title: Characterization of native protein complexes and protein isoform variation using size-fractionation-based quantitative proteomics.
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.
authors.affiliation: Centre for Gene Regulation and Expression, College of Life Sciences, University of Dundee, Dow St., Dundee, DD1 5EH, United Kingdom.
authors.forename: Kathryn J
authors.initials: KJ
authors.lastname: Kirkwood
authors.forename: Yasmeen
authors.initials: Y
authors.lastname: Ahmad
authors.forename: Mark
authors.initials: M
authors.lastname: Larance
authors.forename: Angus I
authors.initials: AI
authors.lastname: Lamond
mesh_terms: D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D045744:Cell Line, Tumor; D002850:Chromatography, Gel; D030562:Databases, Protein; D005786:Gene Expression Regulation; D014644:Genetic Variation; D051548:Histone Acetyltransferases; D006801:Humans; D020407:Internet; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism; D013058:Mass Spectrometry; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism; D010006:Osteoblasts / Q000166:cytology / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D055503:Protein Multimerization; D011499:Protein Processing, Post-Translational; D020543:Proteome / Q000235:genetics / Q000378:metabolism*; D040901:Proteomics / Q000379:methods*; D015203:Reproducibility of Results; D012995:Solubility; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism
chemical_list: D002352:Carrier Proteins; D047908:Intracellular Signaling Peptides and Proteins; C470833:MORF4L1 protein, human; C439067:MRFAP1 protein, human; C479739:MRGBP protein, human; D009687:Nuclear Proteins; D020033:Protein Isoforms; D020543:Proteome; D014157:Transcription Factors; D051548:Histone Acetyltransferases
references.citation: Mol Cell Biol. 2004 Oct;24(19):8366-73
references.pmid: 15367658
references.citation: Proc Natl Acad Sci U S A. 2010 Feb 9;107(6):2431-6
references.pmid: 20133760
references.citation: EMBO J. 1999 Sep 15;18(18):5108-19
references.pmid: 10487762
references.citation: Nature. 2002 Jan 10;415(6868):180-3
references.pmid: 11805837
references.citation: J Proteome Res. 2011 Apr 1;10(4):1794-805
references.pmid: 21254760
references.citation: Mol Cell Proteomics. 2002 May;1(5):376-86
references.pmid: 12118079
references.citation: J Biol Chem. 2013 Mar 22;288(12):8128-8135
references.pmid: 23376345
references.citation: Mol Cell Proteomics. 2012 Mar;11(3):M111.014050
references.pmid: 22278370
references.citation: Structure. 2011 Dec 7;19(12):1807-15
references.pmid: 22153503
references.citation: Biochim Biophys Acta. 2000 Sep 1;1523(1):13-20
references.pmid: 11099853
references.citation: Ann N Y Acad Sci. 1953 Nov 11;57(3):159-76
references.pmid: 13139315
references.citation: Mol Syst Biol. 2011 Nov 08;7:548
references.pmid: 22068331
references.citation: Mol Cell Proteomics. 2013 Mar;12(3):638-50
references.pmid: 23242552
references.citation: Mol Cell Proteomics. 2012 Mar;11(3):M111.014407
references.pmid: 22038470
references.citation: EMBO Rep. 2003 May;4(5):479-83
references.pmid: 12717453
references.citation: Cell. 2012 Aug 31;150(5):1068-81
references.pmid: 22939629
references.citation: Nucleic Acids Res. 2010 Jan;38(Database issue):D497-501
references.pmid: 19884131
references.citation: Oncol Res. 1996;8(10-11):435-47
references.pmid: 9114436
references.citation: Mol Syst Biol. 2007;3:89
references.pmid: 17353931
references.citation: Mol Cell Proteomics. 2006 Jul;5(7):1326-37
references.pmid: 16635985
references.citation: Nucleic Acids Res. 2000 Sep 15;28(18):3442-4
references.pmid: 10982861
references.citation: Protein Sci. 2006 Oct;15(10):2423-34
references.pmid: 17008723
references.citation: Cell Metab. 2012 Oct 3;16(4):538-49
references.pmid: 22982022
references.citation: J Cell Biol. 2006 Feb 27;172(5):679-92
references.pmid: 16492807
references.citation: Nat Protoc. 2009;4(1):44-57
references.pmid: 19131956
references.citation: Nucleic Acids Res. 2013 Jan;41(Database issue):D43-7
references.pmid: 23161681
references.citation: Proteomics. 2010 Nov;10(22):3957-69
references.pmid: 21080489
references.citation: Anal Biochem. 1984 Apr;138(1):141-3
references.pmid: 6731838
references.citation: Mol Cell Biol. 2011 Jan;31(1):54-62
references.pmid: 21041482
references.citation: Nat Biotechnol. 2010 Dec;28(12):1248-50
references.pmid: 21139605
references.citation: Nat Protoc. 2006;1(6):2650-60
references.pmid: 17406521
references.citation: Proteomics. 2012 May;12(10):1576-90
references.pmid: 22611051
references.citation: Mol Cell Proteomics. 2005 Dec;4(12):1920-32
references.pmid: 16127175
references.citation: J Cell Biol. 2008 Oct 20;183(2):223-39
references.pmid: 18936248
references.citation: Proteomics. 2004 Jun;4(6):1551-61
references.pmid: 15174125
references.citation: Nat Methods. 2012 Sep;9(9):907-9
references.pmid: 22863883
references.citation: Curr Opin Genet Dev. 2004 Apr;14(2):147-54
references.pmid: 15196461
references.citation: J Biol Chem. 2010 Feb 26;285(9):6498-507
references.pmid: 20042602
references.citation: Nat Biotechnol. 2008 Dec;26(12):1367-72
references.pmid: 19029910
references.citation: Mol Cell Proteomics. 2010 May;9(5):861-79
references.pmid: 20023298
references.citation: Mol Cell Proteomics. 2010 Jul;9(7):1594-615
references.pmid: 20423899
references.citation: Mol Cell Biol. 2002 Nov;22(22):7868-76
references.pmid: 12391155
references.citation: Nat Rev Mol Cell Biol. 2007 Aug;8(8):645-54
references.pmid: 17593931
references.citation: Nature. 2006 Mar 30;440(7084):631-6
references.pmid: 16429126
references.citation: Structure. 2012 Jan 11;20(1):151-60
references.pmid: 22244764
references.citation: J Biol Chem. 2003 Oct 31;278(44):42733-6
references.pmid: 12963728
references.citation: Cell. 2011 May 27;145(5):787-99
references.pmid: 21620140
references.citation: J Cell Biol. 1999 Mar 22;144(6):1097-112
references.pmid: 10087256
references.citation: Oncol Res. 1997;9(11-12):629-39
references.pmid: 9563011
references.citation: Curr Opin Biotechnol. 2008 Aug;19(4):324-30
references.pmid: 18598764
references.citation: J Biol Chem. 2001 Oct 19;276(42):39171-8
references.pmid: 11500496
references.citation: Nucleic Acids Res. 2011 Jan;39(Database issue):D561-8
references.pmid: 21045058
references.citation: J Mol Biol. 2011 May 20;408(5):987-1000
references.pmid: 21440557
references.citation: Sci Signal. 2009 Sep 08;2(87):ra51
references.pmid: 19738201
references.citation: PLoS Biol. 2006 Aug;4(8):e267
references.pmid: 16869714
references.citation: Mol Cell Proteomics. 2004 Feb;3(2):176-82
references.pmid: 14665681
references.citation: J Cell Sci. 2010 Apr 1;123(Pt 7):1124-30
references.pmid: 20332121
references.citation: J Cell Biol. 2010 May 17;189(4):739-54
references.pmid: 20479470
references.citation: Mol Cell Proteomics. 2012 Mar;11(3):M111.013680
references.pmid: 22002106
references.citation: Protein Expr Purif. 2001 Oct;23(1):75-83
references.pmid: 11570848
references.citation: Structure. 2012 May 9;20(5):814-25
references.pmid: 22503819
references.citation: J Mol Biol. 2006 Dec 15;364(5):1021-33
references.pmid: 17052728
references.citation: J Cell Biol. 1996 May;133(3):495-505
references.pmid: 8636226