( y tandem mass spectrometry on the QSTAR...)Efficient Primary Structure Elucidation of Disulfide-bridged Peptides

y tandem mass spectrometry on the QSTAR Pulsar hybrid quadrupole time-of-flight mass spectrometer2, 4. The purified native peptides were digested by trypsin and Asp-N endopeptidase. The resulting peptides consisted of four to five chains crosslinked by the original disulfide bridges. The peptides were subsequently subjected to MS and MS/MS analysis. Lyophylized samples were redissolved in water/methanol (50/50) and an aliquot of water/methanol/formic acid (49/49/2) was added prior to analysis, resulting in a final peptide concentration of 5 picomoles per microliter. The instrument was tuned for a resolving power of 12'000, which provided sufficient separation of fragment ions of similar m/z and unambiguous assignment of charge states.

Results and Discussion

The complete sequences of CSTX-1 and CSTX-9 with the corresponding overlaps are given in Figure 2. Sequence comparison of CSTX-9 with CSTX-1 revealed an identity of 53% as well as identical positions of all eight Cys residues present in the N-terminal portion of the molecule. Sequence comparison with other spider toxins (Figure 3) is indicative for a disulfide bridge pattern as in the family of ion channel toxins containing the inhibitor cystine knot structural motif. The proximity of the cysteine residues and the absence of suitable cleavage sites within the amino-acid sequences of CSTXpeptides caused the classical approaches for elucidation of the disulfide bridge pattern to fail. As these approaches are based on selective chemical or enzymatic cleavage, they require the presence of defined cleavage sites between two consecutive disulfide bridges. Consequently, the basically unspecific gas-phase dissociation of the disulfide-linked peptides by tandem mass spectrometry is an attractive alternative to the classical procedures. Identification of the disulfide bridge pattern was based on observation of characteristic fragment ions generated by dissociation of
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