Stoichiometry of chromatin-associated protein complexes revealed by label-free quantitative mass spectrometry-based proteomics.
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.
Department of Molecular Cancer Research, UMC Utrecht, Universiteitsweg 100, 3584CG Utrecht, The Netherlands.
Arne H
AH
Smits
Pascal W T C
PW
Jansen
Ina
I
Poser
Anthony A
AA
Hyman
Michiel
M
Vermeulen
D018797:Cell Cycle Proteins / Q000032:analysis; D002843:Chromatin / Q000737:chemistry*; D002868:Chromosomal Proteins, Non-Histone / Q000032:analysis*; D045643:DNA Repair Enzymes / Q000032:analysis; D004268:DNA-Binding Proteins / Q000032:analysis; D006367:HeLa Cells; D006801:Humans; D013058:Mass Spectrometry; D056245:Mi-2 Nucleosome Remodeling and Deacetylase Complex / Q000032:analysis; D009687:Nuclear Proteins / Q000032:analysis; D063151:Polycomb Repressive Complex 2 / Q000032:analysis; D025941:Protein Interaction Mapping / Q000379:methods*; D021122:Protein Subunits / Q000032:analysis; D040901:Proteomics / Q000379:methods*; D000072260:RNA Splicing Factors; D016601:RNA-Binding Proteins / Q000032:analysis
C508357:CDC5L protein, human; D018797:Cell Cycle Proteins; D002843:Chromatin; D002868:Chromosomal Proteins, Non-Histone; D004268:DNA-Binding Proteins; C115369:MBD3 protein, human; D009687:Nuclear Proteins; D021122:Protein Subunits; D000072260:RNA Splicing Factors; D016601:RNA-Binding Proteins; D063151:Polycomb Repressive Complex 2; D056245:Mi-2 Nucleosome Remodeling and Deacetylase Complex; D045643:DNA Repair Enzymes; C417041:PRPF19 protein, human