A new charge derivatization procedure for peptide sequencing

Article abstract of DOI:10.1039/b505950j

The peptide sequencing was deduced by a latest approach of charge derivatization using mass spectrometry. The mass difference between consecutive ions within a series allows the determination of the identity of the consecutive amino acids and thus to dedu

Full text of DOI:10.1039/b505950j

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C O M M U N I C A T I O N
A new charge derivatization procedure for peptide sequencing
Denekamp Chagit,* Emilia Rabkin and Alexander Tsoglin
Department of Chemistry, Technion – Israel Institute of Technology, Haifa 32000, Israel.
E-mail: chchagit@tx.technion.ac.il; Fax: +97248293736
Received 28th April 2005, Accepted 23rd May 2005
First published as an Advance Article on the web 10th June 2005
Derivatization of peptides by a trityl cation-containing
group and collision-induced dissociation measurements of
the cationized peptides results in informative spectra that
simplify peptide sequencing.
The determination of peptide mixtures has become a crucial
step in bioinformatics-related studies and is therefore a major
task in modern analytical biochemistry. Mass spectrometry has
become the most significant tool for proteomics research and is
widely used for the identification and sequencing of peptides.1 It
is a prominent field related, for example, to immunology studies
and protein activity.2 For reliable and confident identification
it is desirable to obtain a simple, yet informative, MS/MS
spectrum of the charged peptide. Despite other approaches
like ‘top down’3 analyses of intact proteins and peptide mass
Scheme 2 Reagents and conditions: i) Succinic anhydride, AlCl3;
fingerprinting4 there is still an indubitable interest in tandem
mass spectrometry of peptide mixtures using HPLC–MS. The
cleavage of a bond in a peptide chain can occur in either of
three types of bonds Ca–C, C–N or N–Ca, which yields six
types of fragments that are respectively labeled an, bn, cn when
a positive charge is kept by the N-terminal side, and xn, yn, zn
when the positive charge is kept by the C-terminal side.5 The
mass difference between consecutive ions within a series allows
the determination of the identity of the consecutive amino acids
and thus to deduce the peptide sequence.
Frequently, however, the collision-induced dissociation (CID)
spectra of protonated or multiply-protonated peptides show two
or more incomplete series of ions that are too complex for inter-
pretation. Consequently, various attempts were made to develop
reagents that incorporate a stable charge into the peptide, a
charge that directs the fragmentation process, prompting the
formation of one informative series of ions.6 Despite previous
work in this field most of the proposed methods form charge-
derivatized peptides that are either unstable under electrospray
ionization conditions or cleave off during CID. It is also apparent
that the proposed methods, for positive ion formation, up to
date, are based on ammonium and phosphonium ions while, to
the best of our knowledge, no charge derivatization procedure
is based on the formation of stable carbenium ions.
ii) NH2NH2, KOH, triethylene glycol; iii) n-BuLi, THF/hexane;
iv) 4,4ꢀ-dimethoxybenzophenone; v) NH2–peptide–OMe, DCC,
N-hydroxysuccinimide, CH2Cl2.8
The synthesis of TAC follows known procedures as described
in Scheme 2a. Reaction of TAC with a tripeptide (Ala–Leu–
PheOCH3) results in a complete transformation giving rise to
the derivatized peptide TAC–tripep. Addition of one equivalent
of perchloric acid before introduction to the electrospray source
results in the formation of the charge-derivatized peptide TAC-
tripep-cat (Scheme 2b).
A CID spectrum (Fig. 1) was measured for the derivatized
peptide TAC-tripep-cat using an LCQDuo ion-trap. The CID
of this ion is very informative, giving rise to the expected series
of bi ions accompanied by the ai satellites. The mass difference
between two such bi ions corresponds to the mass of an amino
acid. For comparison, the CID of the corresponding protonated
peptide was also measured under the same conditions (Fig. 2).
As seen in Fig. 2 the fragmentation product in this case is
an abundant y1 ion. We find that the presence of the cationic
moiety increases the sensitivity and facilitates the formation of
informative fragmentation products.
An MS/MS spectrum was also recorded for a charge-
derivatized octapeptide TAC-octapep-cat at m/z 1275 that
was prepared from an octapeptide with the following se-
quence AFAFAFAFOMe, using infrared multi-photon photo-
dissociation (IRMPD) and a BioAPEXIII FTICR (Fig. 3). In
this case mainly bi ions are identified in the spectrum. When
compared with protonation, an obvious advantage of charge
derivatization is that formation of various charge states is
avoided. This should significantly increase the sensitivity.
A specific advantage of our reagent is that it has a typical
absorption at 500 nm so that the desired modified peptides
can be selected from the HPLC chromatogram (see Fig. 4).
In conclusion, a new derivatization method has been developed
that can provide an exclusive series of b-ions, which makes it very
easy to identify the peptide sequence. We find that the FTICR
is not the best choice for MS/MS measurements of peptides;
however, with short irradiations using maximum laser power
better results are obtained. A CID spectrum was also measured
for the TAC-octapep-cat ion at m/z 1275 using an LCQDuo
Triarylcarbinols give rise to stable trityl cations by the loss
of water under acidic conditions (Scheme 1). These cations are
easily generated in the electrospray source. Here we report an
initial successful attempt to use a new charge derivatization
reagent that has been developed for this purpose. The proposed
reagent TAC (Scheme 2) contains a trityl alcohol that easily
forms the desired stable cation upon acidic loss of water, a
trimethylene spacer and a carboxylic acid moiety that can be
coupled to the peptide by several known methods. The presence
of a trimethylene spacer is essential as it has been shown that an
analogous reagent that lacks the spacer is less reactive towards
the linking step and the CID process that follows.7
Scheme 1
T h i s j o u r n a l i s
T h e R o y a l S o c i e t y o f C h e m i s t r y 2 0 0 5
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 2 5 0 3 – 2 5 0 4
2 5 0 3
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Fig. 3 IRMPD spectrum that was measured for TAC-octapep-cat using
a BioAPEXIII FTICR.
Fig. 1 a) CID spectrum that was measured for the charge-derivatized
tripeptide TAC–Ala–Leu–PheOCH3 using an LCQDuo Ion-trap;
b) CID spectrum that was measured for a free charge-derivatized
pentapeptide TAC–Leu–Pro–Phe–Phe–AspOH.
Fig. 4 HPLC chromatogram that was measured for derivatized
TAC-octapep-cat at 280 and 500 nm. The first peak in the bottom
chromatogram corresponds to the excess of the derivatizing agent TAC.
Notes and references
1 H. Steen and M. Mann, Nature Rev., 2004, 5, 699; J. Lill, Mass
Spectrom. Rev., 2003, 22, 182; M. Hamdan and P. G. Righetti, Mass
Spectrom. Rev., 2002, 21, 287; J. R. J. Yates, Mass Spectrom., 1998,
33, 1; D. F. Hunt, R. A. Henderson, J. Shabanowitz, K. Sakaguchi, H.
Michel, N. Sevilir, A. L. Cox, E. Appella and V. H. Engelhard, Science,
1992, 255, 1261; E. Barnea, I. Beer, R. Patoka, T. Ziv, O. Kessler, E.
Tzehoval, L. Eisenbach, N. Zavazava and A. Admon, Eur. J. Immunol.,
2002, 32, 213 and refs. cited therein.
2 K. M. J. Downard, Mass Spectrom., 2000, 35, 493; F. J. Turecˇek, Mass
Spectrom., 2002, 37, 1.
3 N. L. Kelleher, H. Y. Lin, G. A. Valaskovic, D. J. Aaserud, E. K.
Fridriksson and F. W. McLafferty, J. Am. Chem. Soc., 1999, 121, 806;
G. E. Reid and S. A. McLuckey, J. Mass Spectrom., 2002, 37, 663.
4 W. J. Henzel, C. Watanabe and J. T. Stults, J. Am. Soc. Mass Spectrom.,
2003, 14, 931.
Fig. 2 CID spectrum that was measured for the MH+ ion of
Ala–Leu–PheOCH3 using an LCQDuo Ion-trap.
5 P. Roepstorff and J. Fohlman, J. Biomed. Mass Spectrom., 1984, 11,
601; K. Biemann, Annu. Rev. Biochem., 1992, 61, 977; R. S. Johnson
and K. Biemann, Biomed. Environ. Mass Spectrom., 1989, 18, 945.
6 K. D. Roth, Z.-H. Huang, N. Sadagopan and J. T. Watson, Mass
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(not shown) and a full bi ion series was readily identified.
CID spectra were also measured for derivatized peptides with
a free C-terminus giving rise to similar results as shown above
(Fig. 1b). Peptides with up to 12 amino acids were examined. No
experiments were carried out on peptides longer than 12 amino
acids. Presently our new reagent is being tested with peptides
that contain different mixtures of all amino acids. In order
to use this procedure for a digest mixture it has to be further
optimized for small quantities. However, it is concluded that
charge derivatization with the proposed reagent, followed by
MS/MS measurements contributes to peptide characterization
by introducing informative data that are sometimes lacking in
the MS/MS spectra of protonated peptides.
7 C. Denekamp and E. Rabkin, unpublished results.
8 The peptide was dissolved in water (1 mg mL−1) and 50 lL of the
solution were introduced into an ephendorph tube with 1 mL of
acetonitrile. 50 equiv. of diisopropylethylamine were added to the
solution, followed by addition of 1–3 equiv. of the activated ester
1 (a stock solution was prepared in CH2Cl2, 10 mg mL−1). The
reaction takes 2–6 h depending on the peptide and the quantities.
The yield of the derivatization was 50–100% depending on the
peptide and the quantities. The minimum quantity of peptide used
for derivatization was 50 lg. No purification is needed before analysis
by mass spectrometry.
2 5 0 4
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 2 5 0 3 – 2 5 0 4