Clickable Multitargeting Cross-Linkers Based on SuFEx for In Vivo Cross-Linking Mass Spectrometry

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 depth of the XL-MS analysis and accuracy of the structural resolution. In this work, we designed and synthesized a pair of novel trifunctional cross-linkers, succinimidyl-propargyl-aryl sulfonyl fluoride (SPSF), for multitargeting cross-linking in vivo. The highly reactive succinimide ester reacts rapidly with Lys residues first; then, the less reactive sulfonyl fluoride reacts with multiple nucleophilic amino acid side chains subsequently via a proximity-enhanced sulfur (vi) fluoride exchange (SuFEx) reaction. The compact structures and proper amphipathy of SPSF facilitate its rapid cellular penetration. We demonstrated that SPSF effectively cross-links 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, including Lys-Lys, Lys-Ser, Lys-Thr, Lys-Tyr, and Lys-His. Further analysis of the cross-links revealed that SPSF-mediated multisite 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 phosphorylation sites. Moreover, cross-linking sites were found to be enriched in functional domains such as protein–protein interaction and nucleotide recognition, underscoring the potential of this approach to provide insights into protein functional states. Overall, the novel cross-linkers that 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.