title: Proteome-wide profiling of protein assemblies by cross-linking mass spectrometry.
abstract: 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.
authors.affiliation: Biomolecular Mass Spectrometry and Proteomics, Bijvoet Centre for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, University of Utrecht, Utrecht, the Netherlands.|Netherlands Proteomics Center, Utrecht, the Netherlands.
authors.forename: Fan
authors.initials: F
authors.lastname: Liu
authors.affiliation: Medicinal Chemistry and Chemical Biology, Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Utrecht, the Netherlands.
authors.forename: Dirk T S
authors.initials: DT
authors.lastname: Rijkers
authors.affiliation: Biomolecular Mass Spectrometry and Proteomics, Bijvoet Centre for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, University of Utrecht, Utrecht, the Netherlands.|Netherlands Proteomics Center, Utrecht, the Netherlands.
authors.forename: Harm
authors.initials: H
authors.lastname: Post
authors.affiliation: Biomolecular Mass Spectrometry and Proteomics, Bijvoet Centre for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, University of Utrecht, Utrecht, the Netherlands.|Netherlands Proteomics Center, Utrecht, the Netherlands.
authors.forename: Albert J R
authors.initials: AJ
authors.lastname: Heck
mesh_terms: D003432:Cross-Linking Reagents / Q000737:chemistry*; D030562:Databases, Protein; D006367:HeLa Cells; D006801:Humans; D008958:Models, Molecular; D046912:Multiprotein Complexes / Q000737:chemistry*; D020543:Proteome / Q000737:chemistry*; D040901:Proteomics / Q000295:instrumentation / Q000379:methods*; D015203:Reproducibility of Results; D012269:Ribosomal Proteins / Q000737:chemistry; D053719:Tandem Mass Spectrometry / Q000295:instrumentation / Q000379:methods*
chemical_list: D003432:Cross-Linking Reagents; D046912:Multiprotein Complexes; D020543:Proteome; D012269:Ribosomal Proteins