Clickable Multi-targeting Cross-Linkers Base on SuFEx for In Vivo Cross-Linking Mass Spectrometry

ABSTRACT: Cross-linking mass spectrometry (XL-MS) is a powerful technique to study protein structures and protein–protein interactions. The coverage of amino acids determines the accuracy and depth of XL-MS analysis. In this work, we designed and synthesized a pair of novel tris-functional cross-linkers, succinimidyl-propargyl-aryl sulfonyl fluoride (SPSF), for multi-targeting cross-linking in vivo. The highly reactive succinimide ester reacts rapidly with Lys residues first, then the less reactive sulfonyl fluo-ride reacts with multiple nucleophilic amino acid sidechains subsequently via proximity-enhanced sulfur (vi) fluoride exchange (SuFEx) reaction. The compact structures and proper amphipathy of SPSF facilitate its rapid cellular penetration. We demon-strated that SPSF effectively cross-link proteins in various cellular compartments without apparent perturbation of cellular states. Utilizing an enrichment strategy combining click chemistry with biotin-streptavidin purification, the identification of cross-links was greatly improved, which include Lys-Lys, Lys-Ser, Lys-Thr, Lys-Tyr and Lys-His. Further analysis of the cross-links revealed that SPSF-mediated multi-site cross-linking effectively expands the coverage of proteins or domains with limited lysine residues. Notably, we observed a high degree of overlap between cross-linked Ser, Thr, and Tyr sites and the phosphorylation sites. Moreover, cross-linking sites were found to be enriched in functional domains such as the protein–protein interaction and nucleotide recognition, underscoring the potential of this approach to provide insights into protein func-tional states. Overall, the novel cross-linkers we developed represent a valuable contribution to the XL-MS toolbox, which has great potential to broaden its scope and enhance its capabilities for functional protein structure analysis.

ORGANISM(S): Homo Sapiens

SUBMITTER: Suming Chen  

PROVIDER: PXD063377 | iProX | Sun Apr 27 00:00:00 BST 2025

REPOSITORIES: iProX