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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.
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页数:8
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