Proximity-enhanced SuFEx chemical cross-linker for specific and multitargeting cross-linking mass spectrometry

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 115; no. 44; pp. 11162 - 11167
• Main Authors: Yang, Bing, Wu, Haifan, Schnier, Paul D., Liu, Yansheng, Liu, Jun, Wang, Nanxi, DeGrado, William F., Wang, Lei
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences 30.10.2018
• Subjects: Amino acids; Amino Acids - chemistry; Aromatic compounds; Coding; Cross-Linking Reagents - chemistry; Crosslinking; E coli; Escherichia coli - metabolism; Esters; Fluorides; Fluorides - chemistry; Genetic code; Links; Lysine - chemistry; Mass spectrometry; Mass Spectrometry - methods; Mass spectroscopy; Organic chemistry; Physical Sciences; Proteins; Proteins - chemistry; Proximity; Reagents; Residues; RNA-protein interactions; Spectroscopy; Substrates; Succinimide; Succinimides - chemistry; Sulfur; Sulfur Compounds - chemistry; Variation; protein–protein interaction; proximity-enhanced reactivity; chemical cross-linker; sulfur–fluoride exchange; cross-linking mass spectrometry
• ISSN: 0027-8424; 1091-6490; 1091-6490
• DOI: 10.1073/pnas.1813574115

Chemical cross-linking mass spectrometry (CXMS) is being increasingly used to study protein assemblies and complex protein interaction networks. Existing CXMS chemical cross-linkers target only Lys, Cys, Glu, and Asp residues, limiting the information measurable. Here we report a “plant-and-cast” cross-linking strategy that employs a heterobifunctional cross-linker that contains a highly reactive succinimide ester as well as a less reactive sulfonyl fluoride. The succinimide ester reacts rapidlywith surface Lys residues “planting” the reagent at fixed locations on protein. The pendant aryl sulfonyl fluoride is then “cast” across a limited range of the protein surface, where it can react with multiple weakly nucleophilic amino acid sidechains in a proximity-enhanced sulfur-fluoride exchange (SuFEx) reaction. Using proteins of known structures, we demonstrated that the heterobifunctional agent formed cross-links between Lys residues and His, Ser, Thr, Tyr, and Lys sidechains. This geometric specificity contrasts with current bis-succinimide esters, which often generate nonspecific cross-links between lysines brought into proximity by rare thermal fluctuations. Thus, the current method can provide diverse and robust distance restraints to guide integrative modeling. This work provides a chemical cross-linker targeting unactivated Ser, Thr, His, and Tyr residues using sulfonyl fluorides. In addition, this methodology yielded a variety of cross-links when applied to the complex Escherichia coli cell lysate. Finally, in combination with genetically encoded chemical cross-linking, cross-linking using this reagent markedly increased the identification of weak and transient enzyme–substrate interactions in live cells. Proximity-dependent cross-linking will dramatically expand the scope and power of CXMS for defining the identities and structures of protein complexes.