Novel Urazole-Derived MS-Cleavable Cross-Linkers Targeting Tyrosine/Lysine Residues for Protein Structural Analysis

Chemical cross-linking mass spectrometry (XL-MS) is crucial for probing protein structure and protein-protein interactions. Currently, lysine-reactive cross-linkers have been widely developed and applied. In this study, two urazole-derived MS-cleavable cross-linkers were designed and synthesized, enabling the accurate identification of cross-linked peptides by MSⁿ. A homobifunctional cross-linker (sulfoxide, bis-urazolyl, SBT) was designed to target tyrosine residues through the electrochemical click reaction. Another heterobifunctional cross-linker (sulfoxide, succinimidyl, urazolyl, SCT) was synthesized to target lysine and tyrosine residues. The reaction efficiencies of cross-linkers were systematically evaluated by using them to react with peptides, proteins, and protein complexes, respectively. Additionally, five structures of BSA, derived from AlphaFold 3, were analyzed by comparing them with its crystal structure in the protein database. The results demonstrate that this type of urazole-derived cross-linker is a valuable addition to the existing cross-linker library and enhances the recognition coverage of XL-MS in the structural analysis of proteins.