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Analysis of protein complexes in Arabidopsis leaves using size exclusion chromatography and label-free protein correlation profiling

ABSTRACT: Protein complexes are fundamentally important for diverse cellular functions, and create functionalities that could never be achieved by a single polypeptide. Knowledge of the protein complex assemblies that exist in plant cells are limited. To close this gap, we applied an integrative proteomic approach that combines cell fractionation, protein chromatography and quantitative mass spectrometry (MS) to analyze the oligomerization state of thousands of proteins in a single experiment. Soluble extracts from intact Arabidopsis leaves were fractionated using size exclusion chromatography (SEC), and abundance profiles across the column fractions were quantified using label-free precursor ion (MS1) intensity. In duplicate experiments, we reproducibly detected 1693 proteins, of which 983 proteins were cytosolic. Based on the SEC profiles, approximately one third of all of the soluble proteins were predicted to be oligomeric. Our dataset includes both subunits of previously known complexes as well as hundreds of new protein complexes. The label-free MS1-based quantification method described here produced a highly useful dataset for the plant biology community, and provided a foundation to incorporate orthogonal protein complex separation methods so the composition and dynamics of protein complexes can be analyzed based on LC/MS profile data alone.

INSTRUMENT(S): TripleTOF 5600

ORGANISM(S): Arabidopsis Thaliana (ncbitaxon:3702)

SUBMITTER: Dan Szymanski

PROVIDER: MSV000081206 | MassIVE | Fri Jun 30 06:58:00 BST 2017

REPOSITORIES: MassIVE

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Project description: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.

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Analysis of protein complexes in Arabidopsis leaves using size exclusion chromatography and label-free protein correlation profiling

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