Enhanced Purification of Ubiquitinated Proteins by Engineered Tandem Hybrid Ubiquitin-binding Domains (ThUBDs)
2016; Elsevier BV; Volume: 15; Issue: 4 Linguagem: Inglês
10.1074/mcp.o115.051839
ISSN1535-9484
AutoresYuan Gao, Yanchang Li, Chengpu Zhang, Mingzhi Zhao, Deng Chen, Qiuyan Lan, Zexian Liu, Na Su, Jingwei Wang, Feng Xu, Yongru Xu, Lingyan Ping, Lei Chang, Huiying Gao, Junzhu Wu, Yu Xue, Zixin Deng, Junmin Peng, Ping Xu,
Tópico(s)Peptidase Inhibition and Analysis
ResumoUbiquitination is one of the most common post-translational modifications, regulating protein stability and function. However, the proteome-wide profiling of ubiquitinated proteins remains challenging due to their low abundance in cells. In this study, we systematically evaluated the affinity of ubiquitin-binding domains (UBDs) to different types of ubiquitin chains. By selecting UBDs with high affinity and evaluating various UBD combinations with different lengths and types, we constructed two artificial tandem hybrid UBDs (ThUBDs), including four UBDs made of DSK2p-derived ubiquitin-associated (UBA) and ubiquilin 2-derived UBA (ThUDQ2) and of DSK2p-derived UBA and RABGEF1-derived A20-ZnF (ThUDA20). ThUBD binds to ubiquitinated proteins, with markedly higher affinity than naturally occurring UBDs. Furthermore, it displays almost unbiased high affinity to all seven lysine-linked chains. Using ThUBD-based profiling with mass spectrometry, we identified 1092 and 7487 putative ubiquitinated proteins from yeast and mammalian cells, respectively, of which 362 and 1125 proteins had ubiquitin-modified sites. These results demonstrate that ThUBD is a refined and promising approach for enriching the ubiquitinated proteome while circumventing the need to overexpress tagged ubiquitin variants and use antibodies to recognize ubiquitin remnants, thus providing a readily accessible tool for the protein ubiquitination research community. Ubiquitination is one of the most common post-translational modifications, regulating protein stability and function. However, the proteome-wide profiling of ubiquitinated proteins remains challenging due to their low abundance in cells. In this study, we systematically evaluated the affinity of ubiquitin-binding domains (UBDs) to different types of ubiquitin chains. By selecting UBDs with high affinity and evaluating various UBD combinations with different lengths and types, we constructed two artificial tandem hybrid UBDs (ThUBDs), including four UBDs made of DSK2p-derived ubiquitin-associated (UBA) and ubiquilin 2-derived UBA (ThUDQ2) and of DSK2p-derived UBA and RABGEF1-derived A20-ZnF (ThUDA20). ThUBD binds to ubiquitinated proteins, with markedly higher affinity than naturally occurring UBDs. Furthermore, it displays almost unbiased high affinity to all seven lysine-linked chains. Using ThUBD-based profiling with mass spectrometry, we identified 1092 and 7487 putative ubiquitinated proteins from yeast and mammalian cells, respectively, of which 362 and 1125 proteins had ubiquitin-modified sites. These results demonstrate that ThUBD is a refined and promising approach for enriching the ubiquitinated proteome while circumventing the need to overexpress tagged ubiquitin variants and use antibodies to recognize ubiquitin remnants, thus providing a readily accessible tool for the protein ubiquitination research community. Ubiquitination, a universal post-translational modification, refers to the covalent attachment of ubiquitin to lysine residues or the N terminus of proteins (1.Hershko A. Ciechanover A. Varshavsky A. The ubiquitin system.Nat. Med. 2000; 6: 1073-1081Crossref PubMed Scopus (569) Google Scholar). It is a cascade process catalyzed by ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme (E2), and ubiquitin ligase (E3) (2.Scheffner M. Nuber U. Huibregtse J.M. Protein ubiquitination involving an E1–E2–E3 enzyme ubiquitin thioester cascade.Nature. 1995; 373: 81-83Crossref PubMed Scopus (746) Google Scholar). Ubiquitination is reversible by the action of deubiquitinating enzymes, which cleave ubiquitin moieties from the substrates (3.Komander D. Clague M.J. Urbé S. Breaking the chains: structure and function of the deubiquitinases.Nat. Rev. Mol. Cell Biol. 2009; 10: 550-563Crossref PubMed Scopus (1460) Google Scholar, 4.Reyes-Turcu F.E. Wilkinson K.D. Polyubiquitin binding and disassembly by deubiquitinating enzymes.Chem. Rev. 2009; 109: 1495-1508Crossref PubMed Scopus (109) Google Scholar). 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Different lengths and linkages of ubiquitin modifications are linked to distinct physiological functions in cells. For example, monoubiquitin regulates DNA repair and receptor endocytosis (12.Di Fiore P.P. Polo S. Hofmann K. When ubiquitin meets ubiquitin receptors: a signalling connection.Nat. Rev. Mol. Cell Biol. 2003; 4: 491-497Crossref PubMed Scopus (262) Google Scholar, 13.Hicke L. Protein regulation by monoubiquitin.Nat. Rev. Mol. Cell Biol. 2001; 2: 195-201Crossref PubMed Scopus (994) Google Scholar, 14.Haglund K. Sigismund S. Polo S. Szymkiewicz I. Di Fiore P.P. Dikic I. Multiple monoubiquitination of RTKs is sufficient for their endocytosis and degradation.Nat. Cell Biol. 2003; 5: 461-466Crossref PubMed Scopus (658) Google Scholar), and polyubiquitin chains, such as the well studied Lys-48-linked and Lys-63-linked polyubiquitin chains, function in proteasomal degradation (15.Thrower J.S. Hoffman L. Rechsteiner M. Pickart C.M. Recognition of the polyubiquitin proteolytic signal.EMBO J. 2000; 19: 94-102Crossref PubMed Scopus (1312) Google Scholar), endosomal trafficking to the lysosome, intracellular signaling, and DNA repair (16.Ikeda F. Dikic I. Atypical ubiquitin chains: new molecular signals.EMBO Rep. 2008; 9: 536-542Crossref PubMed Scopus (656) Google Scholar). The specific N-terminal linear polyubiquitin chain activates NF-κB kinase (17.Tokunaga 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-κB activation.Nat. Cell Biol. 2009; 11: 123-132Crossref PubMed Scopus (750) Google Scholar). Recent studies also suggest that Lys-11 linkage plays important roles in the endoplasmic reticulum-associated protein degradation (pathway (18.Xu 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 (849) Google Scholar) and cell cycle (19.Matsumoto M.L. Wickliffe K.E. Dong K.C. Yu C. Bosanac I. Bustos D. Phu L. Kirkpatrick D.S. Hymowitz S.G. Rape M. Kelley R.F. Dixit V.M. K11-linked polyubiquitination in cell cycle control revealed by a K11 linkage-specific antibody.Mol. Cell. 2010; 39: 477-484Abstract Full Text Full Text PDF PubMed Scopus (299) Google Scholar), whereas Lys-33 linkage regulates cell surface receptor-mediated signal transduction (20.Huang H. Jeon M.-S. Liao L. Yang C. Elly C. Yates 3rd, J.R. Liu Y.-C. K33-linked polyubiquitination of T cell receptor-ζ regulates proteolysis-independent T cell signaling.Immunity. 2010; 33: 60-70Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar) and post-Golgi transport (21.Yuan W.-C. Lee Y.-R. Lin S.-Y. Chang L.-Y. Tan Y.P. Hung C.-C. Kuo J.-C. Liu C.-H. Lin M.-Y. Xu M. Chen Z.J. Chen R.H. K33-linked polyubiquitination of coronin 7 by Cul3-KLHL20 ubiquitin E3 ligase regulates protein trafficking.Mol. Cell. 2014; 54: 586-600Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). However, the biological functions of other atypical ubiquitinated chains still remain poorly understood (22.Kulathu Y. Komander D. Atypical ubiquitylation—the unexplored world of polyubiquitin beyond Lys-48 and Lys-63 linkages.Nat. Rev. Mol. Cell Biol. 2012; 13: 508-523Crossref PubMed Scopus (486) Google Scholar). Large scale profiling of ubiquitin conjugates and mapping of ubiquitination sites are instrumental to understanding the ubiquitin regulatory system. However, profiling is still technically challenging due to the low abundance and rapid degradation of the ubiquitin conjugates. To this end, several different strategies have been used to enrich the ubiquitinated proteome or ubiquitinome (23.Shi Y. Xu P. Qin J. Ubiquitinated proteome: ready for global?.Mol. Cell. Proteomics. 2011; 10 (R110.006882)Abstract Full Text Full Text PDF Scopus (43) Google Scholar). One well established method is purifying tagged ubiquitinome from yeast (24.Peng 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 (1307) Google Scholar). Later, this approach has been employed for ubiquitinome profiling in mammalian cells (25.Meierhofer 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 (162) Google Scholar, 26.Franco M. Seyfried N.T. Brand A.H. Peng J. Mayor U. A novel strategy to isolate ubiquitin conjugates reveals wide role for ubiquitination during neural development.Mol. Cell. 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Global analysis of lysine ubiquitination by ubiquitin remnant immunoaffinity profiling.Nat. Biotechnol. 2010; 28: 868-873Crossref PubMed Scopus (395) Google Scholar) developed a di-Gly-lysine-specific antibody against the signature di-glycine residues that are the remnants of ubiquitin after trypsin digestion, to directly enrich the ubiquitinated peptides in a human cell line. This method has been improved and is the primary strategy in ubiquitinome research (32.Kim W. Bennett E.J. Huttlin E.L. Guo A. Li J. Possemato A. Sowa M.E. Rad R. Rush J. Comb M.J. Harper J.W. Gygi S.P. Systematic and quantitative assessment of the ubiquitin-modified proteome.Mol. Cell. 2011; 44: 325-340Abstract Full Text Full Text PDF PubMed Scopus (1183) Google Scholar, 33.Wagner S.A. Beli P. Weinert B.T. Nielsen M.L. Cox J. Mann M. Choudhary C. A proteome-wide, quantitative survey of in vivo ubiquitylation sites reveals widespread regulatory roles.Mol. Cell. 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Chem. Biol. 2013; 17: 49-58Crossref PubMed Scopus (47) Google Scholar, 36.Wagner S.A. Beli P. Weinert B.T. Schölz C. Kelstrup C.D. Young C. Nielsen M.L. Olsen J.V. Brakebusch C. Choudhary C. Proteomic analyses reveal divergent ubiquitylation site patterns in murine tissues.Mol. Cell. Proteomics. 2012; 11: 1578-1585Abstract Full Text Full Text PDF PubMed Scopus (225) Google Scholar). UBDs are small protein module families that can recognize and bind to ubiquitin modifications and are used as alternative tools to enrich ubiquitinated proteomes. To date, more than 20 different UBD families have been identified (5.Dikic I. Wakatsuki S. Walters K.J. Ubiquitin-binding domains—from structures to functions.Nat. Rev. Mol. Cell Biol. 2009; 10: 659-671Crossref PubMed Scopus (624) Google Scholar, 37.Harper J.W. Schulman B.A. Structural complexity in ubiquitin recognition.Cell. 2006; 124: 1133-1136Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). The affinity of different UBDs to ubiquitin spans a wide range, from 2 to 500 μm (6.Hicke L. Schubert H.L. Hill C.P. Ubiquitin-binding domains.Nat. Rev. Mol. Cell Biol. 2005; 6: 610-621Crossref PubMed Scopus (638) Google Scholar), with various preferences for different types of ubiquitin linkages (38.Randles L. Walters K.J. Ubiquitin and its binding domains.Front. Biosci. 2011; 17: 2140-2157Crossref Scopus (36) Google Scholar). For example, the ubiquitin-associated (UBA) domains of hHR23A and MUD1 selectively bind to Lys-48-linked chains (39.Raasi 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 (278) Google Scholar, 40.Trempe J.F. Brown N.R. Lowe E.D. Gordon C. Campbell I.D. Noble M.E. Endicott J.A. Mechanism of Lys-48-linked polyubiquitin chain recognition by the Mud1 UBA domain.EMBO J. 2005; 24: 3178-3189Crossref PubMed Scopus (82) Google Scholar), whereas the Npl4 zinc finger domain of TGFβ-activated kinase 1 (TAK1)-binding protein 2 prefers Lys-63-linked chains (41.Komander D. Reyes-Turcu F. Licchesi J.D. Odenwaelder P. Wilkinson K.D. Barford D. Molecular discrimination of structurally equivalent Lys 63-linked and linear polyubiquitin chains.EMBO Rep. 2009; 10: 466-473Crossref PubMed Scopus (450) Google Scholar), and the UBAN domain of the NF-κB essential modulator (NEMO) specifically binds to linear ubiquitin chains (42.Rahighi S. Ikeda F. Kawasaki M. Akutsu M. Suzuki N. Kato R. Kensche T. Uejima T. Bloor S. Komander D. Randow F. Wakatsuki S. Dikic I. Specific recognition of linear ubiquitin chains by NEMO is important for NF-κB activation.Cell. 2009; 136: 1098-1109Abstract Full Text Full Text PDF PubMed Scopus (578) Google Scholar, 43.Lo Y.-C. Lin S.-C. Rospigliosi C.C. Conze D.B. Wu C.-J. Ashwell J.D. Eliezer D. Wu H. Structural basis for recognition of diubiquitins by NEMO.Mol. Cell. 2009; 33: 602-615Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar). Although single and tandem UBDs have been used to purify ubiquitinated proteins from mammalian cells (44.Layfield R. Tooth D. Landon M. Dawson S. Mayer J. Alban A. Purification of poly-ubiquitinated proteins by S5a-affinity chromatography.Proteomics. 2001; 1: 773-777Crossref PubMed Scopus (49) Google Scholar, 45.Shi Y. Chan D.W. Jung S.Y. Malovannaya A. Wang Y. Qin J. A data set of human endogenous protein ubiquitination sites.Mol. Cell. Proteomics. 2011; 10 (M110.002089)Abstract Full Text Full Text PDF Scopus (0) Google Scholar, 46.Lopitz-Otsoa F. Rodriguez-Suarez E. Aillet F. Casado-Vela J. Lang V. Matthiesen R. Elortza F. Rodriguez M.S. Integrative analysis of the ubiquitin proteome isolated using tandem ubiquitin binding entities (TUBEs).J. Proteomics. 2012; 75: 2998-3014Crossref PubMed Scopus (73) Google Scholar), they are usually restricted to well studied UBA domains (such as UBA from ubiquilin 1 and hHR23A) with ubiquitin chain preference, rather than reflecting the global ubiquitination landscape. To address the bias in UBDs and efficiently recover the ubiquitinome during purification, we systematically evaluated the affinity of a number of UBDs, and we constructed an artificial tandem hybrid UBD (ThUBD) that exhibited no strong bias toward any of the seven tested polyubiquitin chains. By analyzing complex samples of yeast and mammalian cells with ThUBD, we found a dramatic improvement in binding affinity compared with previously reported UBD methods. Our study demonstrates that ThUBDs are promising tools for the efficient and unbiased analysis of the ubiquitinated proteome, especially for animal tissues or pathological specimens. This approach may aid in the discovery of novel biomarkers or therapeutic strategies against human diseases. The UBDs used in this study were DSK2p-derived UBA (DSK2, 327–371 amino acids), ubiquilin 1-derived UBA (UQ1, 541–586 amino acids), ubiquilin 2-derived UBA (UQ2, 575–624 amino acids), RABGEF1-derived A20-ZnF (A20, 9–73 amino acids), and HDAC6-derived ZnF-UBP (HDAC6, 985–1152 amino acids). Five different UBDs were cloned by PCR amplification and inserted into the pGEX-4T-2 vector (GE Healthcare, Chalfont St Giles, UK) using BglII and EcoRI sites. Tandem repeats of UBDs were constructed using BglII and BamHI restriction sites as designed by Hjerpe et al. (47.Hjerpe 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 (343) Google Scholar). hUBDs (DSK2 and UQ2 (termed as UDQ2) and DSK2 and A20 (termed as UDA20)) and ThUBDs (ThUDQ2 and ThUDA20) were constructed in the same way. All of the proteins were overexpressed in Escherichia coli BL21 (DE3) cells, which were induced with 0.5 mm isopropyl 1-thio-β-d-galactopyranoside for 4 h at 30 °C. The harvested cells were lysed by sonication in lysis buffer (1 mm DTT, 1% Triton X-100 in PBS). GST-UBD fusion proteins were purified from cell lysates using glutathione-Sepharose (GSH) 4B beads (Qiagen, Valencia, CA) according to the manufacturer's instructions. The purified GST-UBDs were competitively eluted by reduced glutathione and then resuspended in coupling buffer (0.2 m NaHCO3, 0.5 m NaCl, pH 8.3) before being coupled to NHS-activated Sepharose (GE Healthcare, Munich, Germany) following the manufacturer's instructions. The UBD-conjugated agarose was stored at 4 °C in PBS supplemented with 30% glycine. The yeast strain SUB592 (48.Spence J. Gali R.R. Dittmar G. Sherman F. Karin M. Finley D. Cell cycle–regulated modification of the ribosome by a variant multiubiquitin chain.Cell. 2000; 102: 67-76Abstract Full Text Full Text PDF PubMed Scopus (312) Google Scholar) was grown at 30 °C in yeast extract peptone dextrose (YPD) medium (1% yeast extract, 2% Bacto-peptone, 2% dextrose) and harvested in the early log phase (A600 = 1.0). His-Myc-tagged ubiquitin conjugates were purified from yeast under denaturing conditions with Ni-NTA agarose (24.Peng 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 (1307) Google Scholar) or under native conditions by UBDs (18.Xu 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 (849) Google Scholar). For native conditions, cells were lysed with glass beads in 200 μl of buffer A (50 mm Na2HPO4, pH 8.0, 500 mm NaCl, 0.01% SDS, 5% glycerol). Protein extracts were centrifuged at 70,000 × g for 30 min and then incubated with immobilized GST-UBD beads at 4 °C for 30 min. After incubation, the beads were washed with buffer A and then buffer B (50 mm NH4HCO3 and 5 mm iodoacetamide), followed by 50 mm NH4HCO3 to remove iodoacetamide. Bound ubiquitin conjugates were eluted by boiling in 1× SDS-PAGE loading buffer (50 mm Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, 0.1% bromphenol blue, 1% β-mercaptoethanol). Human hepatocellular carcinoma (HCC) MHCC97-H cells (49.Li Y. Tang Z.-Y. Ye S.-L. Liu Y.-K. Chen J. Xue Q. Chen J. Gao D.-M. Bao W.-H. Establishment of cell clones with different metastatic potential from the metastatic hepatocellular carcinoma cell line MHCC97.World J. Gastroenterol. 2001; 7: 630-636Crossref PubMed Scopus (319) Google Scholar) were cultured to confluence before harvest. Ubiquitin conjugates were purified from cells under native conditions by ThUBDs (ThUDA20) as described above (45.Shi Y. Chan D.W. Jung S.Y. Malovannaya A. Wang Y. Qin J. A data set of human endogenous protein ubiquitination sites.Mol. Cell. Proteomics. 2011; 10 (M110.002089)Abstract Full Text Full Text PDF Scopus (0) Google Scholar). Finally, the purified ubiquitin conjugates were released by boiling in 1× SDS-PAGE loading buffer. Affinity-purified proteins from yeast cells were separated on 10% SDS-polyacrylamide gels, transferred onto nitrocellulose membranes (Bio-Rad), and then probed with antibodies against Myc (Abcam, Cambridge, UK) or visualized by silver staining. Yeast or human ubiquitin conjugates were separated on SDS-polyacrylamide gels or underwent off-line high pH reverse phase LC separation (24.Peng 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 (1307) Google Scholar, 50.Ding C. Jiang J. Wei J. Liu W. Zhang W. Liu M. Fu T. Lu T. Song L. Ying W. Chang C. Zhang Y. Ma J. Wei L. Malovannaya A. et al.A fast workflow for identification and quantification of proteomes.Mol. Cell. Proteomics. 2013; 12: 2370-2380Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). The ubiquitin conjugates separated by SDS-PAGE were sliced into different gel pieces based on molecular weight markers and digested with trypsin overnight. The tryptic peptides were extracted with extraction buffer (5% total cell lysates (FA) + 45% acetonitrile (ACN)) and then ACN and were finally dried using a vacuum dryer (Labconco CentriVap, Kansas City, MO). Peptides were analyzed using an ultra-performance LC-MS/MS platform of hybrid LTQ-Orbitrap Velos mass spectrometer (Thermo Fisher Scientific, San Jose, CA) equipped with a Waters nanoACQUITY ultra-performance liquid chromatography system (Waters). The LC separation was performed on an in-house packed capillary column (75 μm inner diameter × 15 cm) with 3-μm C18 reverse-phase fused silica (Michrom Bioresources, Inc., Auburn, CA). Then the sample was eluted with a 60–140-min nonlinear gradient ramped from 8 to 40% of mobile phase B (phase B, 0.1% FA in ACN; phase A, 0.1% FA + 2% ACN in water) at a flow rate of 0.3 μl/min. Eluting peptides were analyzed using an LTQ-Orbitrap Velos mass spectrometer. The MS1 was analyzed with a mass range of 300–1600 at a resolution of 30,000 at m/z 400. The automatic gain control was set as 1 × 106, and the maximum injection time was 150 ms. The MS2 was analyzed in data-dependent mode for the 20 most intense ions subjected to fragmentation in the linear ion trap (LTQ). For each scan, the automatic gain control was set at 1 × 104, and the maximum injection time was set at 25 ms. The dynamic range was set at 45–60 s to suppress repeated detection of the same ion peaks. The raw data files were searched by the SorcererTM-SEQUEST® (version 4.0.4 build, Sage-N Research, Inc.) and SEQUEST HT algorithms embedded in Proteome Discovery (version 1.4.1.14, Thermo Fisher) against the Swiss-Prot reviewed database (version released in 2013.10 for yeast, contains 6652 entities; version 2014.04 for human, contains 20,266 entities). MS search parameters consisted of full tryptic restriction, fixed modification of cysteine carbamidomethylation, methionine oxidation, and di-Gly-lysine as variable modifications. Peptides were allowed up to three cleavage sites. Precursor mass tolerance was set at 20 ppm and that of the MS2 fragments was set at 0.5 Da. Peptide matches were filtered by a minimum length of seven amino acids. For ubiquitination identification, a re-searching strategy was used for protein identification; di-Gly-lysine was added in the second search. The C-terminal di-Gly-lysine site identifications were removed. The peptides and proteins were filtered until a false discovery rate lower than 1% was estimated using a target-decoy search strategy. To accurately quantify the abundance of the seven polyubiquitin chains from each ubiquitin conjugate purified with different UBDs, ubiquitin conjugates labeled with heavy isotope 13C6-lysine (Lys-6) and 15N413C6-arginine (Arg-10) were purified with nickel beads as a quantification standard to mix equally with each sample (18.Xu 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 (849) Google Scholar). The ubiquitin conjugates were digested as described above, and the resulting peptides were dissolved in a sample buffer (1% FA, 1% ACN) and analyzed by LC-MS/MS. Eluted peptides were detected on the Orbitrap mass spectrometer in a survey scan (300–1600 m/z, resolution 30,000) followed by selective reaction monitoring scans for seven ubiquitin chains ions in the LTQ. The peptide intensity for quantification was manually analyzed by ion chromatograms using Xcalibur version 2.0 software (Thermo Finnigan, San Jose, CA). To accurately compare the binding affinity to ubiquitin of different UBDs, the same amount of each UBD was needed. We selected two typical peptides (supplemental Fig. S1) from glutathione S-transferase (GST) protein as internal standards for quantifying the amount of GST-UBDs coupled to agarose beads. The GST protein was overexpressed and completely labeled with [13C6]lysine (+6.0201 Da) using the SILAC-labeled E. coli BL21 (DE3) cells and was then purified using GSH 4B beads. The amount of purified heavy-labeled GST was measured using the BCA protein assay kit (Thermo Scientific, Rockford, IL) and Coomassie staining (51.Xu P. Duong D.M. Peng J. Systematical optimization of reverse-phase chromatography for shotgun proteomics.
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