Streamlined analysis schema for high-throughput identification of endogenous protein complexes.
Immunoprecipitation followed by mass spectrometry (IP/MS) has recently emerged as a preferred method in the analysis of protein complex components and cellular protein networks. Targeting endogenous protein complexes of higher eukaryotes, particularly in large-scale efforts, has been challenging due to cellular heterogeneity, high proteome complexity, and, compared to lower organisms, lack of efficient in-locus epitope-tagging techniques. It is further complicated by variability in nonspecific identifications and cross-reactivity of primary antibodies. Still, the study of endogenous human protein networks is highly desired despite its challenges. Here we describe a streamlined IP/MS protocol for the purification and identification of extended endogenous protein complexes. We investigate the sources of nonspecific protein binding and develop semiquantitative specificity filters that are based on peptide spectral count measurements. We also outline logical constraints for the derivation of accurate complex composition from IP/MS data and demonstrate the effectiveness of this approach by presenting our analyses of different transcriptional coregulator complexes. We show consistent purification of novel components for the Integrator complex, analyze the composition of the Mediator complex solely from our data to demonstrate the wide usability of spectral counts, and deconvolute heterogeneous HDAC1/2 networks into core complex modules and several novel subcomplex interactions.
Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX 77030, USA.
Anna
A
Malovannaya
Yehua
Y
Li
Yaroslava
Y
Bulynko
Sung Yun
SY
Jung
Yi
Y
Wang
Rainer B
RB
Lanz
Bert W
BW
O'Malley
Jun
J
Qin
D006367:HeLa Cells; D056284:Histone Deacetylase 1 / Q000378:metabolism; D056464:Histone Deacetylase 2 / Q000378:metabolism; D006801:Humans; D047468:Immunoprecipitation / Q000379:methods*; D013058:Mass Spectrometry / Q000379:methods*; D008954:Models, Biological; D046912:Multiprotein Complexes / Q000378:metabolism*; D011485:Protein Binding; D025941:Protein Interaction Mapping / Q000379:methods*; D011506:Proteins / Q000378:metabolism
D046912:Multiprotein Complexes; D011506:Proteins; C110550:HDAC1 protein, human; C533423:HDAC2 protein, human; D056284:Histone Deacetylase 1; D056464:Histone Deacetylase 2