Negative ion fragmentations of disulfide-containing cross-linking reagents are competitive with aspartic acid side-chain-induced cleavages.

It has been shown that the disulfide moiety in the chemical cross-linking reagent dithiobis(succinimidyl)propionate (DSP), which is similar in structure to the natural cystine disulfide, cleaves preferentially to the peptide backbone in the negative ion mode. However, the tandem mass (MS/MS) spectra of peptides in the negative ion mode are often dominated by products arising from low-energy, side-chain-induced processes, which may compete with any facile cross-linker fragmentations and complicate identification of chemical cross-links in a complex mixture. Two disulfide-containing crosslinking reagents similar to DSP, but with varying spacer arm lengths, were synthesized and the MS/MS spectra of several model peptides cross-linked with these reagents were investigated. Theoretical calculations were used to describe the energetics of the cross-linker fragmentations as well as several low-energy side-chain-induced fragmentations which compete with disulfide cleavages. Altering the spacer arm length of the cross-linker, such that there is one methylene group less than in DSP, results in a more facile cleavage process, whilst the opposite is true when a methylene group is added. Of the low-energy side-chain-induced fragmentations studied, only those from aspartic acid compete significantly with those of the cross-linker disulfide. Low-energy cleavage processes from aspartic acid that compete with cross-linker fragmentations occur in the negative ion MS/MS spectra of the cross-linked peptides, irrespective of the spacer arm length. Other fragmentation pathways do not significantly interfere with low-energy disulfide cleavage, making the presence of additional product ions in the MS/MS spectrum diagnostic for the presence of aspartic acid.