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Abstract :
[en] Introduction
Due to the specificity of trypsin, tryptic peptides contain basic residues on the C-terminal side. This feature provides good ionization efficiency, and facilitates fragmentation processes. In the case of non tryptic peptides, the absence of basic residues at one extremity implicates lower fragmentation ratio and poor MS/MS spectra. Several methods have been developed to circumvent this drawback. Derivatization of peptides with compounds containing positive charge has been studied; Chen et al. (RCMS, 2004, 18, 191) demonstrated the simplification of CID spectra of tryptic peptides modified by 4-sulfophenylisothiocyanate. The result is a predominance of y-type ions. In this work, we evaluate the potential of SPITC for the de novo sequencing of unknown non-tryptic peptides containing disulfide bridges, i.e. peptide toxins from animal venoms.
Methods
2µL of peptide solution (100 µM) were diluted in 6µL NH4HCO3 50mM (pH 8.7). As peptide toxins often contain disulfide bridges, reduction (2µL DTT 50mM, 1h at 56°C) and alkylation (2µL IAA 500mM, 1h in darkness at RT) of peptides were performed before the derivatization reaction. Peptides were then adsorbed on a C18 ZipTip micro-column followed by 10 µL of 4-sulfophenylisothiocyanate (SPITC) 50mM. The column was then incubated for 6h at 56°C. Peptides were washed by TFA 0.2% and eluted in 10µL 50/50 ACN/FA 0.1%, before being spotted in 2,5-DHB. MS experiments were performed using a Bruker Ultraflex II MALDI-TOF/TOF. FlexControl 3.0, FlexAnalysis 3.0, BioTools 3.2 and SequenceEditor 3.2 softwares (Bruker Daltonics, Bremen) were used for data acquisition and interpretation.
Preliminary data
According to our first results, SPITC derivatization allows in positive mode to direct the fragmentation thanks to the acidic character of the sulfonate moiety present on the modified molecule. Indeed, a large series of y-type ions is found in the CID spectra allowing determining easily large sequence tags. Moreover, the number of C-terminus ions (b- and a-type ions) decreases, which improve the simplification of MS/MS spectra.
Due to this fragmentation pattern, SPITC derivatization is clearly valuable for the sequencing of peptides that are not described in databases (de novo sequencing). For example, animal venoms are composed of several hundreds of peptides that are poorly studied, up to now. These peptides display a high importance for pharmaceutical applications and their sequencing is, as a consequence, of prime interest. Peptide toxins, which are not resulting from an enzymatic digestion, are however difficult to sequence by classical MS/MS methods. In this work, we demonstrate that the modification of peptide toxins with SPITC reagent is suitable for “real” de novo sequencing. The method was applied to isolated peptides as well as chromatographic fractions that contain up to 30 toxins.
The perspectives of this work rest on the study of the SPITC modified peptides in negative mode. We expect to obtain a better sensitivity due to the presence of the negative sulfonic acid group at the N-terminus extremity, and also interesting MS/MS spectra including mainly a- or b-type ions. The final challenge will be the application of the protocol to high throughput sequencing of peptide toxins from a large variety of animal venoms.
Novel aspect
De novo sequencing of unusual non-tryptic peptides thanks to 4-sulfophenylisothiocyanate derivatization by post-source decay MALDI-MS