Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2012:917:391-407.
doi: 10.1007/978-1-61779-992-1_22.

Complementary proteomic analysis of protein complexes

Todd M Greco  1 , Yana Miteva, Frank L Conlon, Ileana M Cristea
Affiliations

Complementary proteomic analysis of protein complexes

Todd M Greco et al. Methods Mol Biol. 2012.

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.

PubMed Disclaimer

Figures

Fig. 1
Fig. 1
Workflow summary for proteomic analysis of immunoisolated protein complexes; diagram of LTQ-Orbitrap Velos from Thermo Fisher Scientific.
See this image and copyright information in PMC

References

    1. Washburn MP, Wolters D, Yates JR., III Large-scale analysis of the yeast proteome by multidimensional protein identification technology. Nat Biotechnol. 2001;19(3):242–247. - PubMed
    1. Wang H, Chang-Wong T, Tang HY, Speicher DW. Comparison of extensive protein fractionation and repetitive LC-MS/MS analyses on depth of analysis for complex proteomes. J Proteome Res. 2010;9(2):1032–1040. - PMC - PubMed
    1. Gygi SP, Rist B, Gerber SA, Turecek F, Gelb MH, Aebersold R. Quantitative analysis of complex protein mixtures using isotope-coded affinity tags. Nat Biotechnol. 1999;17(10):994–999. - PubMed
    1. Fang X, Zhang WW. Affinity separation and enrichment methods in proteomic analysis. J Proteomics. 2008;71(3):284–303. - PubMed
    1. Kocher T, Swart R, Mechtler K. Ultrahigh-pressure RPLC hyphenated to an LTQ-Orbitrap Velos reveals a linear relation between peak capacity and number of identified peptides. Anal Chem. 2010;83(7):2699–2704. - PubMed

MeSH terms