Expanding the Chemical Cross-linking Tool Kit for Cross-linking Mass Spectrometry

Cross-linking mass spectrometry (XL-MS) is a powerful tool for studying protein-protein interactions and elucidating architectures of protein complexes. However, one of the inherent challenges in MS analysis of cross-linked peptides is their unambiguous identification. To facilitate this process, we have previously developed a series of amine-reactive sulfoxide-containing MS-cleavable cross-linkers. These MS-cleavable reagents have allowed us to establish a common robust XL-MS workflow that enables fast and accurate identification of cross-linked peptides using multistage tandem mass spectrometry (MSn). Although amine reactive reagents targeting lysine residues have been successful, it remains difficult to characterize protein interaction interfaces with little or no lysine residues. To expand the coverage of protein interaction regions, we present here the development of a series novel sulfoxide-containing cross-linker that target acidic residues (dihydrazide sulfoxide (DHSO)), cystine residues (bismaleimidesulfoxide (BMSO)), and finally a heterobifunctional cross-linker that targets lysine on one end and a non-specifically targets residues (Succinimidyl diazirine sulfoxide (SDASO)) on the other. We demonstrate that cross-linkers create cross-linked peptides that display the same predictable and characteristic fragmentation pattern during collision induced dissociation as amine-reactive sulfoxide-containing MS-cleavable cross-linked peptides, thus permitting their simplified analysis and unambiguous identification by MSn. Furthermore, we applied these linkers to either characterize the yeast 26S proteasome (SDASO) or the Cop9 signalosome (DSSO, DHSO and BMSO) demonstrating both the feasibility of SDASO’s photocross-linking of large protein complexes for the first time and the ability of multi-chemistry data for the integrative structure modeling of protein complexes to determine the interaction and structural dynamics of CSN, respectively. Moreover, our platform targeting various chemistries with cross-linking is robust and captures interactions complementary to residue-specific reagents, providing the foundation for future applications of multi-chemistry targeting approach to studying protein complexes