title: Quantitative analysis of global ubiquitination in HeLa cells by mass spectrometry.
abstract: Ubiquitination regulates a host of cellular processes by labeling proteins for degradation, but also by functioning as a regulatory, nonproteolytic posttranslational modification. Proteome-wide strategies to monitor changes in ubiquitination profiles are important to obtain insight into the various cellular functions of ubiquitination. Here we describe generation of stable cell lines expressing a tandem hexahistidine-biotin tag (HB-tag) fused to ubiquitin for two-step purification of the ubiquitinated proteome under fully denaturing conditions. Using this approach we identified 669 ubiquitinated proteins from HeLa cells, including 44 precise ubiquitin attachment sites on substrates and all seven possible ubiquitin chain-linkage types. To probe the dynamics of ubiquitination in response to perturbation of the ubiquitin/proteasome pathway, we combined ubiquitin profiling with quantitative mass spectrometry using the stable isotope labeling with amino acids in cell culture (SILAC) strategy. We compared untreated cells and cells treated with the proteasome inhibitor MG132 to identify ubiquitinated proteins that are targeted to the proteasome for degradation. A number of proteasome substrates were identified. In addition, the quantitative approach allowed us to compare proteasome targeting by different ubiquitin chain topologies in vivo. The tools and strategies described here can be applied to detect changes in ubiquitination dynamics in response to various changes in growth conditions and cellular stress and will contribute to our understanding of the ubiquitin/proteasome system.
authors.affiliation: Department of Biological Chemistry, University of California, Irvine, California 92697, USA.
authors.forename: David
authors.initials: D
authors.lastname: Meierhofer
authors.forename: Xiaorong
authors.initials: X
authors.lastname: Wang
authors.forename: Lan
authors.initials: L
authors.lastname: Huang
authors.forename: Peter
authors.initials: P
authors.lastname: Kaiser
mesh_terms: D000595:Amino Acid Sequence; D005982:Glutathione Transferase / Q000378:metabolism; D006367:HeLa Cells / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D007553:Isotope Labeling; D007976:Leupeptins / Q000378:metabolism; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010455:Peptides / Q000032:analysis* / Q000235:genetics; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D061988:Proteasome Inhibitors; D025801:Ubiquitin / Q000737:chemistry* / Q000378:metabolism
chemical_list: D007976:Leupeptins; D010455:Peptides; D061988:Proteasome Inhibitors; D025801:Ubiquitin; D005982:Glutathione Transferase; D046988:Proteasome Endopeptidase Complex; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde
references.citation: Oncogene. 2007 Jan 25;26(4):604-9
references.pmid: 16862177
references.citation: Proteomics. 2007 Jul;7(14):2375-83
references.pmid: 17623298
references.citation: Mol Cell. 2008 Mar 28;29(6):653-63
references.pmid: 18374642
references.citation: Science. 2001 Apr 20;292(5516):464-8
references.pmid: 11292862
references.citation: Biochemistry. 2007 Mar 20;46(11):3553-65
references.pmid: 17323924
references.citation: Mol Cell Proteomics. 2008 Dec;7(12):2386-98
references.pmid: 18653769
references.citation: Nature. 1995 Jan 5;373(6509):81-3
references.pmid: 7800044
references.citation: J Natl Cancer Inst. 1990 Jul 4;82(13):1107-12
references.pmid: 2359136
references.citation: Cell Div. 2006 Sep 28;1:21
references.pmid: 17007633
references.citation: Annu Rev Biochem. 1998;67:425-79
references.pmid: 9759494
references.citation: Proc Natl Acad Sci U S A. 2003 Oct 28;100(22):12735-40
references.pmid: 14557538
references.citation: Nat Rev Mol Cell Biol. 2001 Mar;2(3):195-201
references.pmid: 11265249
references.citation: Nat Rev Mol Cell Biol. 2003 Nov;4(11):855-64
references.pmid: 14625536
references.citation: J Proteome Res. 2005 Nov-Dec;4(6):2192-200
references.pmid: 16335966
references.citation: Nature. 2007 Aug 9;448(7154):704-8
references.pmid: 17687326
references.citation: Science. 2005 Jul 1;309(5731):127-30
references.pmid: 15994556
references.citation: Methods. 2003 Feb;29(2):124-30
references.pmid: 12606218
references.citation: Mol Cell. 2001 Feb;7(2):249-62
references.pmid: 11239454
references.citation: Cell. 2004 Jan 23;116(2):181-90
references.pmid: 14744430
references.citation: J Proteome Res. 2008 Mar;7(3):1274-87
references.pmid: 18247557
references.citation: Cell. 2003 Oct 3;115(1):71-82
references.pmid: 14532004
references.citation: Mol Cell Proteomics. 2007 Nov;6(11):1885-95
references.pmid: 17644757
references.citation: Nat Cell Biol. 2006 Jul;8(7):700-10
references.pmid: 16799550
references.citation: J Cell Biol. 2006 Apr 10;173(1):19-26
references.pmid: 16606690
references.citation: Nat Biotechnol. 2003 Aug;21(8):921-6
references.pmid: 12872131
references.citation: Nat Cell Biol. 2007 Apr;9(4):422-7
references.pmid: 17310239
references.citation: Proteomics. 2005 Nov;5(16):4145-51
references.pmid: 16196087
references.citation: Methods Enzymol. 2005;402:209-44
references.pmid: 16401511
references.citation: Rapid Commun Mass Spectrom. 2002;16(22):2115-23
references.pmid: 12415544
references.citation: Cell. 2008 May 16;133(4):653-65
references.pmid: 18485873
references.citation: Proteomics. 2005 May;5(8):2104-11
references.pmid: 15852347
references.citation: Mol Cell Proteomics. 2002 May;1(5):376-86
references.pmid: 12118079
references.citation: Mol Cell Proteomics. 2005 Jun;4(6):741-51
references.pmid: 15699485
references.citation: Mol Cell Proteomics. 2006 Apr;5(4):737-48
references.pmid: 16432255
references.citation: Yeast. 2006 Jun;23(8):623-32
references.pmid: 16823883
references.citation: Mol Cell Proteomics. 2006 Feb;5(2):366-78
references.pmid: 16284124
references.citation: Cell. 2007 Apr 20;129(2):289-301
references.pmid: 17412408