biostudies-literatureCiancone AFrederick National Laboratory for Cancer ResearchFoundation for the National Institutes of HealthNational Cancer InstituteNational Institutes of HealthCenter for Cancer Research101315https://www.ebi.ac.uk/biostudies/studies/S-EPMC12682122biostudies-literatureUnknown24(12)Crosslinking mass spectrometry (MS) is a powerful approach for probing protein structures. However, most widely used crosslinkers rely on N-hydroxysuccinimide (NHS) esters, restricting reactivity primarily to lysine residues and protein N-termini, and rendering them incompatible with many amine-containing buffers (e.g., Tris) and key biochemical cofactors (e.g., ATP). To address these limitations, we introduce two novel vinyl-sulfone-based crosslinkers. Alkyne-BVSC is an enrichable, homobifunctional crosslinker featuring an acid-cleavable alkyne handle for downstream peptide enrichment. VSD is a heterobifunctional crosslinker combining a vinyl sulfone with a diazirine moiety for UV-activated photo-crosslinking. Both reagents are synthetically accessible from inexpensive precursors and retaMolecular & cellular proteomics : MCPExpanding the Crosslinking Mass Spectrometry Toolbox With Vinyl Sulfone Crosslinkers.PMC12682122HHSN261200800001EZ01 BC011585 09Swenson REO'Reilly FJSchneekloth JSLee KSibley CCiancone AAtallah-Yunes KSpivey JWu HfalseExpanding the Crosslinking Mass Spectrometry Toolbox With Vinyl Sulfone Crosslinkers.Crosslinking mass spectrometry (MS) is a powerful approach for probing protein structures. However, most widely used crosslinkers rely on N-hydroxysuccinimide (NHS) esters, restricting reactivity primarily to lysine residues and protein N-termini, and rendering them incompatible with many amine-containing buffers (e.g., Tris) and key biochemical cofactors (e.g., ATP). To address these limitations, we introduce two novel vinyl-sulfone-based crosslinkers. Alkyne-BVSC is an enrichable, homobifunctional crosslinker featuring an acid-cleavable alkyne handle for downstream peptide enrichment. VSD is a heterobifunctional crosslinker combining a vinyl sulfone with a diazirine moiety for UV-activated photo-crosslinking. Both reagents are synthetically accessible from inexpensive precursors and reta2025-01-01T00:00:00Z2025 Oct2026-06-05T23:38:09.398Z2026-05-23T03:13:54.978ZS-EPMC126821224113029610.1016/j.mcpro.2025.101315