman’s reagent, phenyl isothiocyanate (PITC). To
understand the mechanism of peptide fragmentation induced by
collision energy during MS/MS analysis, the decomposition of
PITC-derivatized peptides was investigated by Summerfield et al.6
It has been observed that collision-induced dissociation of a doubly
charged peptide derivative resulted in a pair of abundant comple-
mentary b1 and yn-1 ions (Scheme 1). This and additional
experimental evidence lead to the conclusion that gas-phase
fragmentation of a peptide modified by Edman’s reagent proceeds
by a mechanism similar to that of condensed-phase Edman
degradation, where the preferential cleavage of the first peptide
Edman degradation is a widely used method for the N-terminal
sequencing of protein and peptides.5 The N-terminal amino acids
* To whom correspondence should be addressed. E-mail: dov2@cdc.gov.
Fax: 404-639-1331.
(1) Roth, K. D.; Huang, Z. H.; Sadagopan, N.; Watson, J. T. Mass Spectrom.
Rev. 1998, 17 (4), 255–274
(2) Keough, T.; Youngquist, R. S.; Lacey, M. P. Anal. Chem. 2003, 75 (7),
156A–165A
(3) Leo´n, I. R.; Neves-Ferreira, A. G.; Valente, R. H.; Mota, E. M.; Lenzi, H. L.;
Perales, J. J. Mass Spectrom. 2007, 42 (10), 1363–1374
(4) Conrotto, P.; Hellman, U. Rapid Commun. Mass Spectrom. 2008, 22 (12),
1823–1833
(5) Edman, P. Arch. Biochem. 1949, 22 (3), 475–476
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(6) Summerfield, S. G.; Bolgar, M. S.; Gaskell, S. J. J. Mass Spectrom. 1997,
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10.1021/ac8021136 Not subject to U.S. Copyright. Publ. 2009 Am. Chem. Soc.
Published on Web 02/06/2009
Analytical Chemistry, Vol. 81, No. 5, March 1, 2009 1893

bond is promoted by nucleophilic attack of the thiocarbonyl moiety
bearing on the tag on the carbonyl group of the N-terminal peptide
bond.
resulting from trypsin digestion of ubiquitin-modified proteins and
thus lead to the formation of a unique group of signature
fragments that can be used to distinguish these branched peptides
from others.
Gas-phase Edman cleavage and the formation of b1 ions,
typically not observed in the fragmentation of protonated
peptides, can be utilized to facilitate confident protein identifica-
Despite their success in peptide sequencing and protein
identification, some inherent properties of PITC and SPITC limit
their application. The major problem is reduced sensitivity of MS
detection, presumably due to aromatic or sulfonic acid groups of
these reagents. The negative charge feature of sulfonic acid group
is the apparent cause of a 10-fold decrease in positive mode mass
detection of SPITC-derivatized peptides.13 Further decrease in
sensitivity could be expected when two sulfonation tags are
introduced into a peptide as in the case of branched peptide. The
decrease in ionization of PITC-modified peptides is also expected
because the basic amino group is blocked by nonpolar phenyl
group. The loss of PITC-derivatized peptide was reported due to
the precipitation of some modified peptides.7 In addition, it was
observed in our laboratory that excess SPITC reagent could be
eluted out together with some peptides during the separation by
reversed-phase chromatography, presumably due to the hydro-
phobicity of phenyl group, and thus could suppress the detection
of these peptides.
Here we report a study of chemical derivatization with four
novel isothiocyanate analogues in an attempt to overcome prob-
lems with PITC and SPITC described above. All of these
compounds turned out to be effective in the derivatization of model
peptides and in promoting gas-phase Edman cleavage. Most
significantly, modified peptides derivatized with these reagents
displayed higher sensitivity in mass detection than those deriva-
tized by PITC and SPITC.
,8
tion for proteomics.7 The ready identification of N-terminal
residue adds an additional constraint to database searching that
could further enhance interpretation of tandem mass spectra and
increase the confidence level of MS-based peptide sequencing. It
has been demonstrated that PITC derivatization approach can
achieve significant gain in database searching efficiency for
peptide identifications in yeast proteome.9 In a direct analysis of
a mixture of PITC-derivatized tryptic peptides by Fourier transform
ion cyclotron resonance (FTICR) mass spectrometry, van der Rest
et al. demonstrated that as few as one peptide derivative could
give rise to correct identification based on peptide mass and
N-terminal residue identity.7 Beardsley et al. investigated the
possibility of utilizing b1 ions generated from amidinated peptides
as internal calibrants for accurate mass measurements.10
Taking the advantage of the specific fragmentation of PITC
derivatives, a multiple reaction monitoring (MRM) analysis
using either b1 or yn-1 as selected product ion has been
attempted in the detection of prion protein.11
PITC is not the only chemical that induces gas-phase Edman
degradation. It was reported by several groups that a labeling
reagent, 4-sulfophenyl isothiocyanate (SPITC), used for N-terminal
sulfonation, also led to the dominant formation of yn-1 ions
,13
resulting from the fragmentation of derivatized peptides.12
Since the reagent has a structure similar to PITC, and it attaches
to peptides by the same chemistry as the latter does, it was
believed that SPITC-derivatized peptides also undergo preferential
Edman-type cleavages even though it is almost impossible to
detect b1 ions because the positive charge of this ion is
neutralized by negative charge of the sulfonic acid group
(Scheme 1). Gas-phase Edman degradation has been found to
be particularly useful in characterizing branched peptides. Utilizing
SPITC derivatization, Wang and co-workers have recently reported
a novel strategy for identifying protein ubiquitination sites with
high efficiency and specificity using matrix-assisted laser desorp-
tion/ionization (MALDI) time-of-flight (TOF) mass spectrometry
and electrospray ionization (ESI) mass spectrometry.14-16 In this
method, two tags are added to the diglycine-branched peptides
EXPERIMENTAL SECTION
Chemicals. All chemicals used in this study were of analytical
grade. Phenyl isothiocyanate, 4-sulfophenyl isothiocyanate, 3-py-
ridyl isothiocyanate (PYITC), 2-piperidinoethyl isothiocyanate
(PEITC), 3-(4-morpholino) propyl isothiocyanate (MPITC), 3-(di-
ethylamino) propyl isothiocyanate (DEPITC), and bovine serum
albumin (BSA) were obtained from Sigma Company (St. Louis,
MO).
Peptide Synthesis. Peptides were prepared with Fmoc
chemistry using solid-phase peptide synthesis method on an
AAPPTEC APEX 396 multiple peptide synthesizer (Louisville, KY).
Synthetic peptides were purified by reversed-phase (C-18) high-
performance liquid chromatography using a gradient of water-
acetontitrile solvent system containing 0.1% trifluoroacetic acid
(TFA). The correct structures of the peptides were confirmed by
mass spectrometry.
(7) van der Rest, G.; He, F.; Emmett, M. R.; Marshall, A. G.; Gaskell, S. J.
J. Am. Soc. Mass Spectrom. 2001, 12 (3), 288–295
(8) Brancia, F. L.; Butt, A.; Beynon, R. J.; Hubbard, S. J.; Gaskell, S. J.; Oliver,
S. G. Electrophoresis 2001, 22 (3), 552–559
.
.
(9) Sidhu, K. S.; Sangvanich, P.; Brancia, F. L.; Sullivan, A. G.; Gaskell, S. J.;
Wolkenhaue, O.; Oliver, S. G.; Hubbard, S. J. Proteomics 2001, 1, 1368–
Chemical Derivatization. The peptide (100 pmol) was reacted
with derivatization reagent (10 nmol) in 10 µL of solution
containing pyridine, ethanol, and ddH2O (1:1:1 vol/vol/vol) at
55 °C for 60 min. The reaction was terminated by adding 1 µL
of 1% TFA. After reaction, 1 µL of the solution was transferred
to a new 0.6 mL Eppendorf tube and dried completely by
SpeedVac for mass spectrometry analysis. For BSA, 200 µg of
the protein was digested with trypsin (50:1 w/w) in 30 µL of
reaction solution containing 25 mM ammonium bicarbonate
at 37 °C for 18 h. An amount of 1 µL of the reaction (100 pmol/
µL) was then mixed with 10 µL of PYITC (2 nmol/µL) solution
1377
(10) Beardsley, R. L.; Sharon, L. A.; Reilly, J. P. Anal. Chem. 2005, 77 (19),
6300–6309
(11) Onisko, B.; Dynin, I.; Requena, J. R.; Silva, C. J.; Erickson, M.; Carter, J. M.
J. Am. Soc. Mass Spectrom. 2007, 18, 1070–1079
(12) Wang, D.; Kalb, S. R.; Cotter, R. J. Rapid Commun. Mass Spectrom. 2004,
18, 96–102
(13) Lee, Y. H.; Kim, M. S.; Choie, W. S.; Min, H. K.; Lee, S. W. Proteomics
2004, 4, 1684–1694
(14) Wang, D.; Kalume, D.; Pickart, C.; Pandey, A.; Cotter, R. J. Anal. Chem.
2006, 78, 3681–3687
(15) Wang, D.; Xu, W.; McGrath, S. C.; Patterson, C.; Neckers, L.; Cotter, R. J.
J. Proteome Res. 2005, 4, 1554–1560
(16) Wang, D.; Cotter, R. J. Anal. Chem. 2005, 77, 1458–1466
.
.
.
.
.
.
.
.
1894 Analytical Chemistry, Vol. 81, No. 5, March 1, 2009

and underwent derivatization following the same procedure
described.
Scheme 2. Structures of Isothiocyanate
Compounds: Phenyl Isothiocyanate (PITC),
4-Sulfophenyl Isothiocyanate (SPITC), 3-Pyridyl
Isothiocyanate (PYITC), 2-Piperidinoethyl
Isothiocyanate (PEITC), 3-(4-Morpholino) Propyl
Isothiocyanate (MPITC), and
Mass Spectrometry. Derivatized peptide or peptide mixtures
were resuspended in 50% methanol and 1% acetic acid to a final
concentration of 0.5-1 pmol/µL and were introduced into the
mass spectrometer by direct infusion at a flow rate of 0.4 µL/
min. A potential of 3.00 kV was applied to the nanospray emitter
in the ion source. The MS and MS/MS analysis was performed
on a Micromass Q-Tof Ultima mass spectrometer (Waters,
Milford, MA). All mass spectra were obtained in the positive-ion
mode with different collision energies (10, 20, 30, 40, and 50 eV).
In quantitative comparison experiments, the spectra were obtained
by combining MS scans acquired within 1 or 2 min. The
acquisition of data was performed on a MassLynx data system
(version 4.0). Resulting MS/MS data were interpreted manually.
3-(Diethylamino)propyl Isothiocyanate (DEPITC)
RESULTS AND DISCUSSION
In the selection of novel N-terminal derivatization reagents
alternative to PITC and SPITC, several factors should be consid-
ered: promotion or induction of Edman cleavage in MS/MS
fragmentation, enhancement of ionization efficiency of modified
peptides, high modification efficiency, and low side reactions. For
PITC- or SPITC-labeled peptides, the key element for gas-phase
Edman-type cleavage seems to be the thiocarbonyl group formed
from the reaction between the isothiocyanate group of the
reagents and the N-terminal amino group of a peptide. In addition,
the low ionization efficiency of such derivatives likely results not
from the isothiocyanate but from the aromatic and sulfonic acid
groups. For those reasons, we targeted on compounds of isothio-
cyanate. Therefore, we felt we should consider isothiocyanate
analogues containing a basic moiety so that the overall basicity
of peptide derivatives labeled with such reagents would be
maintained and the ionization efficiency could remain as high or
higher than that of its underivatized counterpart. On the basis of
these criteria, we selected four commercially available isothiocy-
anate analogues, PYITC, PEITC, MPITC, and DEPITC, and
examined the feasibility of using them as derivatization reagents
(Scheme 2). In addition to isothiocyanate structure, all of these
reagents contain a tertiary amine group that is expected to
maintain or elevate proton affinity of modified peptides for the
enhancement of their mass sensitivity.
N-Terminal Derivatization of Model Peptides. A synthetic
peptide, IYIGPGRAF, was reacted with six reagents, respectively,
including four newly selected isothiocyanates and two old ones,
PITC and SPITC, for comparison, under identical experimental
conditions. As shown in Figure 1 and Table 1, new mass peaks
corresponding to derivatized peptides were detected from all
reaction mixtures by ESI mass spectrometry, indicating that
selected reagents are all capable of modifying target analyte. The
correct modification at the N-terminus of the peptide was
confirmed by the masses of labeled peptides and tandem mass
sequencing analysis (discussed below). The yields of derivatized
peptides from the original ones ranged from 52-100%, based on
the changes in the intensities of unmodified peptides in each
individual case. Similar to unmodified peptide, doubly protonated
species of the derivatives were dominant in the reaction products
while singly charged forms were detected in some cases. These
results demonstrate that the selected isothiocyanate analogues
are capable of modifying peptides at their N-terminus in the same
way as PITC and SPITC do.
It is not surprising to see that the peak intensities of PITC-
and SPITC-tagged peptides were much smaller than that of the
original peptide not undergoing derivatization, as determined in
the control experiment (Table 1). Although the apparent conver-
sion rate of 83% and 96% from PITC and SPITC reactions,
respectively, seems to imply that the majority of the peptide was
converted to modified derivatives, however, the combined intensi-
ties of doubly and singly charged ions of two derivatives are only
7% and 20%, respectively, of the intensity of the peptide before
derivatization. There was no significant improvement when
samples were desalted prior to MS analysis (data not shown). This
is consistent with the previous observation that the derivatization
by Edman’s reagent PITC, or sulfonation reagent SPITC, dramati-
cally reduces sensitivity in mass detection. In a previous study,8
we had observed that sensitivity to SPITC derivatives could be
influenced significantly by optimization of reaction conditions, but
in every case intensities were less than that of the corresponding,
underivatized peptide.
In contrast, derivatization with four newly selected isothiocy-
anate reagents, PYITC, DEPITC, PEITC, and MPITC, resulted
in intense peaks of the modified peptides. In comparison with the
data for PITC or SPITC reactions, more than 10-fold increases in
the peak intensities of the derivatives were achieved in most cases,
even though less peptide was modified under present conditions.
These results suggest that the basic groups carried by these
isothiocyanate analogues indeed enhance the ionization efficiency
of the analytes of interest. Further improvement could be expected
with the optimization on the derivatization procedure for individual
reactions.
It has been observed by several groups (personal communica-
tions) including ours that a peptide containing an N-terminal
Analytical Chemistry, Vol. 81, No. 5, March 1, 2009 1895

Figure 1. Mass spectrum of the peptide, IYIGPGRAF, after derivatization reaction with 3-(diethylamino)propyl isothiocyanate (DEPITC).
Table 1. Observed Peaks of a Synthetic Peptide, IYIGPGRAF, after Derivatization with Various Isothiocyanate
Reagentsa
unmodified peptide
intensity (count)
809
modified peptide
derivatization reagents
SPITC
mass (m/z)
charge
conversion rate (%)b
96
mass (m/z)
charge
intensity (count)
497.2
2+
615.7c
1208.4
575.7c
1128.5
565.2
1129.5
583.3
582.3
590.3
2+
1+
2+
1+
2+
1+
2+
2+
2+
1028
945
PITC
497.2
497.2
2+
2+
3176
4628
83
75
497
737
9374
878
9095
6616
10711
PYITC
DEPITC
497.2
497.2
497.2
497.2
2+
2+
2+
2+
8789
8052
10
52
56
100
PEITC
MPITC
control (underivatized)
18367
a At least three experiments were conducted for each derivatization reaction. b Conversion rate ) (Int.control - Int.reaction)/Int.control c Sodiated
.
peptide with one sodium attached.
glutamic acid residue can form a truncated peptide (with the loss
of its N-terminal amino acid residue) in addition to the desired
derivative in the SPITC derivatization reaction. This could be
caused by the participation of the carboxylic side chain of the
glutamic acid in Edman-type cleavage, such that some of the
derivatized peptides undergo preferential Edman degradation
which allows an easy departure of SPITC-Glu from the molecule
prior to collision-induced MS/MS environment. It is not clear
whether the side reaction occurred in reaction solution or under
MS condition during mass measurement. To test if this is a
common phenomenon with N-terminal labeling by isothiocyanate
analogues, we examined the reaction of a synthetic peptide,
EGVNDNEEGFFSAR (GFP: Glu-fibrinopeptide, m/z 785.8, 2+),
containing N-terminal glutamic acid residue, with our selected
reagents. It turns out that all reactions yielded both a modified
derivative and an unmodified N-Glu-truncated peptide at the mass
of m/z 721.3 (2+), indicating that this side product is unavoidable
(Figure 2). It was surprising to see that, in the reaction with
PEITC, the major derivatization product was at m/z 806.3 (2+)
which indicates that labeling occurred at the N-terminus of the
truncated peptide instead of the intact one (Figure 2), i.e., that
derivatization reoccurs on the truncated species after initial
labeling and Edman degradation (there was a large excess of
derivatization reagent.) Fortunately, the quantities of the modified
peptides derivatized by the reagents containing basic groups are
still dominant, and the formation of truncated peptide is relatively
low (20% or lower). Given the low yield of the side product and
the low occurrence of glutamic acid adjacent to lysine or arginine
residues in proteins, the generation of N-Glu-truncated peptides
caused by the side reaction during derivatization should not add
significant interference with peptide sequencing and protein
identification as a whole.
Tandem Mass Spectrometry of Peptide Derivatives. To
explore the fragmentation behaviors of the peptide derivatives, a
series of peptides, tag-IYIGPGRAF, modified by four selected
isothiocyanate reagents were subjected to MS/MS analysis in the
Q-Tof mass spectrometer with electrospray ionization at low
collision energy (20 or 10 eV). Fragmentation of underivatized
peptide yielded product ions from random peptide bond cleavage,
but no b1 ion was observed (Figure 3A). Although the yn-1 (y8)
ion was detected, its relative intensity is much lower than those
of other products such as y6 and y7 ions. In contrast, the MS/
MS of the four derivatized peptides all resulted in a pairs of y8
(yn-1) and b1 ions, indicating that derivatizations with those
reagents indeed promoted or induced preferential Edman-type
cleavage during low-energy collision process in a way similar
to PITC or SPITC derivatives. This cleavage occurs for the
PYITC derivative under very low collision energy (10 eV),
which does not usually induce meaningful fragmentation in an
ordinary peptide of similar size and charge state. Interestingly,
the fragmentation of PYITC derivative at 10 eV produced a
pattern similar to those generated at 20 eV for other derivatives.
Whether PYITC has high propensity compared to other
reagents in inducing Edman cleavage needs further investigation.
Unlike PITC-derivatized peptides that usually fail to produce
additional sequence fragments and are therefore limited for
purposes of protein identification via MS/MS, the most interesting
feature of our ITC derivatives is that they do not suppress the
formation of other sequence fragments in MS/MS spectra, even
while inducing N-terminal Edman cleavage. As shown in Figure
1896 Analytical Chemistry, Vol. 81, No. 5, March 1, 2009

Figure 2. MS spectra of the peptide GFP, EGVNDNEEGFFSAR, before (A) and after derivatization with the reagent (B) PYITC, (C) MPITC,
(D) DEPITC, and (E) PEITC. GFP and tGFP represent original and N-terminal Glu truncated peptides, respectively. mGFP and mtGFP stand
for derivatives from intact and N-Glu-truncated peptides, respectively.
4, the MS/MS spectra of PYITC-derivatized peptide (GFP), at a
collision energy of 30 and 40 eV, include all of the major product
ions except b2 seen with the underivatized peptide at a collision
energy of 30 eV, plus the complementary b1 and y13 ion pairs
induced by preferential cleavage. In addition, the b1 ion (m/z
266.0) of PYITC-derivatized GFP could be detected at various
collision energies ranging from 10 to 50 eV, revealing the high
stability of this characteristic ion. The formation of both Edman-
type product and other sequence fragments during peptide
fragmentation is a really significant advantage to peptide
sequencing via MS/MS, because it allows the addition of
another database searching constraint specifying the identity
of the N-terminal amino acid residue, in a way analogous to
that used with PITC-derivatized peptides,8,9 while not sacrificing
the information deduced from the fragmentation of other peptide
bonds. Therefore, we can now truly say that the gas-phase Edman
cleavage caused by peptide derivatization will facilitate protein
identification.
of intense peaks in the spectra (Figure 3B-E, Figure 4C-E) of
the peptide derivatives represented protonated tag molecules (m/z
137.01, 173.10, 171.08, and 187.08) and their counterparts, [MH
- tag] ions. The high intensities of these tag peaks presumably
attribute to their high basicity. The existence of these tag ions
could be used to validate chemical labeling.
Derivatization of Trypsin-Digested Protein. The application
of novel reagents to the chemical derivatization of peptide mixtures
resulting from digested proteins was examined. The result of
derivatization of trypsin-digested BSA by PYITC is depicted in
Figure 5, with the detailed Supporting Information in Table S-1.
Peptide derivatives were determined by mass increases and
confirmed by peptide sequencing by MS/MS analysis (data not
shown). The modifications occurring on most of the tryptic
peptides demonstrate the efficiency of this derivatization. The
intensities of the majority of the derivatized peptides are compa-
rable to those of their underivatized counterparts. Some peptide
derivatives even showed higher intensities than their unmodified
counterparts. Given the fact that the reaction mixtures were
infused directly into the mass spectrometer without preliminary
purification by liquid chromatography and along with the excess
In addition to Edman-type cleavage, the elimination of the
entire tag group form the peptide derivatives was observed as
another preferential cleavage under low collision energy. The pairs
Analytical Chemistry, Vol. 81, No. 5, March 1, 2009 1897

Figure 3. MS/MS spectra of the derivatives of the peptide, IYIGPGRAF: (A) underivatized, (B) PYITC, (C) DEPITC, (D) PEITC, and (E) MPITC.
All but one of the experiments were carried out at 20 eV collision energy. The collision energy used for PYITC derivatives was 10 eV.
of unreacted reagent, one might suspect that the ionization
efficiency of analyte to be adversely affected. These useful, higher
intensities of peptide derivatives might be further optimized by
deliberately adjusting the experiment conditions to that end.
1898 Analytical Chemistry, Vol. 81, No. 5, March 1, 2009

Figure 4. MS/MS spectra of PYITC-derivatized peptide (GFP) obtained at the collision energy of (A) 10, (B) 20, (C) 30, (D) 40, and (E) 50 eV
and (F) the spectrum of underivatized peptide at 30 eV collision energy.
Figure 5. MS spectra of the peptide mixtures resulting from trypsin digestion of bovine serum albumin (top) before and (bottom) after chemical
derivatization with PYITC. The peaks labeled with asterisks represent derivatized peptides. Double asterisks indicate the peptide was labeled
by two PYITC tags, one on N-termini and another one on the amino group of a lysine side chain. The lines between two spectra connect
underivatized peptides to their corresponding derivatives.
These results further confirm that the new derivatization
method, versus conventional PITC and SPITC strategies, signifi-
cantly increases sensitivity in mass detection. The existence of a
few unmodified peptides and the occurrence of several doubly
tagged peptides suggest the necessity for further optimization of
the conditions of the derivatization reaction.
Analytical Chemistry, Vol. 81, No. 5, March 1, 2009 1899

CONCLUSIONS
peptides and a mixture of peptides digested from a protein with
trypsin. In conclusion, novel isothiocyanate derivatization
reagents result in a significant improvement in mass detection
of tagged analytes and should enhance the identification of
proteins and peptides by promoting gas-phase Edman degrada-
tion. We have conducted promising preliminary experiments
with a synthetic branched peptide derivative with a view to
characterizing ubiquitinated proteins. Further studies are on
the way to explore the potential application of the novel
reagents on the direct identification and site mapping of
proteins modified by ubiquitin, SUMO, and other ubiquitin-
like molecules from simple or complex samples. The investiga-
tion on the application of this method in protein characterization
and quantification is also ongoing in our laboratory.
Reduced detection sensitivity of PITC- or SPITC-derivatized
peptides by mass spectrometry likely come from the aromatic or
sulfonic acid groups of these reagents. In order to improve MS
signal intensity, we investigated four carefully selected isothio-
cyanate analogues that contain basic moieties for labeling peptides.
It was observed that all of these reagents were able to label model
peptides by the same mechanism as do PITC and SPITC. In
comparison with PITC and SPITC derivatives, the MS signal
intensities of the derivatives with these four new reagents
increased at least 10-fold, implying that increased overall basicities
of the derivatized peptides indeed enhance the ionization efficiency
of the derivatized analytes. MS/MS analysis revealed that all of
the peptides derivatized with each of the new reagents yielded
complementary b1 and yn-1 ion pairs, induced by preferential
Edman-type cleavage during the low-energy collision process.
While the pair of complementary of b1 and yn-1 ions should
lead to the identification of the N-terminal amino acid residue
and to the addition of a database searching constraint, the other
sequence product ions, which are comparable to these resulted
from underivatized peptides, could be used to provide sequence
information. The method was demonstrated with synthetic
SUPPORTING INFORMATION AVAILABLE
Supplementary table. This material is available free of charge
via the Internet at http://pubs.acs.org.
Received for review October 7, 2008. Accepted January
13, 2009.
AC8021136
1900 Analytical Chemistry, Vol. 81, No. 5, March 1, 2009