title: Evaluation of selected binding domains for the analysis of ubiquitinated proteomes.
abstract: Ubiquitination is an abundant post-translational modification that consists of covalent attachment of ubiquitin to lysine residues or the N-terminus of proteins. Mono- and polyubiquitination have been shown to be involved in many critical eukaryotic cellular functions and are often disrupted by intracellular bacterial pathogens. Affinity enrichment of ubiquitinated proteins enables global analysis of this key modification. In this context, the use of ubiquitin-binding domains is a promising but relatively unexplored alternative to more broadly used immunoaffinity or tagged affinity enrichment methods. In this study, we evaluated the application of eight ubiquitin-binding domains that have differing affinities for ubiquitination states. Small-scale proteomics analysis identified ~200 ubiquitinated protein candidates per ubiquitin-binding domain pull-down experiment. Results from subsequent Western blot analyses that employed anti-ubiquitin or monoclonal antibodies against polyubiquitination at lysine 48 and 63 suggest that ubiquitin-binding domains from Dsk2 and ubiquilin-1 have the broadest specificity in that they captured most types of ubiquitination, whereas the binding domain from NBR1 was more selective to polyubiquitination. These data demonstrate that with optimized purification conditions, ubiquitin-binding domains can be an alternative tool for proteomic applications. This approach is especially promising for the analysis of tissues or cells resistant to transfection, of which the overexpression of tagged ubiquitin is a major hurdle.
authors: Biological Sciences Division, Pacific Northwest National Laboratory, Richland, WA 99352, USA.
authors: Ernesto S
authors: ES
authors: Nakayasu
authors: Charles
authors: C
authors: Ansong
authors: Joseph N
authors: JN
authors: Brown
authors: Feng
authors: F
authors: Yang
authors: Daniel
authors: D
authors: Lopez-Ferrer
authors: Wei-Jun
authors: WJ
authors: Qian
authors: Richard D
authors: RD
authors: Smith
authors: Joshua N
authors: JN
authors: Adkins
mesh_terms: D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport / Q000737:chemistry; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry; D000071183:Autophagy-Related Proteins; D015153:Blotting, Western; D018797:Cell Cycle Proteins / Q000737:chemistry; D002460:Cell Line; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D006868:Hydrolysis; D007120:Immunochemistry; D007202:Indicators and Reagents; D007249:Inflammation / Q000473:pathology; D008264:Macrophages / Q000737:chemistry; D051379:Mice; D008969:Molecular Sequence Data; D011485:Protein Binding; D020543:Proteome / Q000737:chemistry* / Q000302:isolation & purification; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry; D015398:Signal Transduction; D053719:Tandem Mass Spectrometry; D057149:Ubiquitinated Proteins / Q000737:chemistry*; D014452:Ubiquitins / Q000737:chemistry
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport; D000911:Antibodies, Monoclonal; D000071183:Autophagy-Related Proteins; D018797:Cell Cycle Proteins; C100415:DSK2 protein, S cerevisiae; D007202:Indicators and Reagents; D020543:Proteome; D029701:Saccharomyces cerevisiae Proteins; C538987:UBQLN1 protein, mouse; D057149:Ubiquitinated Proteins; D014452:Ubiquitins
references: Mol Cell Proteomics. 2011 May;10(5):M110.002089
references: 20972266
references: Cell. 2008 Aug 22;134(4):668-78
references: 18724939
references: Nat Rev Mol Cell Biol. 2009 Aug;10(8):550-63
references: 19626045
references: Mol Cell Proteomics. 2007 Nov;6(11):1885-95
references: 17644757
references: Plant Mol Biol. 2008 Sep;68(1-2):145-58
references: 18535787
references: Curr Biol. 2003 Feb 4;13(3):258-62
references: 12573224
references: Mol Cell Proteomics. 2011 May;10(5):R110.006882
references: 21339389
references: Mol Cell Proteomics. 2012 May;11(5):148-59
references: 22505724
references: Nat Rev Mol Cell Biol. 2005 Aug;6(8):599-609
references: 16064136
references: Nat Cell Biol. 2008 May;10(5):602-10
references: 18391941
references: Mol Cell. 2009 Feb 27;33(4):505-16
references: 19250911
references: J Biol Chem. 2011 Mar 11;286(10):7905-7916
references: 21220427
references: J Proteome Res. 2005 Nov-Dec;4(6):2192-200
references: 16335966
references: Chem Rev. 2009 Apr;109(4):1481-94
references: 19253967
references: Int J Radiat Biol. 2009 Aug;85(8):643-55
references: 19579069
references: Mol Cell. 2009 Dec 25;36(6):1018-33
references: 20064467
references: J Biol Chem. 2011 Sep 9;286(36):31864-74
references: 21715324
references: Nat Struct Mol Biol. 2008 Dec;15(12):1293-301
references: 18997778
references: Protein Expr Purif. 2005 May;41(1):98-105
references: 15802226
references: PLoS One. 2007 Apr 18;2(4):e381
references: 17440617
references: Nat Biotechnol. 2010 Aug;28(8):868-73
references: 20639865
references: J Biol Chem. 2001 Dec 7;276(49):46655-60
references: 11585840
references: J Am Soc Mass Spectrom. 1994 Nov;5(11):976-89
references: 24226387
references: Anal Chem. 2008 Jan 1;80(1):294-302
references: 18044960
references: J Cell Biol. 2009 Oct 19;187(2):201-17
references: 19822669
references: Mol Cell. 2009 Aug 14;35(3):280-90
references: 19683493
references: Mol Cell Proteomics. 2012 Dec;11(12):1529-40
references: 22729469
references: Nat Struct Mol Biol. 2005 Aug;12(8):708-14
references: 16007098
references: Nat Chem Biol. 2007 Nov;3(11):697-705
references: 17948018
references: Curr Opin Microbiol. 2010 Feb;13(1):41-6
references: 20036613
references: Mol Cell. 2011 Oct 21;44(2):325-40
references: 21906983
references: PLoS Pathog. 2010 Jun 24;6(6):e1000960
references: 20585566
references: Nat Methods. 2011 Jul 10;8(8):691-6
references: 21743460
references: Nat Biotechnol. 2003 Aug;21(8):921-6
references: 12872131
references: Proteomics. 2005 Nov;5(16):4145-51
references: 16196087
references: J Mol Biol. 2007 May 25;369(1):168-76
references: 17408689
references: Mol Cell Biochem. 2008 Nov;318(1-2):129-45
references: 18612801
references: Mol Cell Proteomics. 2012 Dec;11(12):1578-85
references: 22790023
references: Mol Cell Proteomics. 2011 Oct;10(10):M111.013284
references: 21890473
references: Cell. 2003 May 30;113(5):609-20
references: 12787502
references: EMBO Rep. 2009 Nov;10(11):1250-8
references: 19798103
references: J Biol Chem. 2008 Feb 29;283(9):5427-40
references: 18083707
references: Proteomics. 2005 May;5(8):2104-11
references: 15852347
references: Methods. 2011 Aug;54(4):387-95
references: 21382495
references: J Proteomics. 2012 Jun 6;75(10):2998-3014
references: 22178446
references: Mol Cell. 2010 Aug 13;39(3):477-84
references: 20655260
references: J Mol Biol. 2008 Mar 14;377(1):162-80
references: 18241885
references: Cell. 2009 Apr 3;137(1):133-45
references: 19345192
references: Proc Natl Acad Sci U S A. 2007 Feb 27;104(9):3502-7
references: 17360673
references: Mol Cell Proteomics. 2011 May;10(5):M110.002188
references: 20861518
references: mBio. 2010 Oct 26;1(5):
references: 20978539
references: J Proteome Res. 2008 Aug;7(8):3276-81
references: 18605748
references: EMBO J. 2006 May 3;25(9):1934-44
references: 16601676
references: Anal Chem. 2004 Dec 1;76(23):6853-60
references: 15571333
references: Biochem J. 2006 Nov 1;399(3):361-72
references: 17034365
references: Anal Chem. 2012 Nov 20;84(22):10121-8
references: 23101881
references: Bioinformatics. 2008 Jul 1;24(13):1556-8
references: 18453552
references: Nat Methods. 2008 Jun;5(6):459-60
references: 18511913
references: Annu Rev Biochem. 1998;67:425-79
references: 9759494