A Data Set of Human Endogenous Protein Ubiquitination Sites

Lysine ubiquitination is an important and versatile protein post-translational modification. Numerous cellular functions are regulated by ubiquitination, suggesting that extensive numbers of proteins, if not all, are modified with ubiquitin at certain times. However, proteome-wide profiling of ubiquitination sites in the mammalian system is technically challenging. We report the design and characterization of an engineered protein affinity reagent for the isolation of ubiquitinated proteins and the identification of ubiquitination sites with mass spectrometry. This recombinant protein consists of four tandem repeats of ubiquitin-associated domain from UBQLN1 fused to a GST tag. We used this GST-qUBA reagent to isolate polyubiquitinated proteins and identified 294 endogenous ubiquitination sites on 223 proteins from human 293T cells without proteasome inhibitors or overexpression of ubiquitin. Mitochondrial proteins constitute 14.7% of this data set, implicating ubiquitination in a wide range of mitochondrial functions. Lysine ubiquitination is an important and versatile protein post-translational modification. Numerous cellular functions are regulated by ubiquitination, suggesting that extensive numbers of proteins, if not all, are modified with ubiquitin at certain times. However, proteome-wide profiling of ubiquitination sites in the mammalian system is technically challenging. We report the design and characterization of an engineered protein affinity reagent for the isolation of ubiquitinated proteins and the identification of ubiquitination sites with mass spectrometry. This recombinant protein consists of four tandem repeats of ubiquitin-associated domain from UBQLN1 fused to a GST tag. We used this GST-qUBA reagent to isolate polyubiquitinated proteins and identified 294 endogenous ubiquitination sites on 223 proteins from human 293T cells without proteasome inhibitors or overexpression of ubiquitin. Mitochondrial proteins constitute 14.7% of this data set, implicating ubiquitination in a wide range of mitochondrial functions. Post-translational modification of proteins by ubiquitination at lysine residues plays regulatory roles in a broad spectrum of cellular processes including cell cycle progression, DNA damage, and immune response (1Schwartz A.L. Ciechanover A. The ubiquitin-proteasome pathway and pathogenesis of human diseases.Annu. Rev. Med. 1999; 50: 57-74Crossref PubMed Scopus (373) Google Scholar). Deregulation of ubiquitination is linked to many human diseases including cancer and neuronal disorders (2Dikic I. Crosetto N. Calatroni S. Bernasconi P. Targeting ubiquitin in cancers.Eur. J. Cancer. 2006; 42: 3095-3102Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar, 3Shimura H. Hattori N. Kubo S. Mizuno Y. Asakawa S. Minoshima S. Shimizu N. Iwai K. Chiba T. Tanaka K. Suzuki T. Familial Parkinson disease gene product, parkin, is a ubiquitin-protein ligase.Nat. Genet. 2000; 25: 302-305Crossref PubMed Scopus (1682) Google Scholar). In the canonical ubiquitination reaction, a substrate is covalently conjugated with ubiquitin by an enzymatic cascade involving ubiquitin-activating enzyme (E1), ubiquitin conjugation enzyme (E2), and a ubiquitin ligase (E3). Conversely, the ubiquitin moiety can be cleaved off from the substrates by deubiquitinases (DUBs). 1The abbreviations used are:DUBdeubiquitinaseUbubiquitinUBDubiquitin binding domainUBAubiquitin-associated domainqUBAfour tandem repeats of ubiquitin-associated domainUBQLN1ubiquilin-1FAformic acidFDRfalse discovery rateSILACstable isotope labeling by amino acids in cell cultureSTRINGSearch Tool for the Retrieval of Interacting Genes/ProteinsMCODEMolecular Complex Detection. The human genome encodes at least two E1, 53 E2, and ∼500 E3 enzymes and more than 100 DUBs (4Jin J. Li X. Gygi S.P. Harper J.W. Dual E1 activation systems for ubiquitin differentially regulate E2 enzyme charging.Nature. 2007; 447: 1135-1138Crossref PubMed Scopus (257) Google Scholar, 5Semple C.A. The comparative proteomics of ubiquitination in mouse.Genome Res. 2003; 13: 1389-1394Crossref PubMed Scopus (115) Google Scholar); such diversity of the ubiquitination system rivals that of the kinome and offers multiple gateways for regulation of ubiquitination dynamics in a cell.The diversity of the cellular ubiquitination system or ubiquitome is increased by formation of polyubiquitin (poly-Ub) chains with variable lengths and linkages. It is now known that all seven internal lysine residues and the N terminus of ubiquitin can form poly-Ub chains that may impart different functions. For example, proteasome-mediated protein degradation is a well known consequence of Lys-48-linked poly-Ub modification (6Chau V. Tobias J.W. Bachmair A. Marriott D. Ecker D.J. Gonda D.K. Varshavsky A. A multiubiquitin chain is confined to specific lysine in a targeted short-lived protein.Science. 1989; 243: 1576-1583Crossref PubMed Scopus (1106) Google Scholar). Other linkages may also lead to protein degradation (7Xu P. Duong D.M. Seyfried N.T. Cheng D. Xie Y. Robert J. Rush J. Hochstrasser M. Finley D. Peng J. Quantitative proteomics reveals the function of unconventional ubiquitin chains in proteasomal degradation.Cell. 2009; 137: 133-145Abstract Full Text Full Text PDF PubMed Scopus (834) Google Scholar) or may have non-proteolytic consequences. The Lys-63 linkage is best characterized in the inflammatory response where it aids in activation of kinase cascades (8Skaug B. Jiang X. Chen Z.J. The role of ubiquitin in NF-kappaB regulatory pathways.Annu. Rev. Biochem. 2009; 78: 769-796Crossref PubMed Scopus (404) Google Scholar, 9Xia Z.P. Sun L. Chen X. Pineda G. Jiang X. Adhikari A. Zeng W. Chen Z.J. Direct activation of protein kinases by unanchored polyubiquitin chains.Nature. 2009; 461: 114-119Crossref PubMed Scopus (418) Google Scholar), the N-terminal linear poly-Ub has been shown to activate the IκB kinase (10Tokunaga F. Sakata S. Saeki Y. Satomi Y. Kirisako T. Kamei K. Nakagawa T. Kato M. Murata S. Yamaoka S. Yamamoto M. Akira S. Takao T. Tanaka K. Iwai K. Involvement of linear polyubiquitylation of NEMO in NF-kappaB activation.Nat. Cell Biol. 2009; 11: 123-132Crossref PubMed Scopus (735) Google Scholar), and Lys-33 linkage has recently been shown to regulate specific signal transduction in a proteolysis-independent manner (11Huang H. Jeon M.S. Liao L. Yang C. Elly C. Yates 3rd, J.R. Liu Y.C. K33-linked polyubiquitination of T cell receptor-zeta regulates proteolysis-independent T cell signaling.Immunity. 2010; 33: 60-70Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar).Thorough understanding of this complex regulatory system requires identification of ubiquitinated substrates and, preferably, mapping of their ubiquitination sites, which is technically challenging. The most common approach used by laboratories to date is site-directed mutagenesis of putative lysine targets to infer the ubiquitination site(s). Although this is necessary for determining whether ubiquitination has a significant effect on a particular biology of the protein in question, it does not provide direct chemical evidence of ubiquitination. Erroneous conclusions can be made if mutation of lysine residues affects protein structure, folding, or docking site for E2 and E3 enzymes. Mass spectrometry (MS) has emerged as an indispensable tool for direct measurement of the lysine ubiquitination site(s) that complements mutagenesis studies. The ubiquitin conjugation generates an isopeptide bond between the ε-amine of the modified lysine on the substrate and the C terminus of ubiquitin. After trypsin cleavage, ubiquitination can be detected by mass spectrometry as a 114.043-Da mass shift (from the Gly-Gly remnant of the ubiquitin C terminus) on the modified peptides. The primary limitation in proteome-wide identification of ubiquitination sites is the lack of high affinity reagents for isolation of ubiquitinated peptides. Additionally, there are three confounding factors that limit our abilities to enrich ubiquitinated peptides. First, only a small percentage of a given protein is ubiquitinated in the steady state. Second, DUBs have sizable enzymatic activity and further decrease levels of ubiquitinated proteins upon cell lysis. Lastly, ubiquitin is the most abundant ubiquitinated protein in the cell due to the prevalence of poly-Ub chains, masking the identification of other substrates by mass spectrometry. Despite these inherent barriers, several studies have shown moderate success in cataloguing the lysine ubiquitination sites (12Peng J. Schwartz D. Elias J.E. Thoreen C.C. Cheng D. Marsischky G. Roelofs J. Finley D. Gygi S.P. A proteomics approach to understanding protein ubiquitination.Nat. Biotechnol. 2003; 21: 921-926Crossref PubMed Scopus (1292) Google Scholar, 13Mayor T. Lipford J.R. Graumann J. Smith G.T. Deshaies R.J. Analysis of polyubiquitin conjugates reveals that the Rpn10 substrate receptor contributes to the turnover of multiple proteasome targets.Mol. Cell. Proteomics. 2005; 4: 741-751Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 14Tagwerker C. Flick K. Cui M. Guerrero C. Dou Y. Auer B. Baldi P. Huang L. Kaiser P. A tandem affinity tag for two-step purification under fully denaturing conditions: application in ubiquitin profiling and protein complex identification combined with in vivo cross-linking.Mol. Cell. Proteomics. 2006; 5: 737-748Abstract Full Text Full Text PDF PubMed Scopus (301) Google Scholar, 15Meierhofer D. Wang X. Huang L. Kaiser P. Quantitative analysis of global ubiquitination in HeLa cells by mass spectrometry.J. Proteome Res. 2008; 7: 4566-4576Crossref PubMed Scopus (161) Google Scholar, 16Seyfried N.T. Xu P. Duong D.M. Cheng D. Hanfelt J. Peng J. Systematic approach for validating the ubiquitinated proteome.Anal. Chem. 2008; 80: 4161-4169Crossref PubMed Scopus (61) Google Scholar). For example, Peng et al. (12Peng J. Schwartz D. Elias J.E. Thoreen C.C. Cheng D. Marsischky G. Roelofs J. Finley D. Gygi S.P. A proteomics approach to understanding protein ubiquitination.Nat. Biotechnol. 2003; 21: 921-926Crossref PubMed Scopus (1292) Google Scholar) pioneered a yeast ubiquitin deletion strain expressing His6-tagged ubiquitin to isolate ubiquitinated substrates and identified 110 ubiquitination sites in yeast proteins with an LCQ mass spectrometer. Meierhofer et al. (15Meierhofer D. Wang X. Huang L. Kaiser P. Quantitative analysis of global ubiquitination in HeLa cells by mass spectrometry.J. Proteome Res. 2008; 7: 4566-4576Crossref PubMed Scopus (161) Google Scholar) used a similar approach in HeLa cells that stably overexpress tagged ubiquitin and identified ∼50 ubiquitination sites in human proteins with an LTQ-XL-Orbitrap.Eukaryotic cells have evolved protein domain structures, ubiquitin binding domains (UBDs), that can recognize and bind to ubiquitin modifications. More than 20 different families have been identified to date, and most of them bind poly-Ub relatively weakly (17Hicke L. Schubert H.L. Hill C.P. Ubiquitin-binding domains.Nat. Rev. Mol. Cell Biol. 2005; 6: 610-621Crossref PubMed Scopus (635) Google Scholar, 18Dikic I. Wakatsuki S. Walters K.J. Ubiquitin-binding domains—from structures to functions.Nat. Rev. Mol. Cell Biol. 2009; 10: 659-671Crossref PubMed Scopus (614) Google Scholar). The ubiquitin-associated domain (UBA) is the first identified UBD and is one of the best poly-Ub binders (19Raasi S. Varadan R. Fushman D. Pickart C.M. Diverse polyubiquitin interaction properties of ubiquitin-associated domains.Nat. Struct. Mol. Biol. 2005; 12: 708-714Crossref PubMed Scopus (272) Google Scholar, 20Raasi S. Orlov I. Fleming K.G. Pickart C.M. Binding of polyubiquitin chains to ubiquitin-associated (UBA) domains of HHR23A.J. Mol. Biol. 2004; 341: 1367-1379Crossref PubMed Scopus (130) Google Scholar). Although a single UBA domain has been successfully used as an affinity reagent to quantify the poly-Ub chains in a mouse model of Huntington disease (21Bennett E.J. Shaler T.A. Woodman B. Ryu K.Y. Zaitseva T.S. Becker C.H. Bates G.P. Schulman H. Kopito R.R. Global changes to the ubiquitin system in Huntington's disease.Nature. 2007; 448: 704-708Crossref PubMed Scopus (424) Google Scholar), it bears moderate ubiquitin binding affinity that may not be sufficient for proteome-wide isolation of ubiquitinated proteins. Recently, several studies showed that tandem UBDs display avidity in poly-Ub binding (22Sims J.J. Haririnia A. Dickinson B.C. Fushman D. Cohen R.E. Avid interactions underlie the Lys63-linked polyubiquitin binding specificities observed for UBA domains.Nat. Struct. Mol. Biol. 2009; 16: 883-889Crossref PubMed Scopus (71) Google Scholar, 23Sato Y. Yoshikawa A. Mimura H. Yamashita M. Yamagata A. Fukai S. Structural basis for specific recognition of Lys 63-linked polyubiquitin chains by tandem UIMs of RAP80.EMBO J. 2009; 28: 2461-2468Crossref PubMed Scopus (170) Google Scholar, 24Hjerpe R. Aillet F. Lopitz-Otsoa F. Lang V. England P. Rodriguez M.S. Efficient protection and isolation of ubiquitylated proteins using tandem ubiquitin-binding entities.EMBO Rep. 2009; 10: 1250-1258Crossref PubMed Scopus (334) Google Scholar). We have utilized this concept to develop and test the suitability of a tandem UBA protein for large scale characterization of ubiquitination substrates.Here, we report using recombinant GST fusion of four tandem ubiquilin-1 (UBQLN1) UBA domains (GST-qUBA) as a bait to isolate endogenous ubiquitinated proteins from human 293T cells without ubiquitin overexpression and proteasome inhibition. We confirmed that tandem GST-qUBA is more efficient at binding poly-Ub chains than the single GST-UBA. Using GST-qUBA as an affinity purification reagent, we were able to detect ∼300 lysine ubiquitination sites that are supported by high quality mass spectra. Both abundant and low abundance cellular proteins including and of and were We that GST-qUBA is a tool for the affinity purification and identification of endogenous protein ubiquitination sites at the Post-translational modification of proteins by ubiquitination at lysine residues plays regulatory roles in a broad spectrum of cellular processes including cell cycle progression, DNA damage, and immune response (1Schwartz A.L. Ciechanover A. The ubiquitin-proteasome pathway and pathogenesis of human diseases.Annu. Rev. Med. 1999; 50: 57-74Crossref PubMed Scopus (373) Google Scholar). Deregulation of ubiquitination is linked to many human diseases including cancer and neuronal disorders (2Dikic I. Crosetto N. Calatroni S. Bernasconi P. Targeting ubiquitin in cancers.Eur. J. Cancer. 2006; 42: 3095-3102Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar, 3Shimura H. Hattori N. Kubo S. Mizuno Y. Asakawa S. Minoshima S. Shimizu N. Iwai K. Chiba T. Tanaka K. Suzuki T. Familial Parkinson disease gene product, parkin, is a ubiquitin-protein ligase.Nat. Genet. 2000; 25: 302-305Crossref PubMed Scopus (1682) Google Scholar). In the canonical ubiquitination reaction, a substrate is covalently conjugated with ubiquitin by an enzymatic cascade involving ubiquitin-activating enzyme (E1), ubiquitin conjugation enzyme (E2), and a ubiquitin ligase (E3). Conversely, the ubiquitin moiety can be cleaved off from the substrates by deubiquitinases (DUBs). 1The abbreviations used are:DUBdeubiquitinaseUbubiquitinUBDubiquitin binding domainUBAubiquitin-associated domainqUBAfour tandem repeats of ubiquitin-associated domainUBQLN1ubiquilin-1FAformic acidFDRfalse discovery rateSILACstable isotope labeling by amino acids in cell cultureSTRINGSearch Tool for the Retrieval of Interacting Genes/ProteinsMCODEMolecular Complex Detection. The human genome encodes at least two E1, 53 E2, and ∼500 E3 enzymes and more than 100 DUBs (4Jin J. Li X. Gygi S.P. Harper J.W. Dual E1 activation systems for ubiquitin differentially regulate E2 enzyme charging.Nature. 2007; 447: 1135-1138Crossref PubMed Scopus (257) Google Scholar, 5Semple C.A. The comparative proteomics of ubiquitination in mouse.Genome Res. 2003; 13: 1389-1394Crossref PubMed Scopus (115) Google Scholar); such diversity of the ubiquitination system rivals that of the kinome and offers multiple gateways for regulation of ubiquitination dynamics in a ubiquitin ubiquitin binding domain ubiquitin-associated domain four tandem repeats of ubiquitin-associated domain ubiquilin-1 discovery isotope labeling by amino acids in cell Tool for the Retrieval of Interacting Complex Detection. The diversity of the cellular ubiquitination system or ubiquitome is increased by formation of polyubiquitin (poly-Ub) chains with variable lengths and linkages. It is now known that all seven internal lysine residues and the N terminus of ubiquitin can form poly-Ub chains that may impart different functions. For example, proteasome-mediated protein degradation is a well known consequence of Lys-48-linked poly-Ub modification (6Chau V. Tobias J.W. Bachmair A. Marriott D. Ecker D.J. Gonda D.K. Varshavsky A. A multiubiquitin chain is confined to specific lysine in a targeted short-lived protein.Science. 1989; 243: 1576-1583Crossref PubMed Scopus (1106) Google Scholar). Other linkages may also lead to protein degradation (7Xu P. Duong D.M. Seyfried N.T. Cheng D. Xie Y. Robert J. Rush J. Hochstrasser M. Finley D. Peng J. Quantitative proteomics reveals the function of unconventional ubiquitin chains in proteasomal degradation.Cell. 2009; 137: 133-145Abstract Full Text Full Text PDF PubMed Scopus (834) Google Scholar) or may have non-proteolytic consequences. The Lys-63 linkage is best characterized in the inflammatory response where it aids in activation of kinase cascades (8Skaug B. Jiang X. Chen Z.J. The role of ubiquitin in NF-kappaB regulatory pathways.Annu. Rev. Biochem. 2009; 78: 769-796Crossref PubMed Scopus (404) Google Scholar, 9Xia Z.P. Sun L. Chen X. Pineda G. Jiang X. Adhikari A. Zeng W. Chen Z.J. Direct activation of protein kinases by unanchored polyubiquitin chains.Nature. 2009; 461: 114-119Crossref PubMed Scopus (418) Google Scholar), the N-terminal linear poly-Ub has been shown to activate the IκB kinase (10Tokunaga F. Sakata S. Saeki Y. Satomi Y. Kirisako T. Kamei K. Nakagawa T. Kato M. Murata S. Yamaoka S. Yamamoto M. Akira S. Takao T. Tanaka K. Iwai K. Involvement of linear polyubiquitylation of NEMO in NF-kappaB activation.Nat. Cell Biol. 2009; 11: 123-132Crossref PubMed Scopus (735) Google Scholar), and Lys-33 linkage has recently been shown to regulate specific signal transduction in a proteolysis-independent manner (11Huang H. Jeon M.S. Liao L. Yang C. Elly C. Yates 3rd, J.R. Liu Y.C. K33-linked polyubiquitination of T cell receptor-zeta regulates proteolysis-independent T cell signaling.Immunity. 2010; 33: 60-70Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). understanding of this complex regulatory system requires identification of ubiquitinated substrates and, preferably, mapping of their ubiquitination sites, which is technically challenging. The most common approach used by laboratories to date is site-directed mutagenesis of putative lysine targets to infer the ubiquitination site(s). Although this is necessary for determining whether ubiquitination has a significant effect on a particular biology of the protein in question, it does not provide direct chemical evidence of ubiquitination. Erroneous conclusions can be made if mutation of lysine residues affects protein structure, folding, or docking site for E2 and E3 enzymes. Mass spectrometry (MS) has emerged as an indispensable tool for direct measurement of the lysine ubiquitination site(s) that complements mutagenesis studies. The ubiquitin conjugation generates an isopeptide bond between the ε-amine of the modified lysine on the substrate and the C terminus of ubiquitin. After trypsin cleavage, ubiquitination can be detected by mass spectrometry as a 114.043-Da mass shift (from the Gly-Gly remnant of the ubiquitin C terminus) on the modified peptides. The primary limitation in proteome-wide identification of ubiquitination sites is the lack of high affinity reagents for isolation of ubiquitinated peptides. Additionally, there are three confounding factors that limit our abilities to enrich ubiquitinated peptides. First, only a small percentage of a given protein is ubiquitinated in the steady state. Second, DUBs have sizable enzymatic activity and further decrease levels of ubiquitinated proteins upon cell lysis. Lastly, ubiquitin is the most abundant ubiquitinated protein in the cell due to the prevalence of poly-Ub chains, masking the identification of other substrates by mass spectrometry. Despite these inherent barriers, several studies have shown moderate success in cataloguing the lysine ubiquitination sites (12Peng J. Schwartz D. Elias J.E. Thoreen C.C. Cheng D. Marsischky G. Roelofs J. Finley D. Gygi S.P. A proteomics approach to understanding protein ubiquitination.Nat. Biotechnol. 2003; 21: 921-926Crossref PubMed Scopus (1292) Google Scholar, 13Mayor T. Lipford J.R. Graumann J. Smith G.T. Deshaies R.J. Analysis of polyubiquitin conjugates reveals that the Rpn10 substrate receptor contributes to the turnover of multiple proteasome targets.Mol. Cell. Proteomics. 2005; 4: 741-751Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 14Tagwerker C. Flick K. Cui M. Guerrero C. Dou Y. Auer B. Baldi P. Huang L. Kaiser P. A tandem affinity tag for two-step purification under fully denaturing conditions: application in ubiquitin profiling and protein complex identification combined with in vivo cross-linking.Mol. Cell. Proteomics. 2006; 5: 737-748Abstract Full Text Full Text PDF PubMed Scopus (301) Google Scholar, 15Meierhofer D. Wang X. Huang L. Kaiser P. Quantitative analysis of global ubiquitination in HeLa cells by mass spectrometry.J. Proteome Res. 2008; 7: 4566-4576Crossref PubMed Scopus (161) Google Scholar, 16Seyfried N.T. Xu P. Duong D.M. Cheng D. Hanfelt J. Peng J. Systematic approach for validating the ubiquitinated proteome.Anal. Chem. 2008; 80: 4161-4169Crossref PubMed Scopus (61) Google Scholar). For example, Peng et al. (12Peng J. Schwartz D. Elias J.E. Thoreen C.C. Cheng D. Marsischky G. Roelofs J. Finley D. Gygi S.P. A proteomics approach to understanding protein ubiquitination.Nat. Biotechnol. 2003; 21: 921-926Crossref PubMed Scopus (1292) Google Scholar) pioneered a yeast ubiquitin deletion strain expressing His6-tagged ubiquitin to isolate ubiquitinated substrates and identified 110 ubiquitination sites in yeast proteins with an LCQ mass spectrometer. Meierhofer et al. (15Meierhofer D. Wang X. Huang L. Kaiser P. Quantitative analysis of global ubiquitination in HeLa cells by mass spectrometry.J. Proteome Res. 2008; 7: 4566-4576Crossref PubMed Scopus (161) Google Scholar) used a similar approach in HeLa cells that stably overexpress tagged ubiquitin and identified ∼50 ubiquitination sites in human proteins with an cells have evolved protein domain structures, ubiquitin binding domains (UBDs), that can recognize and bind to ubiquitin modifications. More than 20 different families have been identified to date, and most of them bind poly-Ub relatively weakly (17Hicke L. Schubert H.L. Hill C.P. Ubiquitin-binding domains.Nat. Rev. Mol. Cell Biol. 2005; 6: 610-621Crossref PubMed Scopus (635) Google Scholar, 18Dikic I. Wakatsuki S. Walters K.J. Ubiquitin-binding domains—from structures to functions.Nat. Rev. Mol. Cell Biol. 2009; 10: 659-671Crossref PubMed Scopus (614) Google Scholar). The ubiquitin-associated domain (UBA) is the first identified UBD and is one of the best poly-Ub binders (19Raasi S. Varadan R. Fushman D. Pickart C.M. Diverse polyubiquitin interaction properties of ubiquitin-associated domains.Nat. Struct. Mol. Biol. 2005; 12: 708-714Crossref PubMed Scopus (272) Google Scholar, 20Raasi S. Orlov I. Fleming K.G. Pickart C.M. Binding of polyubiquitin chains to ubiquitin-associated (UBA) domains of HHR23A.J. Mol. Biol. 2004; 341: 1367-1379Crossref PubMed Scopus (130) Google Scholar). Although a single UBA domain has been successfully used as an affinity reagent to quantify the poly-Ub chains in a mouse model of Huntington disease (21Bennett E.J. Shaler T.A. Woodman B. Ryu K.Y. Zaitseva T.S. Becker C.H. Bates G.P. Schulman H. Kopito R.R. Global changes to the ubiquitin system in Huntington's disease.Nature. 2007; 448: 704-708Crossref PubMed Scopus (424) Google Scholar), it bears moderate ubiquitin binding affinity that may not be sufficient for proteome-wide isolation of ubiquitinated proteins. Recently, several studies showed that tandem UBDs display avidity in poly-Ub binding (22Sims J.J. Haririnia A. Dickinson B.C. Fushman D. Cohen R.E. Avid interactions underlie the Lys63-linked polyubiquitin binding specificities observed for UBA domains.Nat. Struct. Mol. Biol. 2009; 16: 883-889Crossref PubMed Scopus (71) Google Scholar, 23Sato Y. Yoshikawa A. Mimura H. Yamashita M. Yamagata A. Fukai S. Structural basis for specific recognition of Lys 63-linked polyubiquitin chains by tandem UIMs of RAP80.EMBO J. 2009; 28: 2461-2468Crossref PubMed Scopus (170) Google Scholar, 24Hjerpe R. Aillet F. Lopitz-Otsoa F. Lang V. England P. Rodriguez M.S. Efficient protection and isolation of ubiquitylated proteins using tandem ubiquitin-binding entities.EMBO Rep. 2009; 10: 1250-1258Crossref PubMed Scopus (334) Google Scholar). We have utilized this concept to develop and test the suitability of a tandem UBA protein for large scale characterization of ubiquitination we report using recombinant GST fusion of four tandem ubiquilin-1 (UBQLN1) UBA domains (GST-qUBA) as a bait to isolate endogenous ubiquitinated proteins from human 293T cells without ubiquitin overexpression and proteasome inhibition. We confirmed that tandem GST-qUBA is more efficient at binding poly-Ub chains than the single GST-UBA. Using GST-qUBA as an affinity purification reagent, we were able to detect ∼300 lysine ubiquitination sites that are supported by high quality mass spectra. Both abundant and low abundance cellular proteins including and of and were We that GST-qUBA is a tool for the affinity purification and identification of endogenous protein ubiquitination sites at the We and Kopito for and Yang and A. for of the and with with