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Nucleic Acids Res. 2013 Jan 7;41(1):e28. doi: 10.1093/nar/gks941. Epub 2012 Oct 12.

Stoichiometry of chromatin-associated protein complexes revealed by label-free quantitative mass spectrometry-based proteomics.

Author information

1
Department of Molecular Cancer Research, UMC Utrecht, Universiteitsweg 100, 3584CG Utrecht, The Netherlands.

Abstract

Many cellular proteins assemble into macromolecular protein complexes. The identification of protein-protein interactions and quantification of their stoichiometry is therefore crucial to understand the molecular function of protein complexes. Determining the stoichiometry of protein complexes is usually achieved by mass spectrometry-based methods that rely on introducing stable isotope-labeled reference peptides into the sample of interest. However, these approaches are laborious and not suitable for high-throughput screenings. Here, we describe a robust and easy to implement label-free relative quantification approach that combines the detection of high-confidence protein-protein interactions with an accurate determination of the stoichiometry of the identified protein-protein interactions in a single experiment. We applied this method to two chromatin-associated protein complexes for which the stoichiometry thus far remained elusive: the MBD3/NuRD and PRC2 complex. For each of these complexes, we accurately determined the stoichiometry of the core subunits while at the same time identifying novel interactors and their stoichiometry.

PMID:
23066101
PMCID:
PMC3592467
DOI:
10.1093/nar/gks941
[Indexed for MEDLINE]
Free PMC Article

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