Ionic Reagent for Controlling the Gas-Phase Fragmentation Reactions of Cross-Linked Peptides

Abstract

Chemical cross-linking combined with proteolytic digestion and mass spectrometry (MS) is a promising approach to provide inter- and intramolecular distance constraints for the structural characterization of protein topologies and functional multiprotein complexes. Despite the relative straightforwardness of these methodologies, the identification and characterization of cross-linked proteins presents a significant analytical challenge, due to the complexity of the resultant peptide mixtures, as well as the array of inter-, intra-, or "dead-end"-cross-linked peptides that may be generated from a single cross-linking experiment. To address these issues, we describe here the synthesis, characterization, and initial evaluation of a novel "fixed charge" sulfonium ion-containing crosslinking reagent, S-methyl 5,5'-thiodipentanoylhydroxysuc-cinimide. The peptide products obtained by reaction with this reagent are all shown to fragment exclusively via facile cleavage of the C-S bond directly adjacent to the fixed charge during CID-MS/MS, resulting in the formation of characteristic product ions that enable the presence and type (i.e., inter, intra, or dead-end) of the cross-linked products to be readily determined, independently of the "proton mobility" of the precursor ion. Subsequent isolation and dissociation of these products by MS3 provides additional structural information required for identification of the peptide sequences involved in the cross-linking reactions, as well as for characterization of the specific site(s) at which cross-linking has occurred. The specificity of these gas-phase fragmentation reactions, as well as the solubility and stability of the cross-linking reagent under aqueous conditions, suggests that this strategy holds great promise for use in future studies aimed at the structural analysis of large proteins or multiprotein assemblies.