Chunaram Choudhary

Lysine acetylation is a reversible posttranslational modification of proteins and plays a key role in regulating gene expression. Technological limitations have so far prevented a global analysis of lysine acetylation's cellular roles. We used high-resolution mass spectrometry to identify 3600 lysine acetylation sites on 1750 proteins and quantified acetylation changes in response to the deacetylase inhibitors suberoylanilide hydroxamic acid and MS-275. Lysine acetylation preferentially targets large macromolecular complexes involved in diverse cellular processes, such as chromatin remodeling, cell cycle, splicing, nuclear transport, and actin nucleation. Acetylation impaired phosphorylation-dependent interactions of 14-3-3 and regulated the yeast cyclin-dependent kinase Cdc28. Our data demonstrate that the regulatory scope of lysine acetylation is broad and comparable with that of other major posttranslational modifications.

Selective autophagy can be mediated via receptor molecules that link specific cargoes to the autophagosomal membranes decorated by ubiquitin-like microtubule-associated protein light chain 3 (LC3) modifiers. Although several autophagy receptors have been identified, little is known about mechanisms controlling their functions in vivo. In this work, we found that phosphorylation of an autophagy receptor, optineurin, promoted selective autophagy of ubiquitin-coated cytosolic Salmonella enterica. The protein kinase TANK binding kinase 1 (TBK1) phosphorylated optineurin on serine-177, enhancing LC3 binding affinity and autophagic clearance of cytosolic Salmonella. Conversely, ubiquitin- or LC3-binding optineurin mutants and silencing of optineurin or TBK1 impaired Salmonella autophagy, resulting in increased intracellular bacterial proliferation. We propose that phosphorylation of autophagy receptors might be a general mechanism for regulation of cargo-selective autophagy.

Post-translational modification of proteins by ubiquitin is a fundamentally important regulatory mechanism. However, proteome-wide analysis of endogenous ubiquitylation remains a challenging task, and almost always has relied on cells expressing affinity tagged ubiquitin. Here we combine single-step immunoenrichment of ubiquitylated peptides with peptide fractionation and high-resolution mass spectrometry to investigate endogenous ubiquitylation sites. We precisely map 11,054 endogenous putative ubiquitylation sites (diglycine-modified lysines) on 4,273 human proteins. The presented data set covers 67% of the known ubiquitylation sites and contains 10,254 novel sites on proteins with diverse cellular functions including cell signaling, receptor endocytosis, DNA replication, DNA damage repair, and cell cycle progression. Our method enables site-specific quantification of ubiquitylation in response to cellular perturbations and is applicable to any cell type or tissue. Global quantification of ubiquitylation in cells treated with the proteasome inhibitor MG-132 discovers sites that are involved in proteasomal degradation, and suggests a nonproteasomal function for almost half of all sites. Surprisingly, ubiquitylation of about 15% of sites decreased more than twofold within four hours of MG-132 treatment, showing that inhibition of proteasomal function can dramatically reduce ubiquitylation on many sites with non-proteasomal functions. Comparison of ubiquitylation sites with acetylation sites reveals an extensive overlap between the lysine residues targeted by these two modifications. However, the crosstalk between these two post-translational modifications is significantly less frequent on sites that show increased ubiquitylation upon proteasome inhibition. Taken together, we report the largest site-specific ubiquitylation dataset in human cells, and for the first time demonstrate proteome-wide, site-specific quantification of endogenous putative ubiquitylation sites. Post-translational modification of proteins by ubiquitin is a fundamentally important regulatory mechanism. However, proteome-wide analysis of endogenous ubiquitylation remains a challenging task, and almost always has relied on cells expressing affinity tagged ubiquitin. Here we combine single-step immunoenrichment of ubiquitylated peptides with peptide fractionation and high-resolution mass spectrometry to investigate endogenous ubiquitylation sites. We precisely map 11,054 endogenous putative ubiquitylation sites (diglycine-modified lysines) on 4,273 human proteins. The presented data set covers 67% of the known ubiquitylation sites and contains 10,254 novel sites on proteins with diverse cellular functions including cell signaling, receptor endocytosis, DNA replication, DNA damage repair, and cell cycle progression. Our method enables site-specific quantification of ubiquitylation in response to cellular perturbations and is applicable to any cell type or tissue. Global quantification of ubiquitylation in cells treated with the proteasome inhibitor MG-132 discovers sites that are involved in proteasomal degradation, and suggests a nonproteasomal function for almost half of all sites. Surprisingly, ubiquitylation of about 15% of sites decreased more than twofold within four hours of MG-132 treatment, showing that inhibition of proteasomal function can dramatically reduce ubiquitylation on many sites with non-proteasomal functions. Comparison of ubiquitylation sites with acetylation sites reveals an extensive overlap between the lysine residues targeted by these two modifications. However, the crosstalk between these two post-translational modifications is significantly less frequent on sites that show increased ubiquitylation upon proteasome inhibition. Taken together, we report the largest site-specific ubiquitylation dataset in human cells, and for the first time demonstrate proteome-wide, site-specific quantification of endogenous putative ubiquitylation sites. Ubiquitin is a 76 amino acid long protein that can be conjugated to the ε-amino group of lysines in a process termed ubiquitylation or ubiquitination (1Glickman M.H. Ciechanover A. The ubiquitin-proteasome proteolytic pathway: destruction for the sake of construction.Nat. Rev. 2002; 82: 373-428Google Scholar, 2Weissman A.M. Themes and variations on ubiquitylation.Mol. Cell Biol. 2001; 2: 169-178Google Scholar). Post-translational modification (PTM) 1The abbreviations used are:PTMpost-translational modificationUPSubiquitin proteasome systemSILACstable isotope labeling by amino acids in cell cultureHCDhigher-energy C-trap dissociationGOgene ontologyRTKreceptor tyrosine kinasedi-Glydi-glycine. of proteins by ubiquitin is a reversible regulatory mechanism that is well conserved in eukaryotic organisms. The role of ubiquitylation is extensively studied in the ubiquitin proteasome system (UPS) where substrate-linked ubiquitin provides a signal for proteasomal degradation of target proteins (3Hochstrasser M. Ubiquitin, proteasomes, and the regulation of intracellular protein degradation.Current Opinion Cell Biol. 1995; 7: 215-223Crossref PubMed Scopus (780) Google Scholar). However, ubiquitylation also plays important roles in many other cellular processes including DNA damage repair, DNA replication, cell surface receptor endocytosis, and innate immune signaling (4Chen Z.J. Sun L.J. Nonproteolytic functions of ubiquitin in cell signaling.Mol. Cell. 2009; 33: 275-286Abstract Full Text Full Text PDF PubMed Scopus (684) Google Scholar, 5Grabbe C. Husnjak K. Dikic I. The spatial and temporal organization of ubiquitin networks.Nat. Rev. Mol. Cell. Biol. 2011; 12: 295-307Crossref PubMed Scopus (251) Google Scholar, 6Mukhopadhyay D. Riezman H. Proteasome-independent functions of ubiquitin in endocytosis and signaling.Science. 2007; 315: 201-205Crossref PubMed Scopus (938) Google Scholar). Deregulation of the UPS has been implicated in the development of cancer and neurodegenerative disorders (7Bingol B. Sheng M. Deconstruction for reconstruction: the role of proteolysis in neural plasticity and disease.Neuron. 2011; 69: 22-32Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar, 8Hoeller D. Hecker C.M. Dikic I. Ubiquitin and ubiquitin-like proteins in cancer pathogenesis.Nat. Rev. 2006; 6: 776-788Crossref Scopus (323) Google Scholar, 9Schwartz A.L. Ciechanover A. Targeting proteins for destruction by the ubiquitin system: implications for human pathobiology.Ann. Rev. Pharmacol. Toxicol. 2009; 49: 73-96Crossref PubMed Scopus (352) Google Scholar). The clinical use of the proteasome inhibitor bortezomib, and ongoing clinical trials of several other inhibitors emphasize the therapeutic relevance of the UPS (10Bedford L. Lowe J. Dick L.R. Mayer R.J. Brownell J.E. Ubiquitin-like protein conjugation and the ubiquitin-proteasome system as drug targets.Nat. Rev. 2011; 10: 29-46Google Scholar, 11Hoeller D. Dikic I. Targeting the ubiquitin system in cancer therapy.Nature. 2009; 458: 438-444Crossref PubMed Scopus (460) Google Scholar).Accurate mapping of PTM sites is a key requirement to determine their functional roles and to understand the regulatory complexity of the proteome. Advancements in high resolution mass spectrometry (MS)-based proteomics have enabled the identification of thousands of in vivo PTMs (12Choudhary C. Mann M. Decoding signalling networks by mass spectrometry-based proteomics.Nat. Rev. 2010; 11: 427-439Crossref Scopus (475) Google Scholar). Quantitative proteomics can be used to analyze relative changes in PTM abundance on a global scale, enabling the identification of perturbation-relevant regulatory sites in complex signaling networks.Identification of ubiquitylation sites by mass spectrometry is based on the presence of a di-glycine (di-Gly) remnant on ubiquitylated lysines. The di-Gly remnant is derived from the two C-terminal glycine residues of ubiquitin that remain covalently linked to modified lysines following proteolytic digestion with trypsin. The distinct mass shift (114.0429 Da) caused by the di-Gly remnant enables identification and precise localization of ubiquitylation sites based on peptide fragment masses. Trypsin proteolysis of proteins modified by ubiquitin, NEDD8, or ISG15 generates an identical di-Gly remnant on modified lysines, making it impossible to distinguish among these modifications by mass spectrometry. However, the expression of ISG15 and its conjugation to lysines is relatively low in cells cultured under standard cell culture conditions (13Zhang D. Zhang D.E. Interferon-stimulated gene 15 and the protein ISGylation system.J. Interferon Cytokine Res. 2011; 31: 119-130Crossref PubMed Scopus (224) Google Scholar), and NEDD8 is believed to target primarily cullin family proteins (14Rabut G. Peter M. Function and regulation of protein neddylation. ‘Protein modifications: beyond the usual suspects' review series.EMBO Rep. 2008; 9: 969-976Crossref PubMed Scopus (258) Google Scholar). Consequently, a great majority of cellular peptides containing the di-Gly remnant are believed to stem from ubiquitylated proteins. Therefore, in this paper we refer to all di-Gly modified lysines as “ubiquitylation sites” even though a small fraction of these sites is likely to originate from modification by ISG15 or NEDD8.Large-scale ubiquitylation site mapping by mass spectrometry was first demonstrated in yeast by identifying over 100 ubiquitylation sites (15Peng 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 (1291) Google Scholar). Since then four large-scale ubiquitylation screens have mapped 1,192 sites in human cells (16Danielsen 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. 2011; 10 (M110.003590)Abstract Full Text Full Text PDF PubMed Scopus (247) Google Scholar, 17Xu 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 (386) Google Scholar, 18Meierhofer D. Wang X. Huang L. Kaiser P. Quantitative analysis of global ubiquitination in HeLa cells by mass spectrometry.J. Proteome Res. 2008; 7: 4566-4576Crossref PubMed Scopus (161) Google Scholar, 19Shi Y. Chan D.W. Jung S.Y. Malovannaya A. Wang Y. Qin J. A dataset of human endogenous ubiquitination sites.Mol. Cell. Proteomics. 2010; 10 (M110.002089)PubMed Google Scholar). The methods used in all these studies require enrichment of ubiquitylated proteins. Although, many putatively ubiquitylated proteins were identified (16Danielsen 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. 2011; 10 (M110.003590)Abstract Full Text Full Text PDF PubMed Scopus (247) Google Scholar), only a relatively small number of ubiquitylation sites were mapped. Limitations of previous methods for in-depth ubiquitylation analysis and their incompatibility with a proteome-wide, site-specific quantification highlighted the need to develop more robust methods of ubiquitylation site identification and quantification.In this study we developed a streamlined method in which ubiquitylated peptides are directly enriched from trypsin digested whole cell peptide mixture with a recently developed di-Gly-lysine-specific antibody (17Xu 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 (386) Google Scholar). Direct immunoenrichment of ubiquitylated peptides, together with peptide fractionation and high resolution mass spectrometery, allowed in-depth analysis of putative ubiquitylation sites. Using this method we identified a considerable portion of known human ubiquitylation and more than sites. we method with isotope labeling by amino acids in cell culture to changes in ubiquitylation in response to the proteasome inhibitor The method enables proteome-wide quantification of endogenous ubiquitylation sites in response to cellular is of the extensively studied post-translational modifications in The human about protein G. S. The protein of the human 2002; PubMed Scopus Google Scholar), tyrosine A. J. N. I. I. A. A. J. tyrosine in the human Full Text Full Text PDF PubMed Scopus Google Scholar), and Y. mechanism 2009; Full Text Full Text PDF PubMed Scopus Google Scholar). in human cells and are involved in ubiquitylation Z.J. in innate and 2009; 458: PubMed Scopus Google Scholar), and about 100 this modification Z.J. in innate and 2009; 458: PubMed Scopus Google Scholar, A. A.M. A and functional of Full Text Full Text PDF PubMed Scopus Google Scholar). The presence of this extensive ubiquitylation in human cells suggests that the complexity of protein regulation by ubiquitylation be to that of However, to the number of known ubiquitylation sites was in to the number of known sites. methods identification of ubiquitylation sites enrichment of ubiquitylated modified by ubiquitin. Trypsin digestion of these proteins generates a complex peptide mixture containing a small fraction of ubiquitylated this study we a robust and streamlined method to precisely map endogenous putative ubiquitylation sites in human Our approach provides several over methods for ubiquitylation site it only a single-step affinity enrichment of modified peptides, the enrichment of ubiquitylated peptides enables of low modification it proteome-wide analysis of endogenous ubiquitylation it can be to map ubiquitylation sites in any or and it is with site-specific quantification of ubiquitylation on a proteome-wide this approach we identified 11,054 putative endogenous ubiquitylation sites (diglycine-modified lysines) in human data over of the sites in previous However, more than of the sites in dataset been these data the number of known human ubiquitylation sites. study in of identified as the studied PTM We show that ubiquitylation proteins involved in all cellular functions and that its regulatory is to other PTMs as and The approach enables proteome-wide, analysis of ubiquitylation for the first Our analysis of changes in ubiquitylation following proteasome inhibition demonstrated that about half of all sites show increased a of proteins is by four hours of proteasome inhibition. remains sites that show increased ubiquitylation MG-132 are directly involved in proteasomal However, it is to that sites that show increased ubiquitylation upon proteasome inhibition nonproteasomal regulatory sites identified in this study as a for the functional of many proteins. The method is and can be to map ubiquitylation sites in any cell or and to site-specific quantification of ubiquitylation upon cellular identification of ubiquitylated peptides, as well as all data with this can be from and and all data from contains which all quantification data from The PDF contains of ubiquitylated peptides identified in this and Ubiquitin is a 76 amino acid long protein that can be conjugated to the ε-amino group of lysines in a process termed ubiquitylation or ubiquitination (1Glickman M.H. Ciechanover A. The ubiquitin-proteasome proteolytic pathway: destruction for the sake of construction.Nat. Rev. 2002; 82: 373-428Google Scholar, 2Weissman A.M. Themes and variations on ubiquitylation.Mol. Cell Biol. 2001; 2: 169-178Google Scholar). Post-translational modification (PTM) 1The abbreviations used are:PTMpost-translational modificationUPSubiquitin proteasome systemSILACstable isotope labeling by amino acids in cell cultureHCDhigher-energy C-trap dissociationGOgene ontologyRTKreceptor tyrosine kinasedi-Glydi-glycine. of proteins by ubiquitin is a reversible regulatory mechanism that is well conserved in eukaryotic organisms. The role of ubiquitylation is extensively studied in the ubiquitin proteasome system (UPS) where substrate-linked ubiquitin provides a signal for proteasomal degradation of target proteins (3Hochstrasser M. Ubiquitin, proteasomes, and the regulation of intracellular protein degradation.Current Opinion Cell Biol. 1995; 7: 215-223Crossref PubMed Scopus (780) Google Scholar). However, ubiquitylation also plays important roles in many other cellular processes including DNA damage repair, DNA replication, cell surface receptor endocytosis, and innate immune signaling (4Chen Z.J. Sun L.J. Nonproteolytic functions of ubiquitin in cell signaling.Mol. Cell. 2009; 33: 275-286Abstract Full Text Full Text PDF PubMed Scopus (684) Google Scholar, 5Grabbe C. Husnjak K. Dikic I. The spatial and temporal organization of ubiquitin networks.Nat. Rev. Mol. Cell. Biol. 2011; 12: 295-307Crossref PubMed Scopus (251) Google Scholar, 6Mukhopadhyay D. Riezman H. Proteasome-independent functions of ubiquitin in endocytosis and signaling.Science. 2007; 315: 201-205Crossref PubMed Scopus (938) Google Scholar). Deregulation of the UPS has been implicated in the development of cancer and neurodegenerative disorders (7Bingol B. Sheng M. Deconstruction for reconstruction: the role of proteolysis in neural plasticity and disease.Neuron. 2011; 69: 22-32Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar, 8Hoeller D. Hecker C.M. Dikic I. Ubiquitin and ubiquitin-like proteins in cancer pathogenesis.Nat. Rev. 2006; 6: 776-788Crossref Scopus (323) Google Scholar, 9Schwartz A.L. Ciechanover A. Targeting proteins for destruction by the ubiquitin system: implications for human pathobiology.Ann. Rev. Pharmacol. Toxicol. 2009; 49: 73-96Crossref PubMed Scopus (352) Google Scholar). The clinical use of the proteasome inhibitor bortezomib, and ongoing clinical trials of several other inhibitors emphasize the therapeutic relevance of the UPS (10Bedford L. Lowe J. Dick L.R. Mayer R.J. Brownell J.E. Ubiquitin-like protein conjugation and the ubiquitin-proteasome system as drug targets.Nat. Rev. 2011; 10: 29-46Google Scholar, 11Hoeller D. Dikic I. Targeting the ubiquitin system in cancer therapy.Nature. 2009; 458: 438-444Crossref PubMed Scopus (460) Google Scholar). post-translational modification ubiquitin proteasome system isotope labeling by amino acids in cell culture C-trap gene receptor tyrosine mapping of PTM sites is a key requirement to determine their functional roles and to understand the regulatory complexity of the proteome. Advancements in high resolution mass spectrometry (MS)-based proteomics have enabled the identification of thousands of in vivo PTMs (12Choudhary C. Mann M. Decoding signalling networks by mass spectrometry-based proteomics.Nat. Rev. 2010; 11: 427-439Crossref Scopus (475) Google Scholar). Quantitative proteomics can be used to analyze relative changes in PTM abundance on a global scale, enabling the identification of perturbation-relevant regulatory sites in complex signaling of ubiquitylation sites by mass spectrometry is based on the presence of a di-glycine (di-Gly) remnant on ubiquitylated lysines. The di-Gly remnant is derived from the two C-terminal glycine residues of ubiquitin that remain covalently linked to modified lysines following proteolytic digestion with trypsin. The distinct mass shift (114.0429 Da) caused by the di-Gly remnant enables identification and precise localization of ubiquitylation sites based on peptide fragment masses. Trypsin proteolysis of proteins modified by ubiquitin, NEDD8, or ISG15 generates an identical di-Gly remnant on modified lysines, making it impossible to distinguish among these modifications by mass spectrometry. However, the expression of ISG15 and its conjugation to lysines is relatively low in cells cultured under standard cell culture conditions (13Zhang D. Zhang D.E. Interferon-stimulated gene 15 and the protein ISGylation system.J. Interferon Cytokine Res. 2011; 31: 119-130Crossref PubMed Scopus (224) Google Scholar), and NEDD8 is believed to target primarily cullin family proteins (14Rabut G. Peter M. Function and regulation of protein neddylation. ‘Protein modifications: beyond the usual suspects' review series.EMBO Rep. 2008; 9: 969-976Crossref PubMed Scopus (258) Google Scholar). Consequently, a great majority of cellular peptides containing the di-Gly remnant are believed to stem from ubiquitylated proteins. Therefore, in this paper we refer to all di-Gly modified lysines as “ubiquitylation sites” even though a small fraction of these sites is likely to originate from modification by ISG15 or ubiquitylation site mapping by mass spectrometry was first demonstrated in yeast by identifying over 100 ubiquitylation sites (15Peng 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 (1291) Google Scholar). Since then four large-scale ubiquitylation screens have mapped 1,192 sites in human cells (16Danielsen 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. 2011; 10 (M110.003590)Abstract Full Text Full Text PDF PubMed Scopus (247) Google Scholar, 17Xu 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 (386) Google Scholar, 18Meierhofer D. Wang X. Huang L. Kaiser P. Quantitative analysis of global ubiquitination in HeLa cells by mass spectrometry.J. Proteome Res. 2008; 7: 4566-4576Crossref PubMed Scopus (161) Google Scholar, 19Shi Y. Chan D.W. Jung S.Y. Malovannaya A. Wang Y. Qin J. A dataset of human endogenous ubiquitination sites.Mol. Cell. Proteomics. 2010; 10 (M110.002089)PubMed Google Scholar). The methods used in all these studies require enrichment of ubiquitylated proteins. Although, many putatively ubiquitylated proteins were identified (16Danielsen 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. 2011; 10 (M110.003590)Abstract Full Text Full Text PDF PubMed Scopus (247) Google Scholar), only a relatively small number of ubiquitylation sites were mapped. Limitations of previous methods for in-depth ubiquitylation analysis and their incompatibility with a proteome-wide, site-specific quantification highlighted the need to develop more robust methods of ubiquitylation site identification and this study we developed a streamlined method in which ubiquitylated peptides are directly enriched from trypsin digested whole cell peptide mixture with a recently developed di-Gly-lysine-specific antibody (17Xu 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 (386) Google Scholar). Direct immunoenrichment of ubiquitylated peptides, together with peptide fractionation and high resolution mass spectrometery, allowed in-depth analysis of putative ubiquitylation sites. Using this method we identified a considerable portion of known human ubiquitylation and more than sites. we method with isotope labeling by amino acids in cell culture to changes in ubiquitylation in response to the proteasome inhibitor The method enables proteome-wide quantification of endogenous ubiquitylation sites in response to cellular is of the extensively studied post-translational modifications in The human about protein G. S. The protein of the human 2002; PubMed Scopus Google Scholar), tyrosine A. J. N. I. I. A. A. J. tyrosine in the human Full Text Full Text PDF PubMed Scopus Google Scholar), and Y. mechanism 2009; Full Text Full Text PDF PubMed Scopus Google Scholar). in human cells and are involved in ubiquitylation Z.J. in innate and 2009; 458: PubMed Scopus Google Scholar), and about 100 this modification Z.J. in innate and 2009; 458: PubMed Scopus Google Scholar, A. A.M. A and functional of Full Text Full Text PDF PubMed Scopus Google Scholar). The presence of this extensive ubiquitylation in human cells suggests that the complexity of protein regulation by ubiquitylation be to that of However, to the number of known ubiquitylation sites was in to the number of known sites. methods identification of ubiquitylation sites enrichment of ubiquitylated modified by ubiquitin. Trypsin digestion of these proteins generates a complex peptide mixture containing a small fraction of ubiquitylated this study we a robust and streamlined method to precisely map endogenous putative ubiquitylation sites in human Our approach provides several over methods for ubiquitylation site it only a single-step affinity enrichment of modified peptides, the enrichment of ubiquitylated peptides enables of low modification it proteome-wide analysis of endogenous ubiquitylation it can be to map ubiquitylation sites in any or and it is with site-specific quantification of ubiquitylation on a proteome-wide this approach we identified 11,054 putative endogenous ubiquitylation sites (diglycine-modified lysines) in human data over of the sites in previous However, more than of the sites in dataset been these data the number of known human ubiquitylation sites. study in of identified as the studied PTM We show that ubiquitylation proteins involved in all cellular functions and that its regulatory is to other PTMs as and The approach enables proteome-wide, analysis of ubiquitylation for the first Our analysis of changes in ubiquitylation following proteasome inhibition demonstrated that about half of all sites show increased a of proteins is by four hours of proteasome inhibition. remains sites that show increased ubiquitylation MG-132 are directly involved in proteasomal However, it is to that sites that show increased ubiquitylation upon proteasome inhibition nonproteasomal regulatory sites identified in this study as a for the functional of many proteins. The method is and can be to map ubiquitylation sites in any cell or and to site-specific quantification of ubiquitylation upon cellular identification of ubiquitylated peptides, as well as all data with this can be from and and all data from contains which all quantification data from The PDF contains of ubiquitylated peptides identified in this and is of the extensively studied post-translational modifications in The human about protein G. S. The protein of the human 2002; PubMed Scopus Google Scholar), tyrosine A. J. N. I. I. A. A. J. tyrosine in the human Full Text Full Text PDF PubMed Scopus Google Scholar), and Y. mechanism 2009; Full Text Full Text PDF PubMed Scopus Google Scholar). in human cells and are involved in ubiquitylation Z.J. in innate and 2009; 458: PubMed Scopus Google Scholar), and about 100 this modification Z.J. in innate and 2009; 458: PubMed Scopus Google Scholar, A. A.M. A and functional of Full Text Full Text PDF PubMed Scopus Google Scholar). The presence of this extensive ubiquitylation in human cells suggests that the complexity of protein regulation by ubiquitylation be to that of However, to the number of known ubiquitylation sites was in to the number of known sites. methods identification of ubiquitylation sites enrichment of ubiquitylated modified by ubiquitin. Trypsin digestion of these proteins generates a complex peptide mixture containing a small fraction of ubiquitylated this study we a robust and streamlined method to precisely map endogenous putative ubiquitylation sites in human Our approach provides several over methods for ubiquitylation site it only a single-step affinity enrichment of modified peptides, the enrichment of ubiquitylated peptides enables of low modification it proteome-wide analysis of endogenous ubiquitylation it can be to map ubiquitylation sites in any or and it is with site-specific quantification of ubiquitylation on a proteome-wide Using this approach we identified 11,054 putative endogenous ubiquitylation sites (diglycine-modified lysines) in human data over of the sites in previous However, more than of the sites in dataset been these data the number of known human ubiquitylation sites. study in of identified as the studied PTM We show that ubiquitylation proteins involved in all cellular functions and that its regulatory is to other PTMs as and The approach enables proteome-wide, analysis of ubiquitylation for the first Our analysis of changes in ubiquitylation following proteasome inhibition demonstrated that about half of all sites show increased a of proteins is by four hours of proteasome inhibition. remains sites that show increased ubiquitylation MG-132 are directly involved in proteasomal However, it is to that sites that show increased ubiquitylation upon proteasome inhibition nonproteasomal regulatory functions. The sites identified in this study as a for the functional of many proteins. The method is and can be to map ubiquitylation sites in any cell or and to site-specific quantification of ubiquitylation upon cellular identification of ubiquitylated peptides, as well as all data with this can be from and and all data from contains which all quantification data from The PDF contains of ubiquitylated peptides identified in this and with with