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Characterization of Native Protein Complexes and Protein Isoform Variation Using Size- Fractionation-Based Quantitative Proteomics*□S

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Characterization of Native Protein Complexes and Protein Isoform Variation Using Size- Fractionation-Based Quantitative Proteomics*□S Research Author’s Choice © 2013 by The American Society for Biochemistry and Molecular Biology, Inc. This paper is available on line at http://www.mcponline.org Characterization of Native Protein Complexes and Protein Isoform Variation Using Size- fractionation-based Quantitative Proteomics*□S Kathryn J. Kirkwood‡§, Yasmeen Ahmad‡§, Mark Larance‡§, and Angus I. Lamond‡¶ Proteins form a diverse array of complexes that mediate The majority of proteins function as part of multiprotein cellular function and regulation. A largely unexplored fea- complexes and not as isolated polypeptides. These protein ture of such protein complexes is the selective participa- complexes range from simple homodimers to large structures tion of specific protein isoforms and/or post-translation- composed of many different polypeptides. Protein complexes ally modified forms. In this study, we combined native vary in their size and shape from small globular dimers, such size-exclusion chromatography (SEC) with high-through- as 14-3-3 proteins, to large elongated filaments of variable put proteomic analysis to characterize soluble protein length, such as microtubules. The wide variety of possible complexes isolated from human osteosarcoma (U2OS) protein–protein interactions within multiprotein complexes cells. Using this approach, we have identified over 71,500 contributes to the diversity of functions that are involved in peptides and 1,600 phosphosites, corresponding to over cellular processes and regulatory mechanisms. 8,000 proteins, distributed across 40 SEC fractions. This Another important source of functional diversity and regu- represents >50% of the predicted U2OS cell proteome, lation is the large number of protein isoforms that may be identified with a mean peptide sequence coverage of 27% generated from each gene. Functionally and structurally dis- per protein. Three biological replicates were performed, tinct isoforms can arise via multiple mechanisms, including allowing statistical evaluation of the data and demonstrat- alternative splicing, post-translational modification (PTM),1 ing a high degree of reproducibility in the SEC fraction- ation procedure. Specific proteins were detected interact- and proteolytic cleavage. Distinct isoforms can exhibit radi- ing with multiple independent complexes, as typified by cally different properties. For example, including or excluding the separation of distinct complexes for the MRFAP1- individual exons can either create or remove protein–protein MORF4L1-MRGBP interaction network. The data also re- interaction interfaces for binding specific interaction partners. vealed protein isoforms and post-translational modifica- Similarly, phosphorylation, and other PTMs, can either create tions that selectively associated with distinct subsets of or remove binding sites for interacting proteins, substrates, or protein complexes. Surprisingly, there was clear enrich- ligands. PTMs can also promote structural changes in pro- ment for specific Gene Ontology terms associated with teins and affect catalytic activity. differential size classes of protein complexes. This study The association of protein isoforms and post-translationally demonstrates that combined SEC/MS analysis can be used modified factors in multiprotein complexes can influence their for the system-wide annotation of protein complexes and to subcellular location, activity, and substrate specificity. This predict potential isoform-specific interactions. All of these can be dynamically regulated to modulate protein complex SEC data on the native separation of protein complexes composition, and hence localization and function, to allow have been integrated within the Encyclopedia of Proteome cells to respond to spatial and temporal stimuli. It is therefore Dynamics, an online, multidimensional data-sharing resource important to characterize protein complexes at the level of the available to the community. Molecular & Cellular Proteom- protein isoforms and post-translationally modified forms they ics 12: 10.1074/mcp.M113.032367, 3851–3873, 2013. contain in order to fully decipher the network of signaling and regulatory pathways within cells. Although many types of protein complexes have been stud- From the ‡Centre for Gene Regulation and Expression, College of ied in detail, in-depth analysis of the composition, dynamics, Life Sciences, University of Dundee, Dow St., Dundee, DD1 5EH, United Kingdom and isoform association of protein complexes formed in either Author’s Choice—Final version full access. Received July 4, 2013, and in revised form, August 24, 2013 1 The abbreviations used are: EPD, Encyclopedia of Proteome Dy- Published, MCP Papers in Press, September 16, 2013, DOI namics; GO, gene ontology; HP1BP3, heterochromatin protein 10.1074/mcp.M113.032367 1–binding protein 3; IP, immunoprecipitation; LDS, lithium dodecyl Author contributions: K.K. and M.L. performed SEC experiments. sulfate; MORF4L1, mortality factor 4-like protein 1; MRFAP1, MORF4 K.K. performed the immunoblotting. Y.A. performed the statistical family-associated protein 1; MRGBP, MRG/MORF4L-binding protein; analysis and created the Encyclopedia of Proteome Dynamics. K.K., PTM, post-translational modification; SEC, size-exclusion chroma- Y.A., M.L., and A.I.L. wrote the paper. A.I.L. mentored the project. tography; TCEP, triscarboxyethylphosphine. Molecular & Cellular Proteomics 12.12 3851 Native Protein Complex Analysis by Quantitative Proteomics human cells or model organisms is still not well documented For system-wide studies of the composition and dynamics at a system-wide level. The CORUM database, compiled us- of protein complexes, alternative methods, in addition to im- ing a variety of information from the literature describing pro- mune-affinity purification, are required for convenient separa- tein interactions and assemblies, currently provides the larg- tion, characterization, and comparison of cellular protein est public dataset of protein complexes (1). CORUM contains complexes. To address this, a number of studies have utilized information relating to ϳ1,970 protein complexes identified in various forms of either column chromatography or native gel human cells. However, these complexes are formed from electrophoresis in combination with mass-spectrometry- proteins encoded by only ϳ16% of the known human protein- based proteomics. For example, protein complexes have coding genes, indicating that many forms of protein com- been separated using techniques including blue native poly- plexes still remain to be identified and characterized (1). Fur- acrylamide gel electrophoresis (18, 19), ion-exchange chro- thermore, the CORUM database does not describe how the matography (20), and size-exclusion chromatography (21, 22) protein compositions of the complexes may vary, either dy- prior to MS analysis of proteins in the fractionated complexes. namically or in different subcellular locations, or how this Size-exclusion chromatography (SEC) is a well-established relates to protein isoforms and PTMs. This illustrates that technique used to separate proteins and protein complexes in there is still a major deficit in our knowledge of the structure solution on the basis of their shape/size (rotational cross- and functions of cellular protein complexes and how they section) (23). SEC has been extensively used as an interme- contribute to biological regulatory mechanisms. diate step in conventional multistep biochemical protein pu- The technique that is now most widely used to identify the rification strategies. In contrast, SEC has been less commonly components of protein complexes is affinity purification of an combined with mass-spectrometry-based proteomics for the individual “bait” protein and subsequent analysis of the co- high-throughput characterization of protein complexes. How- isolated proteins, usually via mass spectrometry (2). Affinity ever, this has been demonstrated in previous studies that purification can use antibodies specific for an endogenous analyzed native protein complexes in plant chloroplasts (22) or large cytosolic complexes in mammalian cells (21). target protein (3, 4), if available, or, alternatively, can utilize a In this study, we combined native SEC with high-throughput genetically constructed, epitope-tagged bait protein. The lat- mass-spectrometry-based proteomic analysis to characterize ter procedure is now widely used and is advantageous in that soluble protein complexes isolated from human osteosarcoma many different complexes can be compared using an identical cells. Herein we demonstrate the utility and reproducibility of antibody, or other affinity-purification method, targeted to the this approach for the system-wide characterization of endoge- tag on the bait (for examples, see Refs. 5–7; for reviews, see nous, untagged protein complexes and show how it can be Refs. 8 and 9). In contrast, it is harder to directly compare the used to identify specific protein isoforms and PTMs associated results from immunoprecipitation of different endogenous with distinct protein complexes. The resulting data are available protein complexes because each specific antibody that is to the community in a convenient format in the Encyclopedia of used has different affinities and properties. Nonetheless, al- Proteome Dynamics (EPD) (www.peptracker.com/encyclopedia though epitope-tag “pull-down” techniques are now com- Information/), a user-friendly, searchable online database. monly used, they also have limitations. Not least, the addition of epitope
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