Complementary proteomic analysis of protein complexes

Methods Mol Biol. 2012;917:391-407. doi: 10.1007/978-1-61779-992-1_22.

Abstract

Proteomic characterization of protein complexes leverages the versatile platform of liquid chromatography-tandem mass spectrometry to elucidate molecular and cellular signaling processes underlying the dynamic regulation of macromolecular assemblies. Here, we describe a complementary proteomic approach optimized for immunoisolated protein complexes. As the relative complexity, abundance, and physiochemical properties of proteins can vary significantly between samples, we have provided (1) complementary sample preparation workflows, (2) detailed steps for HPLC and mass spectrometric method development, and (3) a bioinformatic workflow that provides confident peptide/protein identification paired with unbiased functional gene ontology analysis. This protocol can also be extended for characterization of larger complexity samples from whole cell or tissue Xenopus proteomes.

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Chromatography, High Pressure Liquid
  • Electrophoresis, Polyacrylamide Gel / standards
  • Molecular Sequence Data
  • Multiprotein Complexes / chemistry
  • Multiprotein Complexes / isolation & purification
  • Multiprotein Complexes / metabolism*
  • Peptide Fragments / chemistry
  • Peptide Fragments / isolation & purification
  • Peptide Mapping
  • Proteolysis
  • Proteome / chemistry
  • Proteome / isolation & purification
  • Proteome / metabolism*
  • Proteomics
  • Reference Standards
  • Tandem Mass Spectrometry
  • Trypsin / chemistry
  • Xenopus / metabolism*
  • Xenopus Proteins / chemistry
  • Xenopus Proteins / isolation & purification
  • Xenopus Proteins / metabolism*

Substances

  • Multiprotein Complexes
  • Peptide Fragments
  • Proteome
  • Xenopus Proteins
  • Trypsin