Development of a Novel Sulfoxide-Containing MS-Cleavable Homobifunctional Cysteine-Reactive Cross-Linker for Studying Protein-Protein Interactions

Anal Chem. 2018 Jun 19;90(12):7600-7607. doi: 10.1021/acs.analchem.8b01287. Epub 2018 Jun 5.

Abstract

Cross-linking mass spectrometry (XL-MS) has become an emerging technology for defining protein-protein interactions (PPIs) and elucidating architectures of large protein complexes. Up to now, the most widely used cross-linking reagents target lysines. Although such reagents have been successfully applied to map PPIs at the proteome-wide scale, comprehensive PPI profiling would require additional cross-linking chemistries. Cysteine is one of the most reactive amino acids and an attractive target for cross-linking owing to its unique role in protein structures. Although sulfhydryl-reactive cross-linkers are commercially available, their applications in XL-MS studies remain sparse, likely due to the difficulty in identifying cysteine cross-linked peptides. Previously, we developed a new class of sulfoxide-containing MS-cleavable cross-linkers to enable fast and accurate identification of cross-linked peptides using multistage tandem mass spectrometry (MS n). Here, we present the development of a new sulfoxide-containing MS-cleavable homobifunctional cysteine-reactive cross-linker, bismaleimide sulfoxide (BMSO). We demonstrate that BMSO-cross-linked peptides display the same characteristic fragmentation pattern during collision-induced dissociation (CID) as other sulfoxide-containing MS-cleavable cross-linked peptides, thus permitting their simplified analysis and unambiguous identification by MS n. Additionally, we show that BMSO can complement amine- and acidic-residue-reactive reagents for mapping protein-interaction regions. Collectively, this work not only enlarges the toolbox of MS-cleavable cross-linkers with diverse chemistries, but more importantly expands our capacity and capability of studying PPIs in general.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cattle
  • Cross-Linking Reagents / chemistry*
  • Cysteine / chemistry*
  • Molecular Structure
  • Protein Binding
  • Protein Interaction Mapping*
  • Serum Albumin, Bovine / chemistry*
  • Sulfoxides / chemical synthesis
  • Sulfoxides / chemistry*
  • Tandem Mass Spectrometry

Substances

  • Cross-Linking Reagents
  • Sulfoxides
  • Serum Albumin, Bovine
  • Cysteine