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.

交联质谱（Crosslinking mass spectrometry, MS）是用于解析蛋白质空间结构的高效技术手段。然而，目前应用最广泛的交联剂多以N-羟基琥珀酰亚胺（N-hydroxysuccinimide, NHS）酯为活性基团，其反应活性主要局限于赖氨酸残基与蛋白质N末端，且无法兼容多数含胺缓冲体系（如Tris）及关键生化辅因子（如ATP）。为解决上述局限性，我们开发了两款新型乙烯砜基交联剂。炔基-BVSC（Alkyne-BVSC）是一种可富集型同双功能交联剂，带有酸裂解型炔基手柄，可用于下游肽段富集流程。VSD则为异双功能交联剂，将乙烯砜基团与双吖丙啶（diazirine）模块相结合，可实现紫外激活的光交联反应。两款试剂均可由廉价前驱体合成，且在富胺生化环境中仍可保持良好的反应活性。实验证实，乙烯砜可与半胱氨酸、组氨酸及赖氨酸残基发生交联反应，将可交联残基范围拓展至N-羟基琥珀酰亚胺酯类无法覆盖的位点。此外，我们开发了一种基于短片段的检索后过滤策略，利用乙烯砜连接键的MS裂解特性，提升了交联肽段鉴定的灵敏度。综上，这些研究进展确立了乙烯砜基交联剂作为结构蛋白质组学领域通用且互补的研究工具的地位。