Proteome-wide profiling of protein assemblies by cross-linking mass spectrometry

Nat Methods. 2015 Dec;12(12):1179-84. doi: 10.1038/nmeth.3603. Epub 2015 Sep 28.


We describe an integrated workflow that robustly identifies cross-links from endogenous protein complexes in human cellular lysates. Our approach is based on the application of mass spectrometry (MS)-cleavable cross-linkers, sequential collision-induced dissociation (CID)-tandem MS (MS/MS) and electron-transfer dissociation (ETD)-MS/MS acquisitions, and a dedicated search engine, XlinkX, which allows rapid cross-link identification against a complete human proteome database. This approach allowed us to detect 2,179 unique cross-links (1,665 intraprotein cross-links at a 5% false discovery rate (FDR) and 514 interprotein cross-links at 1% FDR) in HeLa cell lysates. We validated the confidence of our cross-linking results by using a target-decoy strategy and mapping the observed cross-link distances onto existing high-resolution structures. Our data provided new structural information about many protein assemblies and captured dynamic interactions of the ribosome in contact with different elongation factors.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cross-Linking Reagents / chemistry*
  • Databases, Protein
  • HeLa Cells
  • Humans
  • Models, Molecular
  • Multiprotein Complexes / chemistry*
  • Proteome / chemistry*
  • Proteomics / instrumentation
  • Proteomics / methods*
  • Reproducibility of Results
  • Ribosomal Proteins / chemistry
  • Tandem Mass Spectrometry / instrumentation
  • Tandem Mass Spectrometry / methods*


  • Cross-Linking Reagents
  • Multiprotein Complexes
  • Proteome
  • Ribosomal Proteins