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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.

Author information

1
Department of Pharmaceutical Chemistry, University of California, San Francisco, CA 94158.
2
The Cardiovascular Research Institute, University of California, San Francisco, CA 94158.
3
Institute for Neurodegenerative Diseases, University of California, San Francisco, CA 94158.
4
Yale Cancer Center, Yale University, West Haven, CT 06516.
5
Department of Pharmaceutical Chemistry, University of California, San Francisco, CA 94158; william.degrado@ucsf.edu lei.wang2@ucsf.edu.

Abstract

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.

KEYWORDS:

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

PMID:
30322930
PMCID:
PMC6217395
[Available on 2019-04-30]
DOI:
10.1073/pnas.1813574115

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