Developing a Novel Sulfoxide-containing MS-cleavable Homobifunctional Cysteine Reactive Cross-linker for Studying Protein-Protein Interactions

Cross-linking mass spectrometry (XL-MS) has become an emerging technology for defining protein-protein interactions (PPIs) and elucidating architectures of large protein complexes. Up to now, the most widely used cross-linking reagents target lysines. While such reagents have been successfully applied to map PPIs at the proteome-wide scale, comprehensive PPI profiling would require additional cross-linking chemistries. Cysteine is one of the most reactive amino acids and an attractive target for cross-linking owing to its unique role in protein structures. Although sulfhydryl-reactive cross-linkers are commercially available, their applications in XL-MS studies remain sparse–likely due to the difficulty in identifying cysteine cross-linked peptides. Previously, we have developed a new class of sulfoxide-containing MS-cleavable cross-linkers to enable fast and accurate identification of cross-linked peptides using multistage tandem mass spectrometry (MS n ). Here, we present the development of a new sulfoxide-containing MS-cleavable homobifunctional cysteine reactive cross-linker, B is m aleimide S ulf o xide (BMSO). We demonstrate that BMSO cross-linked peptides display the same characteristic fragmentation pattern during collision induced dissociation (CID) as other sulfoxide-containing MS-cleavable cross-linked peptides, thus permitting their simplified analysis and unambiguous identification by MS n . Additionally, we show that BMSO can complement amine- and acidic residue- reactive reagents for mapping protein interaction regions. Collectively, this work not only enlarges the toolbox of MS-cleavable cross-linkers with diverse chemistries, but more importantly expands our capacity and capability of studying PPIs in general.