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...

Full description

Saved in:
Bibliographic Details
Published in:Proceedings of the National Academy of Sciences - PNAS 2010-02, Vol.107 (6), p.2431-2436
Main Authors: Malovannaya, Anna, Li, Yehua, Bulynko, Yaroslava, Jung, Sung Yun, Wang, Yi, Lanz, Rainer B, O'Malley, Bert W, Qin, Jun
Format: Article
Language:English
Subjects:
Analysis
Antibodies
Antigens
Biochemistry
Biological Sciences
Cross reaction
Datasets
Eukaryotes
Gene expression regulation
HeLa Cells
Histone Deacetylase 1 - metabolism
Histone Deacetylase 2 - metabolism
Humans
Immunoprecipitation - methods
Mass spectrometry
Mass Spectrometry - methods
Models, Biological
Multiprotein Complexes - metabolism
Peptides
Protein Binding
Protein Interaction Mapping - methods
Proteins
Proteins - metabolism
Proteomes
Stem cells
Yeasts
Citations: Items that this one cites
Items that cite this one
Online Access:Get full text
Tags: Add Tag
No Tags, Be the first to tag this record!
Description
Summary: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.
ISSN:0027-8424
1091-6490
DOI:10.1073/pnas.0912599106