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