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

摘要

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 cross-linking 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 MS~(3) 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.