title: Large-scale top-down proteomics of the human proteome: membrane proteins, mitochondria, and senescence.
abstract: Top-down proteomics is emerging as a viable method for the routine identification of hundreds to thousands of proteins. In this work we report the largest top-down study to date, with the identification of 1,220 proteins from the transformed human cell line H1299 at a false discovery rate of 1%. Multiple separation strategies were utilized, including the focused isolation of mitochondria, resulting in significantly improved proteome coverage relative to previous work. In all, 347 mitochondrial proteins were identified, including ~50% of the mitochondrial proteome below 30 kDa and over 75% of the subunits constituting the large complexes of oxidative phosphorylation. Three hundred of the identified proteins were found to be integral membrane proteins containing between 1 and 12 transmembrane helices, requiring no specific enrichment or modified LC-MS parameters. Over 5,000 proteoforms were observed, many harboring post-translational modifications, including over a dozen proteins containing lipid anchors (some previously unknown) and many others with phosphorylation and methylation modifications. Comparison between untreated and senescent H1299 cells revealed several changes to the proteome, including the hyperphosphorylation of HMGA2. This work illustrates the burgeoning ability of top-down proteomics to characterize large numbers of intact proteoforms in a high-throughput fashion.
authors.affiliation: Departments of Chemistry and Molecular Biosciences, the Chemistry of Life Processes Institute, the Proteomics Center of Excellence, and the Robert H. Lurie Comprehensive Cancer Center, Northwestern University, Evanston, Illinois 60208.
authors.forename: Adam D
authors.initials: AD
authors.lastname: Catherman
authors.forename: Kenneth R
authors.initials: KR
authors.lastname: Durbin
authors.forename: Dorothy R
authors.initials: DR
authors.lastname: Ahlf
authors.forename: Bryan P
authors.initials: BP
authors.lastname: Early
authors.forename: Ryan T
authors.initials: RT
authors.lastname: Fellers
authors.forename: John C
authors.initials: JC
authors.lastname: Tran
authors.forename: Paul M
authors.initials: PM
authors.lastname: Thomas
authors.forename: Neil L
authors.initials: NL
authors.lastname: Kelleher
mesh_terms: D002166:Camptothecin / Q000494:pharmacology; D002458:Cell Fractionation; D002461:Cell Line, Transformed; D016922:Cellular Senescence / Q000187:drug effects / Q000235:genetics*; D002853:Chromatography, Liquid; D004847:Epithelial Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D005786:Gene Expression Regulation; D057166:High-Throughput Screening Assays; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D008745:Methylation; D008928:Mitochondria / Q000737:chemistry / Q000378:metabolism; D024101:Mitochondrial Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D058977:Molecular Sequence Annotation; D009097:Multienzyme Complexes / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D040901:Proteomics / Q000379:methods; D015398:Signal Transduction; D053719:Tandem Mass Spectrometry
chemical_list: D008565:Membrane Proteins; D024101:Mitochondrial Proteins; D009097:Multienzyme Complexes; D002166:Camptothecin
references.citation: J Biol Chem. 2004 May 21;279(21):21883-7
references.pmid: 15010464
references.citation: Anal Chem. 2012 Jan 3;84(1):209-15
references.pmid: 22103811
references.citation: J Biol Chem. 2012 Jan 6;287(2):848-57
references.pmid: 22052912
references.citation: Proc Natl Acad Sci U S A. 2006 Oct 31;103(44):16170-5
references.pmid: 17060615
references.citation: Proc Natl Acad Sci U S A. 2013 Jun 18;110(25):10153-8
references.pmid: 23720318
references.citation: Mol Cell Proteomics. 2010 May;9(5):791-803
references.pmid: 20093275
references.citation: Nature. 2011 Oct 30;480(7376):254-8
references.pmid: 22037311
references.citation: Anal Chem. 2004 Jun 1;76(11):197A-203A
references.pmid: 15190879
references.citation: J Proteome Res. 2002 Sep-Oct;1(5):451-8
references.pmid: 12645917
references.citation: Mol Biol Cell. 2005 Nov;16(11):5236-46
references.pmid: 16162815
references.citation: Nat Protoc. 2009;4(12):1798-806
references.pmid: 20010931
references.citation: J Am Chem Soc. 2004 Mar 24;126(11):3386-7
references.pmid: 15025441
references.citation: Mol Cell Proteomics. 2006 Apr;5(4):608-19
references.pmid: 16415296
references.citation: Expert Rev Proteomics. 2006 Dec;3(6):585-96
references.pmid: 17181473
references.citation: Anal Biochem. 1984 Apr;138(1):141-3
references.pmid: 6731838
references.citation: Cell. 2006 Apr 7;125(1):173-86
references.pmid: 16615898
references.citation: Proc Natl Acad Sci U S A. 2007 Sep 4;104(36):14330-5
references.pmid: 17720804
references.citation: Proc Int Conf Intell Syst Mol Biol. 1998;6:175-82
references.pmid: 9783223
references.citation: Proteomics. 2010 Oct;10(20):3644-56
references.pmid: 20845333
references.citation: J Biol Chem. 2009 Nov 6;284(45):30965-73
references.pmid: 19635794
references.citation: Proc Natl Acad Sci U S A. 2003 Nov 11;100(23):13207-12
references.pmid: 14576278
references.citation: Dev Cell. 2005 Jan;8(1):19-30
references.pmid: 15621527
references.citation: Nat Biotechnol. 2001 Oct;19(10):952-7
references.pmid: 11581661
references.citation: Cell. 2003 Nov 26;115(5):629-40
references.pmid: 14651853
references.citation: Science. 2011 Feb 11;331(6018):778-82
references.pmid: 21311024
references.citation: Proc Natl Acad Sci U S A. 2003 Aug 5;100(16):9440-5
references.pmid: 12883005
references.citation: Science. 2006 Oct 6;314(5796):65-6
references.pmid: 17023639
references.citation: Anal Chem. 2013 Feb 5;85(3):1880-8
references.pmid: 23305238
references.citation: Proteomics. 2010 Oct;10(20):3589-97
references.pmid: 20848673
references.citation: Exp Nephrol. 1998 Mar-Apr;6(2):148-51
references.pmid: 9567221
references.citation: Nat Methods. 2013 Mar;10(3):186-7
references.pmid: 23443629
references.citation: EMBO J. 2003 Jun 2;22(11):2729-40
references.pmid: 12773388
references.citation: J Proteome Res. 2012 Aug 3;11(8):4308-14
references.pmid: 22746247
references.citation: J Proteome Res. 2008 Apr;7(4):1761-6
references.pmid: 18284188