ABSTRACT: Proteins form a diverse array of complexes that mediate cellular function and regulation. A largely unexplored feature of such protein complexes is the selective participation of specific protein isoforms and/or post-translationally modified forms. In this study, we combined native size-exclusion chromatography (SEC) with high-throughput proteomic analysis to characterize soluble protein complexes isolated from human osteosarcoma (U2OS) cells. Using this approach, we have identified over 71,500 peptides and 1,600 phosphosites, corresponding to over 8,000 proteins, distributed across 40 SEC fractions. This represents >50% of the predicted U2OS cell proteome, identified with a mean peptide sequence coverage of 27% per protein. Three biological replicates were performed, allowing statistical evaluation of the data and demonstrating a high degree of reproducibility in the SEC fractionation procedure. Specific proteins were detected interacting with multiple independent complexes, as typified by the separation of distinct complexes for the MRFAP1-MORF4L1-MRGBP interaction network. The data also revealed protein isoforms and post-translational modifications that selectively associated with distinct subsets of protein complexes. Surprisingly, there was clear enrichment for specific Gene Ontology terms associated with differential size classes of protein complexes. This study demonstrates that combined SEC/MS analysis can be used for the system-wide annotation of protein complexes and to predict potential isoform-specific interactions. All of these SEC data on the native separation of protein complexes have been integrated within the Encyclopedia of Proteome Dynamics, an online, multidimensional data-sharing resource available to the community. U2OS cells were grown in DMEM supplemented with 10% FCS, 100 U/l penicillin, and 100 μg/l streptomycin at 37 °C in 10% CO2 and passaged at ~80% confluence. U2OS cells expressing LAP1-tagged MRFAP1 were grown in the same medium but with the addition of 150 μg/ml hygromycin B and 15 μg/ml blasticidine HCl. The cells for each condition were harvested separately via trypsinization, washed in PBS, and lysed in IP buffer (1% Nonidet P-40, 50 mm Tris-HCl, pH 7.4, 10% glycerol, 150 mm NaCl, complete protease inhibitor mixture (Roche Applied Science), PhosStop, 50 mm N-ethylmaleimide). The lysates were sonicated for 10 s at 10% power (three times in total) and then centrifuged for 10 min at 17,000g at 4 °C. Equal protein amounts of each sample were then combined with GFP-trap agarose beads from ChromoTek (Martinsried, Germany) that had been washed once in IP buffer (40 μl of 50% GFP-trap bead slurry per IP) and incubated for 2 h at 4 °C with rotation. The beads were then washed three times with IP buffer via centrifugation at 2,000g for 2 min at 4 °C.
Vackar Afzal 
