Protocol

Xenopus Protocols

Volume 917 of the series Methods in Molecular Biology pp 391-407

Date:

Complementary Proteomic Analysis of Protein Complexes

  • Todd M. GrecoAffiliated withDepartment of Molecular Biology, Princeton University
  • , Yana MitevaAffiliated withDepartment of Molecular Biology, Princeton University
  • , Frank L. ConlonAffiliated withDepartment of Genetics, University of North Carolina
  • , Ileana M. CristeaAffiliated withDepartment of Molecular Biology, Princeton University Email author 

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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.

Key words

Xenopus Proteomics Protein complex Protein interactions In-gel digestion In-solution digestion Mass spectrometry HPLC Bioinformatics