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.

A New in Vivo Cross-linking Mass Spectrometry Platform to Define Protein–Protein Interactions in Living Cells

“…Various efforts have been made to address the limitations of XL-MS studies, resulting in new developments in bioinformatics tools for improved data interpretation (28 -32) and new designs of cross-linking reagents for enhanced MS analysis of cross-linked peptides (24,(33)(34)(35)(36)(37)(38)(39). Among these approaches, the development of new cross-linking reagents holds great promise for mapping PPIs on the systems level.…”

‘Fixed charge’ chemical derivatization and data dependant multistage tandem mass spectrometry for mapping protein surface residue accessibility

“…To overcome this problem, we have developed a chemical derivatization and multistage MS/MS (MS n ) based analysis strategy, involving the introduction of a 'fixed-charge' sulfonium ion to peptides or proteins containing certain structural features (e.g., the side chains of selected amino acids such as methionine [28 -31], or cysteine [32], or within a cross-linking reagent targeting lysine residues [33]. CID-MS/MS of these peptide ions results in the exclusive loss of a dialkylsulfide moiety from the modified side chain under low-energy CID-MS/MS conditions, independently of the amino acid composition and precursor ion charge state (i.e., proton mobility) of the peptide [34 -37].…”

An Isotopically Coded CID-cleavable Biotinylated Cross-linker for Structural Proteomics

“…The method is potentially applicable in LC-ESI mode, although as in other labeling studies with deuterium attention needs to be paid to possible differences in retention time of the light and heavy isotopic forms of the cross-links. This problem may be eliminated in a future version of the cross-linker with 13 C isotopic labels instead of deuterium.…”

“…Because the cleaved fragments are still isotopically coded, they can be easily detected in the spectra, and their relationships to the uncleaved parent crosslink can be determined based on mass differences. Cleavage of cross-linkers can be either done chemically (10), photoinduced (10), or done using CID (11)(12)(13). CID cleavage of crosslinks has the advantage that the cleavage reaction occurs inside the mass spectrometer and can be performed individually by automatically mass-selecting each cross-link using an "include list.…”