title: A method to resolve the composition of heterogeneous affinity-purified protein complexes assembled around a common protein by chemical cross-linking, gel electrophoresis and mass spectrometry.
abstract: Protein complexes form, dissociate and re-form in order to perform specific cellular functions. In this two-pronged protocol, noncovalent protein complexes are initially isolated by affinity purification for subsequent identification of the components by liquid chromatography high-resolution mass spectrometry (LC-MS) on a hybrid LTQ Orbitrap Velos. In the second prong of the approach, the affinity-purification strategy includes a chemical cross-linking step to 'freeze' a series of concurrently formed, heterogeneous protein subcomplex species that are visualized by gel electrophoresis. This branch of the methodology amalgamates standard and well-practiced laboratory methods to reveal compositional changes that occur in protein complex architecture. By using mouse N-terminally tagged streptavidin-binding peptide-hemagglutinin-TANK-binding kinase 1 (SH-TBK1), we chemically cross-linked the affinity-purified complex of SH-TBK1 with the homobifunctional lysine-specific reagent bis(sulfosuccinimidyl) suberate (BS(3)), and we separated the resultant protein complexes by denaturation and by silver-stained one- and two-dimensional SDS-PAGE. We observed a range of cross-linked TBK1 complexes of variable pI and M(r) and confirmed them by immunoblotting. LC-MS analysis of in situ-digested cross-linked proteins shows differences in the composition of the TBK1 subcomplexes. The protocol is inherently simple and can be readily extended to the investigation of a range of protein complexes. From cell lysis to data generation by LC-MS, the protocol takes approximately 2.5 to 5.5 d to perform.
authors.affiliation: CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna, Austria.
authors.forename: Elena L
authors.initials: EL
authors.lastname: Rudashevskaya
authors.forename: Roberto
authors.initials: R
authors.lastname: Sacco
authors.forename: Klaus
authors.initials: K
authors.lastname: Kratochwill
authors.forename: Marie L
authors.initials: ML
authors.lastname: Huber
authors.forename: Matthias
authors.initials: M
authors.lastname: Gstaiger
authors.forename: Giulio
authors.initials: G
authors.lastname: Superti-Furga
authors.forename: Keiryn L
authors.initials: KL
authors.lastname: Bennett
mesh_terms: D000465:Algorithms; D000818:Animals; D002352:Carrier Proteins; D002846:Chromatography, Affinity / Q000379:methods*; D002853:Chromatography, Liquid / Q000379:methods; D003432:Cross-Linking Reagents; D030562:Databases, Protein; D004591:Electrophoresis, Polyacrylamide Gel; D057809:HEK293 Cells; D006388:Hemagglutinins / Q000737:chemistry*; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods; D051379:Mice; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry*; D011506:Proteins / Q000737:chemistry*; D014638:Vanadates / Q000737:chemistry
chemical_list: D002352:Carrier Proteins; D003432:Cross-Linking Reagents; D006388:Hemagglutinins; D011506:Proteins; C452700:streptavidin-binding peptide; D014638:Vanadates; C498588:Tbk1 protein, mouse; D017346:Protein Serine-Threonine Kinases