Impact of proline and aspartic acid residues on the dissociation of intermolecularly crosslinked peptides

Abstract

The dissociation of intermolecularly crosslinked peptides was evaluated for a series of peptides with proline or aspartic acid residues positioned adjacent to the crosslinking sites (lysine residues). The peptides were crosslinked with either disuccinimidyl suberate (DSS) or disuccinimidyl L-tartrate (DST), and the influence of proline and aspartic acid residues on the fragmentation patterns were investigated for precursor ions with and without a mobile proton. Collisionally activated dissociation (CAD) spectra of aspartic acid-containing crosslinked peptide ions, doublycharged with both protons sequestered, were dominated by cleavage C-terminal to the Asp residue, similar to that of unmodified peptides. The proline-containing crosslinked peptides exhibited a high degree of internal ion formation, with the resulting product ions having an N-terminal proline residue. Upon dissociation of the doubly-charged crosslinked peptides, twenty to fifty percent of the fragment ion abundance was accounted for by multiple cleavage products. Crosslinked peptides possessing a mobile proton yielded almost a full series of b-and y-type fragment ions, with only proline-directed fragments still observed at high abundances. Interestingly, the crosslinked peptides exhibited a tendency to dissociate at the amide bond C-terminal to the crosslinked lysine residue, relative to the N-terminal side. One could envision updating computer algorithms to include these crosslinker specific product ions-particularly for precursor ions with localized protons-that provide complementary and confirmatory information, to offer more confident identification of both the crosslinked peptides and the location of the crosslink, as well as affording predictive guidelines for interpretation of the product-ion spectra of crosslinked peptides. (J

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