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.