GPMDB00114OtherUdeshi ND, et al.InstrumentNot AvailableNot availableHuman, Human_adenovirus_54, Human_adenovirus_a, Human_adenovirus_b, Human_adenovirus_c, Human_adenovirus_d, Human_adenovirus_e, Human_adenovirus_f, Human_adenovirus_g, Human_astrovirus, Human_bocavirus, Human_bocavirus_2, Human_bocavirus_3, Human_bocavirus_4, Human_coronavirus_229e, Human_coronavirus_hku1, Human_coronavirus_nl63, Human_coronavirus_oc43, Human_cosavirus_a, Human_cosavirus_b, Human_cosavirus_d, Human_cosavirus_e, Human_enteric_coronavirus_4408, Human_enterovirus_100, Human_enterovirus_a, Human_enterovirus_b, Human_enterovirus_c, Human_enterovirus_d, Human_erythrovirus_v9, Human_herpesvirus_1, Human_herpesvirus_2, Human_herpesvirus_3, Human_herpesvirus_4_type_1, Human_herpesvirus_4_type_2, Human_herpesvirus_5, Human_herpesvirus_6a, Human_herpesvirus_6b, Human_herpesvirus_7, Human_herpesvirus_8_type_p, Human_immunodeficiency_virus_1, Human_immunodeficiency_virus_2, Human_metapneumovirus, Human_papillomavirus_fa75_ki88_03, Human_papillomavirus_rtrx7, Human_papillomavirus_type_10, Human_papillomavirus_type_100, Human_papillomavirus_type_101, Human_papillomavirus_type_103, Human_papillomavirus_type_104, Human_papillomavirus_type_105, Human_papillomavirus_type_108, Human_papillomavirus_type_109, Human_papillomavirus_type_112, Human_papillomavirus_type_113, Human_papillomavirus_type_16, Human_papillomavirus_type_24, Human_papillomavirus_type_26, Human_papillomavirus_type_32, Human_papillomavirus_type_34, Human_papillomavirus_type_4, Human_papillomavirus_type_41, Human_papillomavirus_type_48, Human_papillomavirus_type_49, Human_papillomavirus_type_5, Human_papillomavirus_type_50, Human_papillomavirus_type_53, Human_papillomavirus_type_60, Human_papillomavirus_type_63, Human_papillomavirus_type_6b, Human_papillomavirus_type_7, Human_papillomavirus_type_71, Human_papillomavirus_type_88, Human_papillomavirus_type_9, Human_papillomavirus_type_92, Human_papillomavirus_type_96, Human_papillomavirus_type_98, Human_papillomavirus_type_99, Human_papillomavirus___1, Human_papillomavirus___18, Human_papillomavirus___2, Human_papillomavirus___54, Human_papillomavirus___61, Human_papillomavirus___cand90, Human_parainfluenza_virus_1, Human_parainfluenza_virus_2, Human_parainfluenza_virus_3, Human_parechovirus, Human_parvovirus_4, Human_parvovirus_b19, Human_picobirnavirus, Human_respiratory_syncytial_virus, Human_rhinovirus_a, Human_rhinovirus_b, Human_rhinovirus_c, Human_tmev_like_cardiovirus, Human_t_lymphotropic_virus_1, Human_t_lymphotropic_virus_2, Human_t_lymphotropic_virus_4scarr@broad.mit.edu22505724http://gpmdb.thegpm.org/~/dblist_gpmnum/gpmnum=GPM11210018389http://gpmdb.thegpm.org/~/dblist_gpmnum/gpmnum=GPM11210018378http://gpmdb.thegpm.org/~/dblist_gpmnum/gpmnum=GPM11210018379http://gpmdb.thegpm.org/~/dblist_gpmnum/gpmnum=GPM11210018387http://gpmdb.thegpm.org/~/dblist_gpmnum/gpmnum=GPM11210018376http://gpmdb.thegpm.org/~/dblist_gpmnum/gpmnum=GPM11210018377http://gpmdb.thegpm.org/~/dblist_gpmnum/gpmnum=GPM11210018388http://gpmdb.thegpm.org/~/dblist_gpmnum/gpmnum=GPM11210018392http://gpmdb.thegpm.org/~/dblist_gpmnum/gpmnum=GPM11210018381http://gpmdb.thegpm.org/~/dblist_gpmnum/gpmnum=GPM11210018393http://gpmdb.thegpm.org/~/dblist_gpmnum/gpmnum=GPM11210018382http://gpmdb.thegpm.org/~/dblist_gpmnum/gpmnum=GPM11210018390http://gpmdb.thegpm.org/~/dblist_gpmnum/gpmnum=GPM11210018380http://gpmdb.thegpm.org/~/dblist_gpmnum/gpmnum=GPM11210018391http://gpmdb.thegpm.org/~/dblist_gpmnum/gpmnum=GPM11210018385http://gpmdb.thegpm.org/~/dblist_gpmnum/gpmnum=GPM11210018374http://gpmdb.thegpm.org/~/dblist_gpmnum/gpmnum=GPM11210018396http://gpmdb.thegpm.org/~/dblist_gpmnum/gpmnum=GPM11210018386http://gpmdb.thegpm.org/~/dblist_gpmnum/gpmnum=GPM11210018394http://gpmdb.thegpm.org/~/dblist_gpmnum/gpmnum=GPM11210018383http://gpmdb.thegpm.org/~/dblist_gpmnum/gpmnum=GPM11210018373http://gpmdb.thegpm.org/~/dblist_gpmnum/gpmnum=GPM11210018384http://gpmdb.thegpm.org/~/dblist_gpmnum/gpmnum=GPM11210018395Broad Institute of MIT and HarvardNot availableGPMDBUbiquitination plays a key role in protein degradation and signal transduction. Ubiquitin is a small protein modifier that is adducted to lysine residues by the combined function of E1, E2, and E3 enzymes and is removed by deubiquitinating enzymes. Characterization of ubiquitination sites is important for understanding the role of this modification in cellular processes and disease. However, until recently, large-scale characterization of endogenous ubiquitination sites has been hampered by the lack of efficient enrichment techniques. The introduction of antibodies that specifically recognize peptides with lysine residues that harbor a di-glycine remnant (K-ε-GG) following tryptic digestion has dramatically improved the ability to enrich and identify ubiquitination sites from cellular lysates. We used this enrichment technique to study the effects of proteasome inhibition by MG-132 and deubiquitinase inhibition by PR-619 on ubiquitination sites in human Jurkat cells by quantitative high performance mass spectrometry. Minimal fractionation of digested lysates prior to immunoaffinity enrichment increased the yield of K-ε-GG peptides three- to fourfold resulting in detection of up to ~3300 distinct K-GG peptides in SILAC triple encoded experiments starting from 5 mg of protein per label state. In total, we identify 5533 distinct K-ε-GG peptides of which 4907 were quantified in this study, demonstrating that the strategy presented is a practical approach to perturbational studies in cell systems. We found that proteasome inhibition by MG-132 and deubiquitinase inhibition by PR-619 induces significant changes to the ubiquitin landscape, but that not all ubiquitination sites regulated by MG-132 and PR-619 are likely substrates for the ubiquitin-proteasome system. Additionally, we find that the proteasome and deubiquitinase inhibitors studied induced only minor changes in protein expression levels regardless of the extent of regulation induced at the ubiquitin site level. We attribute this finding to the low stoichiometry of the majority ubiquitination sites identified in this study.Methods for quantification of in vivo changes in protein ubiquitination following proteasome and deubiquitinase inhibition.Udeshi Namrata D ND,Mani D R DR,Eisenhaure Thomas T,Mertins Philipp P,Jaffe Jacob D JD,Clauser Karl R KR,Hacohen Nir N,Carr Steven A SA,Udeshi Namrata D ND, Mani D R DR, Eisenhaure Thomas T, Mertins Philipp P, Jaffe Jacob D JD, Clauser Karl R KR, Hacohen Nir N, Carr Steven A SAprotein ubiquitinylation, Study, proteasome, UBP, ubiquitin-specific protease activity, ubiquitin hydrolase activity, deubiquitinating enzyme, deubiquitinase activity, Methodological Studies, Procedures, protein ubiquitylation, Method, deubiquitinase, Studies, 26S proteasome, techniques, Methodological, procedures, Procedure, Methodological Study, UCH2., deubiquitylase, methodology, ubiquitin C-terminal hydrolase activityGlycine <subgenus>, Glycine Carbonate (2:1), human being, Calcium Salt Glycine, Sodium Hydrogen Carbonate, Lysine Hydrochloride, mol, number, glycine, 1110004P21Rik, 9030402K04Rik, Monoammonium, Spectrum Analyses, Homo sapiense, Monosodium, presence, deubiquitylase, Importin-alpha re-exporter, GLYCINE, Polypeptides, Homo spaiens, Lysine Acetate, deubiquitinase activity, Homo sapien, Calcium Salt (2:1), Homo sapians, OTTMUSG00000009329, Monoammonium Salt Glycine, Mass, 2, Lysine, Glycine Hydrochloride (2:1), Analysis, Mass Spectroscopy, L Lysine, Mass Spectrum Analysis, study, JM, ubiquitin-specific protease activity, ubiquitin hydrolase activity, Ubiquitylation, Homo sapients, Glycine Hydrochloride, Analyses, cell, Glycine, Monosodium Salt, epsilon-diaminocaproic acid, Phosphate, D4Ertd314e, C6H14N2O2, man, K2p3.1, UBP, Salt Glycine, Homo sapience, Lysin, Homo sampiens, REP1, Monopotasssium Salt, 26S proteasome, RCB0806, 2-aminoacetic acid, ubiquitination, Home sapiens, ubiquitin C-terminal hydrolase activity, 5730453G22Rik, Monoammonium Salt, Monopotasssium Salt Glycine, Cobalt Salt Glycine, data, Cellular apoptosis susceptibility protein, Glycine Phosphate (1:1), OAT1, Glycine Phosphate, PR-619, Copper Salt, Monosodium Salt Glycine, antibodies, Spectrum Analysis, Cell, Copper Salt Glycine, Spectroscopy, TASK, PSF, AU021830, count in organism, Experiment, RCB3052, Glycine Carbonate (1:1), TBAK1, count, Acetate, Homo sapian, Hydrochloride, Homo sapeins, L-Lysine, Mass Spectrum Analyses, Jurkat Cell, proteasome, Acid, Mass Spectrum, Aminoacetic Acid, PPH4, Gm12940, Glycine Sulfate (3:1), Spectrometry, Humo sapiens, Monopotassium Salt, alpha, Cobalt Salt, 2810416M14Rik, Monolithium Salt, Homo sapines, human, UCH2, deubiquitinating enzyme, Homo spiens, "human" EXACT genbank_common_name [], Aminoacetic, JURKAT, deubiquitinase, Cells, Mass., Exp2, JURKAT cell, aminoacetic acid, quantitative, TASK-1, 6-diaminohexanoic acid, Proteomes, Jurkat, Enisyl, Chromosome segregation 1-like protein, JM cell, Calcium Salt, presence or absence in organismprotein ubiquitinylation, Study, proteasome, UBP, ubiquitin-specific protease activity, ubiquitin hydrolase activity, deubiquitinating enzyme, deubiquitinase activity, Methodological Studies, Procedures, protein ubiquitylation, Method, deubiquitinase, Studies, 26S proteasome, techniques, Methodological, procedures, Procedure, Methodological Study, UCH2., deubiquitylase, methodology, ubiquitin C-terminal hydrolase activityprojections, extent, Signal Transductions, scale tissue, Glycine Carbonate (2:1), Calcium Salt Glycine, Lysine Hydrochloride, protein breakdown, CSMF, Monoammonium, Homo sapiense, Monosodium, GLYCINE, Receptor Mediated Signal Transduction, Polypeptides, Readability, Lysine Acetate, Homo sapien, Roles, Calcium Salt (2:1), Homo sapians, Monoammonium Salt Glycine, Concepts, 2, Analysis, protein degradation, L Lysine, Mass Spectrum Analysis, increased, human disease, ubiquitin hydrolase activity, me75, Ubiquitylation, Homo sapients, High Mobility Protein 20, enzymes, Analyses, Glycine, plant peltate hair, hypoplasia, epsilon-diaminocaproic acid, Pathways, Ubiquitin-related 1, proteins, Phosphate, D17Mit170, T1, APF-1, NOR-1, Homo sapience, Lysin, papilla, Role Concepts, 26S proteasome, Homo sapiens disease, Signal Transduction Pathways, ubiquitination, Transductions, Home sapiens, ubiquitin C-terminal hydrolase activity, Diagnostic Findings, Deubiquitinating, SIGNS SYMPTOMS, Monopotasssium Salt Glycine, Cobalt Salt Glycine, Pathway, anatomical protrusion, CHN, Glycine Phosphate, completeness, DESC, lamina, flanges, Monosodium Salt Glycine, antibodies, Tl3, CEP52, Tl2, Spectrum Analysis, Copper Salt Glycine, Spectroscopy, signaling cascade, Role Concept, count, Homo sapian, shelf, Diseases, Role, Transduction, Ubiquitin, proteasome, Acid, Glycine Sulfate (3:1), Deubiquitinases, System, shelves, Human Ubiquitin, expanded, Spectrometry, alpha, Cobalt Salt, projection, Homo sapines, ridge, human, organ system, multicellular organismal protein catabolic process, signaling pathway, enlarged, spine, Cells, Clinical Finding, Jurkat, accessory, Glycine <subgenus>, big, Signal Transduction Systems, human being, Sodium Hydrogen Carbonate, Biocatalysts, lamellae, peltate hair, Receptor-Mediated, number, Signal Transduction System, glycine, ATP Dependent Proteolysis Factor 1, Ubiquitin carboxyl extension protein 80, Spectrum Analyses, process of organ, presence, supernumerary, body system, deubiquitylase, Symptoms and Signs, Human, protrusion, lamella, Signal Transduction, large, Homo spaiens, deubiquitinase activity, reduced, Mass, HMG-20, system, Lysine, Low, tiny, Glycine Hydrochloride (2:1), ubiquitin-like protein modifier, Finding, Mass Spectroscopy, study, ubiquitin-specific protease activity, anatomical systems, pheromone catabolism, Glycine Hydrochloride, AI573420, cell, Monosodium Salt, Signal, study., Signal Pathways, ridges, C6H14N2O2, Signal Transduction Pathway, man, protein catabolism, UBP, 40S ribosomal protein S27a, signalling pathway, Salt Glycine, Homo sampiens, great, ubiquitin, Monopotasssium Salt, 2-aminoacetic acid, protein tagging activity, laminae, Signal Pathway, small, Monoammonium Salt, NOR1, Glycine Phosphate (1:1), Nor1, cou, anatomical process, PR-619, Copper Salt, Receptor-Mediated Signal Transductions, enzyme activity, function, Cell, Concept, Enzymes, polypeptide, count in organism, Signs and Symptoms, Ubiquitin A-52 residue ribosomal protein fusion product 1, Glycine Carbonate (1:1), Lr, Ubiquitin-related 2, Acetate, Receptor-Mediated Signal Transduction, Systems, Hydrochloride, ATP-Dependent Proteolysis Factor 1, scales, Homo sapeins, connected anatomical system, signalling cascade, L-Lysine, flange, Mass Spectrum Analyses, organ process, Jurkat Cell, covalent modifier, Mass Spectrum, pheromone catabolic process, Aminoacetic Acid, scale, distinct, underdeveloped, MINOR, increased number, Humo sapiens, Monopotassium Salt, Understanding, Monolithium Salt, UCH2, process, processes, present in greater numbers in organism, deubiquitinating enzyme, Homo spiens, "human" EXACT genbank_common_name [], 60S ribosomal protein L40, Aminoacetic, deubiquitinase, Bra, processus, Ubiquitin-related, TEC, regulation, aminoacetic acid, quantitative, 6-diaminohexanoic acid, Enisyl, Minor, Calcium Salt, presence or absence in organism1104http://gpmdb.thegpm.org/~/dblist_gpmnum/gpmnum=GPM11210018396dbgap_ncbi~0patentfamilies~0rfam~0merops~0complex-portal~0uniprot~0wormbaseparasite~0embl-covid19~0reactome~0emdb~0wgs_masters~0ebiweb_resources~0opentargets_genetics~0biomodels_all~0ipd-mhc~0ebiweb_teams~0taxonomy~0genome_assembly~0sc-experiments~0ebiweb_people~0enzymeportal_enzymes~0ipd-nhkir~0cellosaurus~0pdbe~0chebi~0patentproteins~0interpro7~0uniref~0chembl~0pdbekb~0gpcrdb~0hgnc~0sc-genes~0intact~0rhea~0ebiweb_training~0alphafold~0imgt-hla~0patentnucleotides~0ensemblroot~0eva_studies~0non-coding~0europepmc~0pubmed~1identifiers_registry~0pdbechem~0massive~1hpa-covid19~0eva-variants-covid19~0biosamples~0gwas_catalog~0biotools~0tls_masters~0mesh~0coding~0sra~0opentargets~0efo~0embl-pathogen~0project~0human_diseases~0geo_datasets~0embl~0treefam~0uniparc~0ols~0dgva~0intenz~0go~0tsa_masters~0biosamples-covid19~0ebiweb_corporate~0omim~0lrg~0earlycause-molecular-sequences~0ipd-kir~0empiar~0rnacentral~0orcid_data_claims~0gpmdb~2lineage-covid19~0metagenomics~0pfam~0varsite~00.00.00.0033929673041332510.001.0falseMethods for Quantification of in vivo Changes in Protein Ubiquitination following Proteasome and Deubiquitinase InhibitionData from MassIVE, [[http://proteomics.ucsd.edu/ProteoSAFe/status.jsp?task=TRANCHE-MSV000077546 MSV000077546]]. Experiment: exp2_rep1_Proteome, file: K20110724_NU_Jurkat_rep1B_Proteome_SILAC_L-no_M-17uMPR619_H-5uMMG13217uM_SCXFxn24.mzml. Published as part of Mol Cell Proteomics. 2012 May;11(5):148-59 . From the Abstract: {{i}} ... The introduction of antibodies that specifically recognize peptides with lysine residues that harbor a di-glycine remnant (K-eplison-GG) following tryptic digestion has dramatically improved the ability to enrich and identify ubiquitination sites from cellular lysates. We used this enrichment technique to study the effects of proteasome inhibition by MG-132 and deubiquitinase inhibition by PR-619 on ubiquitination sites in human Jurkat cells by quantitative high performance mass spectrometry. ... {{/i}}2013-06-26GPM1121001839622505724MSV000077546