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Proc Natl Acad Sci U S A. 2018 Oct 30;115(44):11162-11167. doi: 10.1073/pnas.1813574115. Epub 2018 Oct 15.

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

Yang B1,2, Wu H1,2, Schnier PD3, Liu Y4, Liu J1,2, Wang N1,2, DeGrado WF5,2, Wang L5,2.

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Department of Pharmaceutical Chemistry, University of California, San Francisco, CA 94158.
The Cardiovascular Research Institute, University of California, San Francisco, CA 94158.
Institute for Neurodegenerative Diseases, University of California, San Francisco, CA 94158.
Yale Cancer Center, Yale University, West Haven, CT 06516.
Department of Pharmaceutical Chemistry, University of California, San Francisco, CA 94158;


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 rapidly with 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.


chemical cross-linker; cross-linking mass spectrometry; protein–protein interaction; proximity-enhanced reactivity; sulfur–fluoride exchange

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