Expanding 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 retain reactivity in amine-rich biochemical environments. We show that vinyl sulfones react with cysteine, histidine, and lysine residues, thereby expanding crosslinkable residues beyond those accessible to NHS-esters. Moreover, we develop a stub-based post-search filtering strategy that leverages the MS-cleavable nature of vinyl sulfone linkages to improve crosslink identification sensitivity. Together, these advances establish vinyl-sulfone-based crosslinkers as versatile and complementary tools for structural proteomics.