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

被引:187
作者
Smits, Arne H. [1 ]
Jansen, Pascal W. T. C. [1 ]
Poser, Ina [2 ]
Hyman, Anthony A. [2 ]
Vermeulen, Michiel [1 ]
机构
[1] UMC Utrecht, Dept Mol Canc Res, NL-3584 CG Utrecht, Netherlands
[2] Max Planck Inst Mol Cell Biol & Genet, D-01307 Dresden, Germany
关键词
BAC TRANSGENEOMICS; ABSOLUTE PROTEIN; QUANTIFICATION; PEPTIDES;
D O I
10.1093/nar/gks941
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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.
引用
收藏
页数:8
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