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

Nat Protoc. 2013 Jan;8(1):75-97. doi: 10.1038/nprot.2012.133. Epub 2012 Dec 13.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Algorithms
  • Animals
  • Carrier Proteins
  • Chromatography, Affinity / methods*
  • Chromatography, Liquid / methods
  • Cross-Linking Reagents
  • Databases, Protein
  • Electrophoresis, Polyacrylamide Gel
  • HEK293 Cells
  • Hemagglutinins / chemistry*
  • Humans
  • Mass Spectrometry / methods
  • Mice
  • Protein-Serine-Threonine Kinases / chemistry*
  • Proteins / chemistry*
  • Vanadates / chemistry

Substances

  • Carrier Proteins
  • Cross-Linking Reagents
  • Hemagglutinins
  • Proteins
  • streptavidin-binding peptide
  • Vanadates
  • Tbk1 protein, mouse
  • Protein-Serine-Threonine Kinases