Refined Preparation and Use of Anti-diglycine Remnant (K-ε-GG) Antibody Enables Routine Quantification of 10,000s of Ubiquitination Sites in Single Proteomics Experiments

Abstract

Detection of endogenous ubiquitination sites by mass spectrometry has dramatically improved with the commercialization of anti-di-glycine remnant (K-ε-GG) antibodies. Here, we describe a number of improvements to the K-ε-GG enrichment workflow, including optimized antibody and peptide input requirements, antibody cross-linking, and improved off-line fractionation prior to enrichment. This refined and practical workflow enables routine identification and quantification of ∼20,000 distinct endogenous ubiquitination sites in a single SILAC experiment using moderate amounts of protein input. Detection of endogenous ubiquitination sites by mass spectrometry has dramatically improved with the commercialization of anti-di-glycine remnant (K-ε-GG) antibodies. Here, we describe a number of improvements to the K-ε-GG enrichment workflow, including optimized antibody and peptide input requirements, antibody cross-linking, and improved off-line fractionation prior to enrichment. This refined and practical workflow enables routine identification and quantification of ∼20,000 distinct endogenous ubiquitination sites in a single SILAC experiment using moderate amounts of protein input. The commercialization of antibodies that recognize lysine residues modified with a di-glycine remnant (K-ε-GG) 1The abbreviations used are:K-ε-GGdi-glycine remnantSILACstable isotope labeling by amino acids in cell cultureDMPdimethyl pimelimidatebasic RPbasic pH reversed phaseHCDhigh energy collision-induced dissociationFAformic acid. 1The abbreviations used are:K-ε-GGdi-glycine remnantSILACstable isotope labeling by amino acids in cell cultureDMPdimethyl pimelimidatebasic RPbasic pH reversed phaseHCDhigh energy collision-induced dissociationFAformic acid. has significantly transformed the detection of endogenous protein ubiquitination sites by mass spectrometry (1Emanuele 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, 2Wagner 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. Proteomics. 2011; 10M111.013284Abstract Full Text Full Text PDF PubMed Google Scholar, 3Udeshi 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, 4Xu G. Paige J.S. Jaffrey S.R. Global analysis of lysine ubiquitination by ubiquitin remnant immunoaffinity profiling.Nat. Biotechnol. 2010; 28: 868-873Crossref PubMed Scopus (395) Google Scholar, 5Kim 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). Prior to the development of these highly specific reagents, proteomics experiments were limited to identification of up to only several hundred ubiquitination sites, which severely limited the scope of global ubiquitination studies (6Danielsen J.M. Sylvestersen K.B. Bekker-Jensen S. Szklarczyk D. Poulsen J.W. Horn H. Jensen L.J. Mailand N. Nielsen M.L. Mass spectrometric analysis of lysine ubiquitylation reveals promiscuity at site level.Mol. Cell. Proteomics. 2010; 10M110.003590Abstract Full Text Full Text PDF PubMed Scopus (248) Google Scholar). Recent proteomic studies employing anti-K-ε-GG antibodies have enhanced our understanding of ubiquitin biology through the identification of thousands of ubiquitination sites and the analysis of the change in relative abundance of these sites after chemical or biological perturbation (1Emanuele 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, 2Wagner 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. Proteomics. 2011; 10M111.013284Abstract Full Text Full Text PDF PubMed Google Scholar, 3Udeshi 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, 5Kim 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, 7Ong S.-E. Blagoev B. Kratchmarova I. Kristensen D.B. Steen H. Pandey A. Mann M. Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics.Mol. Cell. Proteomics. 2002; 1: 376-386Abstract Full Text Full Text PDF PubMed Scopus (4577) Google Scholar). Use of stable isotope labeling by amino acids in cell culture (SILAC) for quantification has enabled researchers to better understand the extent of ubiquitin regulation upon proteasome inhibition and precisely identify those protein classes, such as newly synthesized proteins or chromatin-related proteins, that see overt changes in their ubiquitination levels upon drug treatment (2Wagner 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. Proteomics. 2011; 10M111.013284Abstract Full Text Full Text PDF PubMed Google Scholar, 3Udeshi 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, 5Kim 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). Emanuel et al. (1Emanuele 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) have combined genetic and proteomics assays implementing the anti-K-ε-GG antibody to identify hundreds of known and putative Cullin-RING ligase substrates, which has clearly demonstrated the extensive role of Cullin-RING ligase ubiquitination on cellular protein regulation. di-glycine remnant stable isotope labeling by amino acids in cell culture dimethyl pimelimidate basic pH reversed phase high energy collision-induced dissociation formic acid. di-glycine remnant stable isotope labeling by amino acids in cell culture dimethyl pimelimidate basic pH reversed phase high energy collision-induced dissociation formic acid. Despite the successes recently achieved with the use of the anti-K-ε-GG antibody, increased sample input (up to ∼35 mg) and/or the completion of numerous experimental replicates have been necessary to achieve large numbers of K-ε-GG sites (>5,000) in a single SILAC-based experiment (1Emanuele 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, 2Wagner 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. Proteomics. 2011; 10M111.013284Abstract Full Text Full Text PDF PubMed Google Scholar, 3Udeshi 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, 5Kim 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 example, it has been recently shown that detection of more than 20,000 unique ubiquitination sites is possible from the analysis of five different murine tissues (8Wagner 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). However, as the authors indicate, only a few thousands sites are detected in any single analysis of an individual tissue sample (8Wagner 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). It is recognized that there is need for further improvements in global ubiquitin technology to increase the depth-of-coverage attainable in quantitative proteomic experiments using moderate amounts of protein input (9Bustos D. Bakalarski C.E. Yang Y. Peng J. Kirkpatrick D.S. Characterizing ubiquitination sites by peptide-based immunoaffinity enrichment.Mol. Cell. Proteomics. 2012; 11: 1529-1540Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). Through systematic study and optimization of key pre-analytical variables in the preparation and use of the anti-K-ε-GG antibody as well as the proteomic workflow, we have now achieved, for the first time, routine quantification of ∼20,000 nonredundant K-ε-GG sites in a single SILAC triple encoded experiment starting with 5 mg of protein per SILAC channel. This represents a 10-fold improvement over our previously published method (3Udeshi 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). To generate titration curve data, Jurkat E6-1 cells (ATCC) were grown in Roswell Park Memorial Institute (RPMI) 1640 media (Invitrogen) supplemented with 10% dialyzed fetal bovine serum (Sigma-Aldrich), penicillin, streptomycin, and glutamine (Invitrogen). For titration data where peptide input was held constant and antibody amount was varied, Jurkat cells were treated for 4 h with 2 μm MG-132 (Calbiochem) prior to harvest. For SILAC experiments, Jurkat cells were cultured in RPMI media (custom preparation from Caisson Laboratories, North Logan, UT) deficient in l-arginine and l-lysine and supplemented with 10% dialyzed fetal bovine serum (Sigma-Aldrich), penicillin, streptomycin, and glutamine (Invitrogen). For each experiment, SILAC labeling was completed such that cells were grown with l-arginine (Arg-0) and l-lysine (Lys-0), l-arginine [13C6]HCl (Arg-6) and l-lysine-4,4,5,5-d4 (Lys-4), or l-arginine [13C6,15N4]HCl (Arg-10) and l-lysine [13C6,15N2]HCl (Lys-8) (Sigma-Aldrich). SILAC cells underwent ∼6 doublings and were treated for 4 h with either 5 μm MG132 (Calbiochem), 5 μm PR-619 (LifeSensors), or 0.5% dimethyl sulfoxide (DMSO) (3Udeshi 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). Following treatment, cells were washed twice with 1× PBS and pelleted for cell lysis. SILAC-labeled cell pellets were lysed at 4°C in denaturing conditions. Specifically, the cell lysis buffer contained 8 m urea, 50 mm Tris-HCl, pH 7.5, 150 mm NaCl, 1 mm EDTA, 2 μg/ml aprotinin (Sigma-Aldrich), 10 μg/ml leupeptin (Roche Applied Science), 1 mm phenylmethylsulfonyl fluoride (PMSF), 50 μm PR-619, and 1 mm chloroacetamide. Following lysis, cells were centrifuged at 20,000 × g for at 4°C to were using a protein SILAC sample was by 5 mg of protein input per SILAC were with 5 mm for at and using 10 mm for at in the were to 2 m with 50 mm Tris-HCl, pH 7.5, and at with at an to of Following were with formic and using a were with 5 of by 5 of and of was the washed with of and with of were to in a basic fractionation was completed using a × 5 on an sample was in of basic A 5 mm pH and with A at a of A basic method was for off-line The of an increase to 5 mm pH by a from to and to and A of was used for the of the sample basic were in a For K-ε-GG peptide basic were in a using the in Specifically, to generate the first for K-ε-GG and were The were for K-ε-GG enrichment with a were to in a the anti-K-ε-GG antibody was from the ubiquitin remnant (K-ε-GG) and was as previously 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 antibody cross-linking, the anti-K-ε-GG antibody were washed with 1 of mm pH was completed by antibody in 1 of mm dimethyl pimelimidate and at for were washed with 1 of mm pH and in 1 of mm for 2 h at 4°C with the antibody were washed in of buffer mm pH 10 mm 50 mm in and at 4°C for peptide were in of buffer and with anti-K-ε-GG antibody for 1 h on a at For titration experiments, were with either or of antibody, and for SILAC experiments, each basic was with of were washed with of K-ε-GG were by 50 of were using J. Mann M. Y. for and of for proteomics using PubMed Scopus Google Scholar). was with high and with of of and of were washed with of and with 50 of were to For were in and by using a mass on to a were a μm × μm with an to with μm and to The A was and the was were the mass at a of using a of following by a to The was including sample and The was in the after each on the using an of × and an of 5 × The for was the energy was to the was to and the peptide and isotope were data were using the J. Mann M. enables high peptide identification mass and protein Biotechnol. PubMed Scopus Google Scholar, J. N. A. Olsen J.V. Mann M. A peptide the 2011; PubMed Scopus Google Scholar) and the A of by was to the For the was to with the number of to The mass was to for the first for mass and was to for the to and protein were as of was as a The for and site identification was to the peptide was to and the amino and peptide were K-ε-GG site identification and quantification were from the sites and For titration curve experiments, and K-ε-GG were on as previously (3Udeshi 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). For the SILAC-based experiments, site was from the sites of to a peptide lysine were from the K-ε-GG sites were using a from the in the as previously (3Udeshi 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, and for expression in PubMed Scopus Google Scholar). biological enrichment was completed using the W. B.T. Systematic and analysis of large using PubMed Scopus Google Scholar). 1 and 2 that to a an for K-ε-GG using on for data are at with the anti-K-ε-GG antibody that proteins and were in and were on our over the detection of K-ε-GG by mass spectrometry and in are from the anti-K-ε-GG antibody at pH using (3Udeshi 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). of these that large amounts of and antibody are from the anti-K-ε-GG antibody that is to protein A prior to of K-ε-GG a for mass spectrometric were using J. Y. Mann M. and for and sample in PubMed Scopus Google Scholar). we that antibody from K-ε-GG the that the and are than in and the buffer only after in the on the mass and the detection of K-ε-GG the of the anti-K-ε-GG antibody using up to of the antibody for each K-ε-GG enrichment of antibody, the amount of and the by than which the of K-ε-GG to a J. Y. Mann M. and for and sample in PubMed Scopus Google Scholar). To the that have on the enrichment and detection of K-ε-GG we chemical of the anti-K-ε-GG antibody to that chemical of the antibody to the protein A with of the of and upon pH and the number of K-ε-GG by D. A Scholar). This increase is of the amount of starting anti-K-ε-GG antibody used over a of or enrichment is completed using 1 mg of protein input and that chemical of the anti-K-ε-GG antibody significantly to K-ε-GG the of with antibody are after The need to have antibody to is well such a study has been for the anti-K-ε-GG Here, we the amount of anti-K-ε-GG antibody, and in experiments, the amount of input protein from cells treated with the proteasome MG-132 to study the of these on the number of K-ε-GG and was by study that only of is for a single in the of either or antibody is to achieve the of distinct K-ε-GG from 1 mg of protein and of antibody is to K-ε-GG from up to 10 mg of starting and that are achieved only 10 mg of peptide input and from our titration experiments the number of significantly change antibody input is constant and only peptide input is and we that the number of detected K-ε-GG significantly as the input amount of antibody was increased the number of in each experiment that amounts of antibody input the number of This a high of of to protein A and/or the antibody which detection of K-ε-GG that it is to the antibody and protein input to the of K-ε-GG in a This is for peptide and enrichment further our antibody titration study to the depth-of-coverage from 1 mg of protein input from cells that been treated with MG-132 and endogenous cellular levels of the number of K-ε-GG by in the of proteasome we an of K-ε-GG starting with 1 mg of input and of antibody 2 and This that optimized global ubiquitination assays completed in that have proteasome treatment that is to ubiquitination sites in those that to by proteasome J.W. Cullin-RING Cell. Proteomics. 2012; 11: Full Text Full Text PDF PubMed Scopus Google Scholar). refined and optimized workflow for of K-ε-GG from cellular is shown in The is for a SILAC experiment using 5 mg of input protein per workflow, SILAC-labeled cells are lysed denaturing and combined in amounts on protein Following and the proteins are with to generate as well as the di-glycine we used off-line fractionation to increase the number of K-ε-GG detected by relative to (3Udeshi 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). of our optimized we have basic to generate for K-ε-GG enrichment Y. Yang Y. T. T. Y. M.E. D. T. with for of 2011; 11: PubMed Scopus Google Scholar, Y. with for proteomic Proteomics. 2012; PubMed Scopus Google Scholar). that basic is to our previously published method of peptide is prior to K-ε-GG enrichment as a of the which sample and peptide of basic highly and nonredundant for K-ε-GG enrichment that enables depth-of-coverage for K-ε-GG Y. Yang Y. T. T. Y. M.E. D. T. with for of 2011; 11: PubMed Scopus Google Scholar, Y. with for proteomic Proteomics. 2012; PubMed Scopus Google Scholar). The basic are for K-ε-GG using of anti-K-ε-GG antibody per each was by using a mass the refined and optimized K-ε-GG workflow using biological to those in our previously published (3Udeshi 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). Specifically, SILAC-labeled Jurkat cells were treated for 4 h with either 5 μm of the deubiquitinase M. S.J. R. R. J. B. chemical proteomics development for 2011; Full Text Full Text PDF PubMed Scopus Google 5 μm of the proteasome or were The workflow, which an optimized amount of antibody to K-ε-GG from basic in detection of K-ε-GG sites from a single SILAC completed biological replicates with SILAC and we that in a single we an of distinct K-ε-GG per basic and an of distinct K-ε-GG sites of which were with a of in for the and and of of are shown in and This represents a 10-fold increase in the number of K-ε-GG sites for a single SILAC sample relative to our previously published where only and sites were detected and in a single (3Udeshi 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). biological we that K-ε-GG sites are in at and sites are in replicates the improvement in the enrichment and detection of K-ε-GG is achieved through a number of including the use of optimized antibody antibody, improved off-line fractionation and and the previously for quantification and detection of K-ε-GG sites from a single SILAC-labeled sample where starting is limited to 5 mg per (1Emanuele 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, 2Wagner 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. Proteomics. 2011; 10M111.013284Abstract Full Text Full Text PDF PubMed Google Scholar, 3Udeshi 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, 5Kim 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). to we that proteasome inhibition by MG-132 and deubiquitinase inhibition by PR-619 changes to the ubiquitin MG-132 is more at the levels of ubiquitination sites relative to PR-619 4 and (2Wagner 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. Proteomics. 2011; 10M111.013284Abstract Full Text Full Text PDF PubMed Google Scholar, 3Udeshi 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). Following cellular treatment with of K-ε-GG sites were of sites were following PR-619 reagents, we that significantly K-ε-GG sites were on proteins in cell K-ε-GG sites were on proteins in regulation. that our data that proteasome inhibition with MG-132 significantly the levels of and is more at the levels of than deubiquitinase inhibition by PR-619 (3Udeshi 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, 5Kim 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). we have a number of to the K-ε-GG analysis have in the and practical method to for the analysis of endogenous ubiquitination sites from cellular The specific improvements we have including of the antibody, optimization of the amount of antibody for enrichment of as well as use of peptide that of sample prior to analysis by to of the technology for of have for the first time, that the depth-of-coverage from moderate amounts of starting is on with the of using highly optimized to protein sites J. Gygi S.P. The enrichment approach for global analysis by mass PubMed Scopus Google Scholar, G. Y. for and optimization of for PubMed Scopus (123) Google the to the of and ubiquitin sites from amounts of protein input using the anti-K-ε-GG antibody (9Bustos D. Bakalarski C.E. Yang Y. Peng J. Kirkpatrick D.S. Characterizing ubiquitination sites by peptide-based immunoaffinity enrichment.Mol. Cell. Proteomics. 2012; 11: 1529-1540Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). that further in the number of ubiquitination sites detected and in single experiments are the use of more than 5 mg of input protein per combined with further in sample for by use of SILAC, or by peptide fractionation a number of prior to enrichment. that the and in limited in to only experiments using the anti-K-ε-GG antibody highly in the for optimization of antibodies. and of for with