Title: Using the Ubiquitin-modified Proteome to Monitor Distinct and Spatially Restricted Protein Homeostasis Dysfunction
Abstract: Protein homeostasis dysfunction has been implicated in the development and progression of aging related human pathologies. There is a need for the establishment of quantitative methods to evaluate global protein homoeostasis function. As the ubiquitin (ub) proteasome system plays a key role in regulating protein homeostasis, we applied quantitative proteomic methods to evaluate the sensitivity of site-specific ubiquitylation events as markers for protein homeostasis dysfunction. Here, we demonstrate that the ub-modified proteome can exceed the sensitivity of engineered fluorescent reporters as a marker for proteasome dysfunction and can provide unique signatures for distinct proteome challenges which is not possible with engineered reporters. We demonstrate that combining ub-proteomics with subcellular fractionation can effectively separate degradative and regulatory ubiquitylation events on distinct protein populations. Using a recently developed potent inhibitor of the critical protein homeostasis factor p97/VCP, we demonstrate that distinct insults to protein homeostasis function can elicit robust and largely unique alterations to the ub-modified proteome. Taken together, we demonstrate that proteomic approaches to monitor the ub-modified proteome can be used to evaluate global protein homeostasis and can be used to monitor distinct functional outcomes for spatially separated protein populations. Protein homeostasis dysfunction has been implicated in the development and progression of aging related human pathologies. There is a need for the establishment of quantitative methods to evaluate global protein homoeostasis function. As the ubiquitin (ub) proteasome system plays a key role in regulating protein homeostasis, we applied quantitative proteomic methods to evaluate the sensitivity of site-specific ubiquitylation events as markers for protein homeostasis dysfunction. Here, we demonstrate that the ub-modified proteome can exceed the sensitivity of engineered fluorescent reporters as a marker for proteasome dysfunction and can provide unique signatures for distinct proteome challenges which is not possible with engineered reporters. We demonstrate that combining ub-proteomics with subcellular fractionation can effectively separate degradative and regulatory ubiquitylation events on distinct protein populations. Using a recently developed potent inhibitor of the critical protein homeostasis factor p97/VCP, we demonstrate that distinct insults to protein homeostasis function can elicit robust and largely unique alterations to the ub-modified proteome. Taken together, we demonstrate that proteomic approaches to monitor the ub-modified proteome can be used to evaluate global protein homeostasis and can be used to monitor distinct functional outcomes for spatially separated protein populations. Maintenance of the collective proteome is achieved through the careful balance of protein synthesis and degradation (1.Rodrigo-Brenni M.C. Hegde R.S. Design principles of protein biosynthesis-coupled quality control.Dev. Cell. 2012; 23: 896-907Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar, 2.Wolff S. Weissman J.S. Dillin A. Differential scales of protein quality control.Cell. 2014; 157: 52-64Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar). As the primary protein degradation system within the cell, the ubiquitin proteasome system (UPS) plays a key role in maintaining proper protein homeostasis and responding to internal and external insults to proteome fidelity (3.Lykke-Andersen J. Bennett E.J. Protecting the proteome: Eukaryotic cotranslational quality control pathways.J. Cell Biol. 2014; 204: 467-476Crossref PubMed Scopus (103) Google Scholar). Separate from overseeing the regulated degradation of critical cell signaling factors, the UPS plays an essential quality-control role by identifying and destroying misfolded or otherwise nonfunctional proteins (4.Comyn S.A. Chan G.T. Mayor T. False start: cotranslational protein ubiquitination and cytosolic protein quality control.J. Proteomics. 2014; 100: 92-101Crossref PubMed Scopus (29) Google Scholar). Defects in protein quality control function have been widely hypothesized to contribute to the pathology of a wide array of human aging-associated disorders (5.Koga H. Kaushik S. Cuervo A.M. Protein homeostasis and aging: The importance of exquisite quality control.Ageing Res. Rev. 2011; 10: 205-215Crossref PubMed Scopus (308) Google Scholar, 6.Labbadia J. Morimoto R.I. The biology of proteostasis in aging and disease.Annu. Rev. Biochem. 2015; 84: 435-464Crossref PubMed Scopus (762) Google Scholar). For instance, cells containing visible protein aggregates that are associated with neurodegeneration display impaired protein homeostasis function and manipulating protein homeostasis pathways modulates neurodegenerative phenotypes in mouse models (7.Bence N.F. Sampat R.M. Kopito R.R. Impairment of the ubiquitin-proteasome system by protein aggregation.Science. 2001; 292: 1552-1555Crossref PubMed Scopus (1821) Google Scholar, 8.Bennett E.J. Bence N.F. Jayakumar R. Kopito R.R. Global impairment of the ubiquitin-proteasome system by nuclear or cytoplasmic protein aggregates precedes inclusion body formation.Mol. Cell. 2005; 17: 351-365Abstract Full Text Full Text PDF PubMed Scopus (411) Google Scholar, 9.Chu J. Hong N.A. Masuda C.A. Jenkins B.V. Nelms K.A. Goodnow C.C. Glynne R.J. Wu H. Masliah E. Joazeiro C.A. Kay S.A. A mouse forward genetics screen identifies LISTERIN as an E3 ubiquitin ligase involved in neurodegeneration.Proc. Natl. Acad. Sci. U S A. 2009; 106: 2097-2103Crossref PubMed Scopus (148) Google Scholar, 10.Das I. Krzyzosiak A. Schneider K. Wrabetz L. D'Antonio M. Barry N. Sigurdardottir A. Bertolotti A. Preventing proteostasis diseases by selective inhibition of a phosphatase regulatory subunit.Science. 2015; 348: 239-242Crossref PubMed Scopus (277) Google Scholar, 11..Lee, J. W., Beebe, K., Nangle, L. A., Jang, J., Longo-Guess, C. M., Cook, S. A., Davisson, M. T., Sundberg, J. P., Schimmel, P., and Ackerman, S. L., Editing-defective tRNA synthetase causes protein misfolding and neurodegeneration. Nature 443, 50–55,Google Scholar, 12.Ishimura R. Nagy G. Dotu I. Zhou H. Yang X.L. Schimmel P. Senju S. Nishimura Y. Chuang J.H. Ackerman S.L. RNA function. Ribosome stalling induced by mutation of a CNS-specific tRNA causes neurodegeneration.Science. 2014; 345: 455-459Crossref PubMed Scopus (268) Google Scholar). Further, protein homeostasis dysfunction occurs in various cancer models and tumor cells have been hypothesized to experience chronically elevated levels of protein homeostasis stress (13.Deshaies R.J. Proteotoxic crisis, the ubiquitin-proteasome system, and cancer therapy.BMC Biol. 2014; 12: 94Crossref PubMed Scopus (229) Google Scholar). This observation suggests that the acquisition of an elevated protein homeostasis capacity may be a key event during tumorigenesis (14.Bhat M. Robichaud N. Hulea L. Sonenberg N. Pelletier J. Topisirovic I. Targeting the translation machinery in cancer.Nat. Rev. Drug Discov. 2015; 14: 261-278Crossref PubMed Scopus (500) Google Scholar, 15.Silvera D. Formenti S.C. Schneider R.J. Translational control in cancer.Nat. Rev. Cancer. 2010; 10: 254-266Crossref PubMed Scopus (622) Google Scholar). Thus, there is a need to precisely monitor protein homeostasis function to both evaluate the contribution of protein homeostasis dysfunction to disease progression, and to detect the distinct protein homeostasis impairment that accompanies human aging-associated disorders. One approach to monitor protein homeostasis is to use engineered fluorescent sensors as protein homeostasis reporters (16.Bence N.F. Bennett E.J. Kopito R.R. Application and analysis of the GFPu family of ubiquitin-proteasome system reporters.Methods Enzymol. 2005; 399: 481-490Crossref PubMed Scopus (72) Google Scholar, 17.Salomons F.A. Acs K. Dantuma N.P. Illuminating the ubiquitin/proteasome system.Exp. Cell. Res. 2010; 316: 1289-1295Crossref PubMed Scopus (30) Google Scholar, 18.Salomons F.A. Verhoef L.G. Dantuma N.P. Fluorescent reporters for the ubiquitin-proteasome system.Essays Biochem. 2005; 41: 113-128Crossref PubMed Google Scholar). These optical UPS sensors report on protein homeostasis function in live cells and have been widely utilized to examine protein homeostasis impairment in cell culture and mouse models upon expression of aggregation prone proteins (7.Bence N.F. Sampat R.M. Kopito R.R. Impairment of the ubiquitin-proteasome system by protein aggregation.Science. 2001; 292: 1552-1555Crossref PubMed Scopus (1821) Google Scholar, 8.Bennett E.J. Bence N.F. Jayakumar R. Kopito R.R. Global impairment of the ubiquitin-proteasome system by nuclear or cytoplasmic protein aggregates precedes inclusion body formation.Mol. Cell. 2005; 17: 351-365Abstract Full Text Full Text PDF PubMed Scopus (411) Google Scholar, 19.Dantuma N.P. Lindsten K. Stressing the ubiquitin-proteasome system.Cardiovasc. Res. 2010; 85: 263-271Crossref PubMed Scopus (64) Google Scholar, 20.Dantuma N.P. Lindsten K. Glas R. Jellne M. Masucci M.G. Short-lived green fluorescent proteins for quantifying ubiquitin/proteasome-dependent proteolysis in living cells.Nat. Biotechnol. 2000; 18: 538-543Crossref PubMed Scopus (470) Google Scholar, 21.Lindsten K. Menendez-Benito V. Masucci M.G. Dantuma N.P. A transgenic mouse model of the ubiquitin/proteasome system.Nat. Biotechnol. 2003; 21: 897-902Crossref PubMed Scopus (194) Google Scholar). However, these engineered reporters require exogenous expression and the abundance of a single fluorescent reporter protein is often governed by a relatively small number of protein degradation factors which limits the ability of a single reporter protein to examine global protein homeostasis function. The development of quantitative proteomic approaches to interrogate the ubiquitin (ub) 1The abbreviations used are:ububiquitinUPSubiquitin-proteome systemdiGLYdiGlycinePCFpurified cytosolic fractionUPRunfolded protein responseERADER associated degradation.-modified proteome provides an opportunity to globally monitor protein homeostasis function without exogenous expression of reporter proteins (22.Carrano A.C. Bennett E.J. Using the ubiquitin-modified proteome to monitor protein homeostasis function.Mol. Cell Proteomics. 2013; 12: 3521-3531Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar, 23.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). One advantage of the ub-proteomics approach is the ability to interrogate a wide-array of endogenous ubiquitylation events that either target proteins for degradation or regulate protein function without proteasomal targeting. This approach has been employed to examine alterations to the ub-modified proteome upon exposure to cell stressors (23.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, 24.Elia A.E. Boardman A.P. Wang D.C. Huttlin E.L. Everley R.A. Dephoure N. Zhou C. Koren I. Gygi S.P. Elledge S.J. Quantitative proteomic atlas of ubiquitination and acetylation in the DNA damage response.Mol. Cell. 2015; 59: 867-881Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar, 25.Higgins R. Gendron J.M. Rising L. Mak R. Webb K. Kaiser S.E. Zuzow N. Riviere P. Yang B. Fenech E. Tang X. Lindsay S.A. Christianson J.C. Hampton R.Y. Wasserman S.A. Bennett E.J. The unfolded protein response triggers site-specific regulatory ubiquitylation of 40S ribosomal proteins.Mol. Cell. 2015; 59: 35-49Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 26.Povlsen L.K. Beli P. Wagner S.A. Poulsen S.L. Sylvestersen K.B. Poulsen J.W. Nielsen M.L. Bekker-Jensen S. Mailand N. Choudhary C. Systems-wide analysis of ubiquitylation dynamics reveals a key role for PAF15 ubiquitylation in DNA-damage bypass.Nat. Cell Biol. 2012; 14: 1089-1098Crossref PubMed Scopus (197) Google Scholar, 27.Sarraf S.A. Raman M. Guarani-Pereira V. Sowa M.E. Huttlin E.L. Gygi S.P. Harper J.W. Landscape of the PARKIN-dependent ubiquitylome in response to mitochondrial depolarization.Nature. 2013; 496: 372-376Crossref PubMed Scopus (711) Google Scholar, 28.Udeshi N.D. Mani D.R. Eisenhaure T. Mertins P. Jaffe J.D. Clauser K.R. Hacohen N. Carr S.A. Methods for quantification of in vivo changes in protein ubiquitination following proteasome and deubiquitinase inhibition.Mol. Cell. Proteomics. 2012; 11: 148-159Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar), during differentiation (29.Buckley S.M. Aranda-Orgilles B. Strikoudis A. Apostolou E. Loizou E. Moran-Crusio K. Farnsworth C.L. Koller A.A. Dasgupta R. Silva J.C. Stadtfeld M. Hochedlinger K. Chen E.I. Aifantis I. Regulation of pluripotency and cellular reprogramming by the ubiquitin-proteasome system.Cell Stem Cell. 2012; 11: 783-798Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar), and to identify substrates that are targeted by specific ubiquitin ligases (30.Emanuele M.J. Elia A.E. Xu Q. Thoma C.R. Izhar L. Leng Y. Guo A. Chen Y.N. Rush J. Hsu P.W. Yen H.C. Elledge S.J. Global identification of modular cullin-RING ligase substrates.Cell. 2011; 147: 459-474Abstract Full Text Full Text PDF PubMed Scopus (318) Google Scholar, 31.Kronke J. Fink E.C. Hollenbach P.W. MacBeth K.J. Hurst S.N. Udeshi N.D. Chamberlain D.R. Mani H.W. Man A.K. Gandhi T. Svinkina R.K. Schneider M. McConkey P.P. Jaras M. Griffiths E. Wetzler M. Bullinger L. Cathers B.E. Carr S.A. Chopra R. Ebert B.L. Lenalidomide induces ubiquitination and degradation of CK1alpha in del(5q) MDS.Nature. 2015; 523: 183-188Crossref PubMed Scopus (524) Google Scholar, 32.Kronke J. Udeshi N.D. Narla A. Grauman P. Hurst S.N. McConkey M. Svinkina T. Heckl D. Comer E. Li X. Ciarlo C. Hartman E. Munshi N. Schenone M. Schreiber S.A. Carr S.L. Ebert B.L. Lenalidomide causes selective degradation of IKZF1 and IKZF3 in multiple myeloma cells.Science. 2014; 343: 301-305Crossref PubMed Scopus (1084) Google Scholar, 33.Theurillat J.P. Udeshi N.D. Errington W.J. Svinkina T. Baca S.C. Pop M. Wild P.J. Blattner M. Groner A.C. Rubin M.A. Moch H. Prive G.G. Carr S.A. Garraway L.A. Prostate cancer. Ubiquitylome analysis identifies dysregulation of effector substrates in SPOP-mutant prostate cancer.Science. 2014; 346: 85-89Crossref PubMed Scopus (158) Google Scholar, 34.Tong Z. Kim M.S. Pandey A. Espenshade P.J. Identification of candidate substrates for the Golgi Tul1 E3 ligase using quantitative diGly proteomics in yeast.Mol. Cell. Proteomics. 2014; 13: 2871-2882Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). Interestingly, a large fraction of the ub-modified proteome that is stabilized upon proteasome inhibition arises from newly synthesized proteins (23.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). This observation suggests that the quality control function of the UPS is continuously targeting defective translation products for destruction. Further, the ability to monitor and quantify these diverse quality control substrates may be an effective strategy to globally monitor protein homeostasis during disease progression. ubiquitin ubiquitin-proteome system diGlycine purified cytosolic fraction unfolded protein response ER associated degradation. Protein ubiquitylation can occur in many forms and different types of ubiquitylation events can impart distinct functional outputs on substrate proteins (35.Komander D. Rape M. The ubiquitin code.Annu. Rev. Biochem. 2012; 81: 203-229Crossref PubMed Scopus (2241) Google Scholar, 36.Williamson A. Werner A. Rape M. The Colossus of ubiquitylation: decrypting a cellular code.Mol. Cell. 2013; 49: 591-600Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). For example, canonical polyubiquitylation mediated through lysine 48 linkages target substrate proteins for degradation where polyubiquitylation through lysine 63 linkages generally alter substrate function or localization. Further, proteins can be monoubiquitylated which can impact protein activity or protein complexes with the core histone proteins H2A and H2B serving as examples of this type of ubiquitylation (37.Sun Z.W. Allis C.D. Ubiquitination of histone H2B regulates H3 methylation and gene silencing in yeast.Nature. 2002; 418: 104-108Crossref PubMed Scopus (828) Google Scholar). Regulatory and quality control dependent ubiquitylation of the same protein is likely to be spatially separated both in the cell and on the substrate protein. For instance, polyubiquitylation of transmembrane proteins at the endoplasmic reticulum or within the cytosol likely occurs as a quality control event to target defective proteins for degradation, whereas monoubiquitylation of the same transmembrane protein at the cell surface regulates its intracellular trafficking (38.Tanno H. Komada M. The ubiquitin code and its decoding machinery in the endocytic pathway.J. Biochem. 2013; 153: 497-504Crossref PubMed Scopus (46) Google Scholar). Currently, we lack tools to monitor or differentiate between these functionally distinct ubiquitylation events. Here, we benchmark the sensitivity of the ub-modified proteome to proteasome inhibition. We find that utilization of proteomic methods to monitor the ub-modified proteome is, at minimum as sensitive and more robust to stochastic environmental noise as engineered fluorescent reporters as a marker for proteasome dysfunction. We also find that a narrow range of proteasome inhibition is required to impact overall protein homeostasis function. Surprisingly, regulatory, nondegradative ubiquitylation events are among the most abundant ubiquitylation events in the cell. These site-specific regulatory events mark distinct functional outputs and combining ub-proteomics with subcellular fractionation can effectively separate degradative and regulatory ubiquitylation events on distinct protein populations. To establish if different protein homeostasis stressors induce general or specific alterations within the ub-modified proteome we used a recently developed potent inhibitor of the critical protein homeostasis factor, p97/VCP (hereafter referred to as VCP) (39.Anderson D.J. Le Moigne R. Djakovic S. Kumar B. Rice J. Wong S. Wang J. Yao B. Valle E. Kiss von Soly S. Madriaga A. Soriano F. Menon M.K. Wu Z.Y. Kampmann M. Chen Y. Weissman J.S. Aftab B.T. Yakes F.M. Shawver L. Zhou H. Wustrow D.J. Rolfe M. Targeting the AAA ATPase p97 as an approach to treat cancer through disruption of protein homeostasis.Cancer Cell. 2009; 28: 653-665Abstract Full Text Full Text PDF Scopus (246) Google Scholar). We demonstrate that VCP inhibition, despite similarly impacting global ubiquitin homeostasis, results in largely distinct alterations of the ub-modified proteome compared with proteasome inhibition. Taken together, we demonstrate that the ub-modified proteome can serve as a sensitive reporter for protein homeostasis and can be used to monitor distinct functional outcomes for spatially separated protein populations. Epoxomicin (EMD Millipore, Billerica, MA) and MG132 (Enzo, Farmingdale, NY) were resuspended in DMSO and used at 1 μm or 10 μm, respectively unless otherwise noted. Dithiothreitol (DTT, ACROS Organics, Geel, Belgium) was resuspended in water and used at 5 mm. The following antibodies were utilized in this study. CB-5083 (Cleave, Burlingame, CA) was resuspended in DMSO and used at 1 μm. Antibodies for Nrf2 (ab62352) were from Abcam Inc., Cambridge, MA. Antibodies for α-tubulin (3873), HDAC2 (5113P), Ubiquityl-Histone H2B (5546), COX IV (4850), Calnexin (2433), E-Cadherin (3195), were from Cell Signaling Technology, Danvers, MA. Ubiquitin antibody (MAB1510) was from EMD Millipore/Chemicon. Antibodies for c-Myc (sc-40) were from Santa Cruz Biotechnology Inc., Dallas, TX. Antibodies for GFP were from Roche, Indianapolis, IN (11814460001). HCT116 cell lines were both purchased from American Type Culture Collection (ATCC) and grown in complete DMEM media (Gibco, Waltham, MA) containing 10%FBS (Omega Scientific, Tarzana, CA), penicillin (50 I.U./ml), and streptomycin (50 μg/ml) (Mediatech, Manassas, VA). For stable isotope labeling by amino acids in cell culture (SILAC) experiments, cells were cultured in custom DMEM without arginine or lysine (Mediatech) supplemented with 10% dialyzed FBS (Life Technologies), penicillin (50 I.U./ml) streptomycin (50 μg/ml) (Mediatech), l-Arginine hydrochloride (85 μg/ml Sigma, St. Louis, MO) and either “light” l-Lysine hydrochloride (50 μg/ml Sigma) or heavy 13C6,15N2 l-Lysine-hydrochloride (50 μg/ml Cambridge Isotopes, Tewesbury, MA) and 292 μg/ml l-Glutamine (Mediatech). All cell lines were grown at 37 °C in the presence of 5% CO2. Plasmids containing the coding sequences for GFPu, Ub-M-GFP, Ub-R-GFP, and UFD-GFP (7.Bence N.F. Sampat R.M. Kopito R.R. Impairment of the ubiquitin-proteasome system by protein aggregation.Science. 2001; 292: 1552-1555Crossref PubMed Scopus (1821) Google Scholar, 16.Bence N.F. Bennett E.J. Kopito R.R. Application and analysis of the GFPu family of ubiquitin-proteasome system reporters.Methods Enzymol. 2005; 399: 481-490Crossref PubMed Scopus (72) Google Scholar, 20.Dantuma N.P. Lindsten K. Glas R. Jellne M. Masucci M.G. Short-lived green fluorescent proteins for quantifying ubiquitin/proteasome-dependent proteolysis in living cells.Nat. Biotechnol. 2000; 18: 538-543Crossref PubMed Scopus (470) Google Scholar) were amplified by PCR and cloned into pDONR223 using recombination-based gateway™ cloning (Invitrogen, Carlsbad, CA). The alpha S2 isoform (accession: BC008855) of GNAS in pDONR was obtained as part of the orfeome collection (Dharmacon, Lafayette, CO). Subsequent LR reactions into retroviral expression vectors, viral production, and transduction of 293T cell lines were done as previously described (25.Higgins R. Gendron J.M. Rising L. Mak R. Webb K. Kaiser S.E. Zuzow N. Riviere P. Yang B. Fenech E. Tang X. Lindsay S.A. Christianson J.C. Hampton R.Y. Wasserman S.A. Bennett E.J. The unfolded protein response triggers site-specific regulatory ubiquitylation of 40S ribosomal proteins.Mol. Cell. 2015; 59: 35-49Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). Samples to be analyzed for protein or ub-modified peptide abundance changes were processed as follows. HCT116 cells were grown in media containing either light (K0) lysine or 13C15N-labeled (K8) lysine. Cell pellets containing a 1:1 mixture of heavy and light cells were lysed in 4 ml of denaturing lysis buffer (8 m urea, 75 mm NaCl, 50 mm Tris-Cl pH 8.2, Roche complete protease inhibitor, 1 mm NaF, 1 mm β-glycerophosphate, 1 mm sodium orthovanadate, 1 mm PMSF, 5 mm NEM). Lysates were sonicated twice for 5s with 30s rest on ice between cycles. Clarified lysates were digested with Lys-C (Wako, Richmond, VA) at final concentration of 10 ng/μl for 4 h at 37 °C. Lysates were then diluted to 2 m urea with 50 mm Tris-Cl pH 7.2. Trypsin (Sigma) was added at a ratio of 1:100 enzyme/substrate and allowed to incubate overnight at 37 °C. Trypsin digestion was halted by addition of Trifluoroacetic acid (TFA, Sigma) to a final concentration of 0.4%. Digested samples were clarified by centrifugation (2500 × g for 15 min) at room temperature and the supernatant was collected. Digested peptides were desalted with C18 solid-phase extraction cartridges (Waters). Eluted peptides were flash frozen with liquid nitrogen dried down to completion in a lyophilizer. For protein level analysis, peptides were resuspended in 5% formic acid, 5% CAN. For diGLY-modified peptide enrichment, dried peptides were resuspended in 1.3 ml of 2 × IAP buffer (50 mm MOPS-NaOH pH 7.5, 10 mm Na2HPO4, 50 mm NaCl). Resuspended peptides were incubated with α-diGly antibody (Cell Signaling Technologies) preconjugated to Protein-A (Thermo) beads at 4 °C for 2 h with rotating. Beads were then washed 4× with 1 ml IAP buffer with rotating for 10 min between each wash. Peptides were eluted with 5% formic acid. The resulting peptides were desalted with in-house prepared C18 stage-tips and dried in a vacuum centrifuge. Samples were resuspended in 10 μl 5% formic acid, 5% ACN and transferred to autosampler vials. Samples were analyzed in triplicate by LC-MS/MS using a Q-Exactive mass spectrometer (Thermo Scientific, San Jose, CA) with the following conditions. The following is a generalized nHPLC and instrument method that is representative of individual analyses. Peptides were first separated by reverse-phase chromatography using a fused silica microcapillary column (100 μm ID, 20 cm) packed with C18 reverse-phase resin (XSELECT CSH 130 C18 2.5 μm, Waters Co., Wilford, MA) using an in-line nano-flow EASY-nLC 1000 UHPLC (Thermo Scientific). Peptides were eluted over a 2 min 0–5% ACN gradient, followed by a 158 min 5–30% ACN gradient, a 15 min 30–45% ACN gradient, a 1 min 45–98% gradient, with a final 14 min isocratic step at 98% ACN for a total run time of 190 min at a flow rate of 250 nl/min. All gradient mobile phases contained 0.1% formic acid. MS/MS data were collected in a data-dependent fashion using a top 10 method with a full MS mass range from 300–1750 m/z, 70,000 resolution, and an AGC target of 3e6. MS2 scans were triggered when an ion intensity threshold of 1e5 was reached with a maximum injection time of 250 ms. Peptides were fragmented using a normalized collision energy setting of 22. A dynamic exclusion time of 40 s was used and the peptide match setting was disabled. Singly charged ions, charge states above 8 and unassigned charge states were excluded. All mass spectrometry data files are available through the MassIVE archive (massive.ucsd.edu) ID: MSV000079454. Annotated MS/MS spectra for all ubiquitin-modified peptides identified in this study can be viewed with MS-Viewer (40.Kisselev A.F. Goldberg A.L. Monitoring activity and inhibition of 26S proteasomes with fluorogenic peptide substrates.Methods Enzymol. 2005; 398: 364-378Crossref PubMed Scopus (264) Google Scholar) (http://prospector2.ucsf.edu/prospector/cgi-bin/msform.cgi?form=msviewer) using the search key j5hxyl3atu. The resultant RAW files were converted into mzXML format using the ReadW.exe (version 4.3.1) program. The SEQUEST search algorithm (version 28) was used to search MS/MS spectra against a concatenated target-decoy database comprised of forward and reversed sequences from the reviewed UniprotKB/Swiss-Prot FASTA Human database (downloaded 3/4/2015) with GFP and common contaminants appended (∼26,000 entries). The search parameters used are as follows: 50 ppm precursor ion tolerance and 0.01 Da fragment ion tolerance; Trypsin (1 1 KR P) was set as the enzyme; up to three missed cleavages were allowed; dynamic modifications of 15.99491 Da on methionine (oxidation), and 114.04293 Da on lysine (diGLY), and 42.010564 on peptide N-term (acetylation), and static modification of 125.047679 on cysteines (NEM alkylation). For SILAC labeled samples, each RAW file was searched separately with a “light-lysine” database containing the parameters above, and a “heavy-lysine” database containing a static (8.0141988132 Da) modification on lysines. Peptide matches were filtered to a peptide false discovery rate of 1% for all diGLY-modified enrichment samples using the linear discriminant analysis. For protein-level analysis, a protein false discovery rate of less than 2% was used and peptides were assembled into proteins using maximum parsimony and only unique and razor peptides were retained for subsequent analysis. diGLY-modified lysines were localized on peptides using ModScore and sites with a localization score above 13 were retained for further analysis. C-terminal modified lysines were excluded from further analysis. Unique diGLY-modified sites were filtered and quantified as described previously (23.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). For label-free data, spectral counts for ub-modified peptides from triplicate runs for each epoxomicin concentration was first normalized to the CUL5_K724 peptide. Spectral counts from triplicate runs were then summed and made relative to spectral counts in the untreated sample. p value determination for the volcano plots was determined used a two-tailed t test compared with the untreated sample. Proteasome activity assays were performed as described (41.Tsirigos K.D. Peters C. Shu N. Kall L. Elofsson A. The TOPCONS web server for consensus prediction of membrane protein topology and signal peptides.Nucleic Acids Res. 2015; 43: W401-W407Crossref PubMed Scopus (536) Google Scholar). The chymotryptic activity was measured using the fluorogenic peptide substrate Suc-LLVY-AMC (Boston Biochem, Cambridge, MA). AMC fluorescence was measured over a 60 min time course in a SpectraMAX plate reader. Slopes of the progress curves was used to determine percent proteasome inhibition with the slope of the progress curve from untreated cell lysates set to 0% and the same lysates with exogenous addition of 1 μm epoxomicin set t