Systematic analysis of human nuclear protein complexes by quantitative mass spectrometry profiling

ABSTRACT: Many proteins form stable complexes that coordinate diverse and complex cellular processes. Systematic analysis of protein complexes provides insights into how the ensemble of expressed proteome is organized into functional units. Protein complexes are usually identified using either yeast-two-hybrid (Y2H) or affinity purification-mass spectrometry (AP-MS); however, these methods are low throughput and require genetic manipulation. While there have been advances in techniques for proteome-wide profiling of cytoplasmic protein complexes, information about human nuclear protein complexes are very limited. To close this gap, we combined native size exclusion chromatographic (SEC) with label-free quantitative liquid chromatography tandem-MS (LC-MS/MS) profiling to characterize hundreds of nuclear protein complexes isolated from human glioblastoma multiforme (GBM) cells. We identified 1794 proteins that overlapped between two biological replicates, of which 1244 proteins were characterized as existing within stably associated putative complexes. We demonstrated a high degree of SEC reproducibility by comparing protein elution profiles between the two independent fractionation procedures, and co-elution of several chromatin remodeling and DNA damage repair complexes was confirmed by independent co-immunoprecipitation (co-IP) and immunoblot analyses. Co-IP experiments confirmed the interaction of PARP1 with Ku70/Ku80 proteins, and between HDAC1 and CHD4. HDAC1/2 also co-migrated with various SIN3a and NuRD components in SEC fractionation including SIN3A, SAP30, RBBP4, RBBP7, and NCOR1. Co-elution of HDAC1/2/3 with both the KDM1A and RCOR1 further confirmed that these proteins are integral components of human deacetylase complexes. Our approach also demonstrated the ability to identify potential moonlighting complexes and novel complexes containing uncharacterized proteins. Overall, the results demonstrated the utility of SEC fractionation and LC-MS analysis for system-wide profiling of proteins to predict the existence of distinct forms of nuclear protein complexes.

INSTRUMENT(S): Q-Exactive Orbitrap HF

ORGANISM(S): Human Gbm Cell Lines

SUBMITTER: Uma K Aryal  

PROVIDER: MSV000081520 | MassIVE |

REPOSITORIES: MassIVE