title: Lysine-specific chemical cross-linking of protein complexes and identification of cross-linking sites using LC-MS/MS and the xQuest/xProphet software pipeline.
abstract: Chemical cross-linking in combination with LC-MS/MS (XL-MS) is an emerging technology to obtain low-resolution structural (distance) restraints of proteins and protein complexes. These restraints can also be used to characterize protein complexes by integrative modeling of the XL-MS data, either in combination with other types of structural information or by themselves, to establish spatial relationships of subunits in protein complexes. Here we present a protocol that has been successfully used to generate XL-MS data from a multitude of native proteins and protein complexes. It includes the experimental steps for performing the cross-linking reaction using disuccinimidyl suberate (a homobifunctional, lysine-reactive cross-linking reagent), the enrichment of cross-linked peptides by peptide size-exclusion chromatography (SEC; to remove smaller, non-cross-linked peptides), instructions for tandem MS analysis and the analysis of MS data via the open-source computational software pipeline xQuest and xProphet (available from http://proteomics.ethz.ch). Once established, this robust protocol should take ∼4 d to complete, and it is generally applicable to purified proteins and protein complexes.
authors.affiliation: 1] Department of Biology, Institute of Molecular Systems Biology, Eidgenössische Technische Hochschule (ETH) Zürich, Zurich, Switzerland. [2].
authors.forename: Alexander
authors.initials: A
authors.lastname: Leitner
authors.affiliation: 1] Department of Biology, Institute of Molecular Systems Biology, Eidgenössische Technische Hochschule (ETH) Zürich, Zurich, Switzerland. [2] PhD Program in Molecular Life Sciences, University of Zurich-ETH Zurich, Zurich, Switzerland. [3].
authors.forename: Thomas
authors.initials: T
authors.lastname: Walzthoeni
authors.affiliation: 1] Department of Biology, Institute of Molecular Systems Biology, Eidgenössische Technische Hochschule (ETH) Zürich, Zurich, Switzerland. [2] Faculty of Science, University of Zurich, Zurich, Switzerland.
authors.forename: Ruedi
authors.initials: R
authors.lastname: Aebersold
mesh_terms: D000804:Angiotensin II / Q000737:chemistry; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D002853:Chromatography, Liquid / Q000379:methods*; D003432:Cross-Linking Reagents; D007328:Insulin / Q000737:chemistry; D008239:Lysine / Q000737:chemistry*; D020539:Sequence Analysis, Protein / Q000379:methods; D012984:Software; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D003432:Cross-Linking Reagents; D007328:Insulin; D000804:Angiotensin II; D008239:Lysine