Systematic and Quantitative Assessment of the Ubiquitin-Modified Proteome
Despite the diverse biological pathways known to be regulated by ubiquitylation, global identification of substrates that are targeted for ubiquitylation has remained a challenge. To globally characterize the human ubiquitin-modified proteome (ubiquitinome), we utilized a monoclonal antibody that re...
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| Veröffentlicht in: | Molecular cell 2011-10, Vol.44 (2), p.325-340 |
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| creator | Kim, Woong Bennett, Eric J. Huttlin, Edward L. Guo, Ailan Li, Jing Possemato, Anthony Sowa, Mathew E. Rad, Ramin Rush, John Comb, Michael J. Harper, J. Wade Gygi, Steven P. |
| description | Despite the diverse biological pathways known to be regulated by ubiquitylation, global identification of substrates that are targeted for ubiquitylation has remained a challenge. To globally characterize the human ubiquitin-modified proteome (ubiquitinome), we utilized a monoclonal antibody that recognizes diglycine (diGly)-containing isopeptides following trypsin digestion. We identify ∼19,000 diGly-modified lysine residues within ∼5000 proteins. Using quantitative proteomics we monitored temporal changes in diGly site abundance in response to both proteasomal and translational inhibition, indicating both a dependence on ongoing translation to observe alterations in site abundance and distinct dynamics of individual modified lysines in response to proteasome inhibition. Further, we demonstrate that quantitative diGly proteomics can be utilized to identify substrates for cullin-RING ubiquitin ligases. Interrogation of the ubiquitinome allows for not only a quantitative assessment of alterations in protein homeostasis fidelity, but also identification of substrates for individual ubiquitin pathway enzymes.
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► α-diGly was used to identify/quantify 19,000 ubiquitylation sites in 5,000 proteins ► Kinetics of ubiquitylation allows for classification of distinct substrate types ► Ubiquitinome formation largely requires ongoing protein synthesis ► diGly proteomics can be used to identify cullin-RING ligase substrates |
| doi_str_mv | 10.1016/j.molcel.2011.08.025 |
| format | Article |
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► α-diGly was used to identify/quantify 19,000 ubiquitylation sites in 5,000 proteins ► Kinetics of ubiquitylation allows for classification of distinct substrate types ► Ubiquitinome formation largely requires ongoing protein synthesis ► diGly proteomics can be used to identify cullin-RING ligase substrates</description><identifier>ISSN: 1097-2765</identifier><identifier>EISSN: 1097-4164</identifier><identifier>DOI: 10.1016/j.molcel.2011.08.025</identifier><identifier>PMID: 21906983</identifier><language>eng</language><publisher>United States: Elsevier Inc</publisher><subject>Cells, Cultured ; Cullin Proteins - metabolism ; digestion ; Glycylglycine - genetics ; HCT116 Cells ; homeostasis ; Humans ; inhibition ; lysine ; Lysine - genetics ; monoclonal antibodies ; proteasome endopeptidase complex ; proteome ; Proteome - metabolism ; Proteomics ; temporal variation ; translation (genetics) ; trypsin ; ubiquitin ; Ubiquitin - metabolism ; ubiquitin-protein ligase ; Ubiquitination</subject><ispartof>Molecular cell, 2011-10, Vol.44 (2), p.325-340</ispartof><rights>2011 Elsevier Inc.</rights><rights>Copyright © 2011 Elsevier Inc. All rights reserved.</rights><rights>2011 Elsevier Inc. All rights reserved. 2011</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c585t-756aeaf75e30f2b326c013ea78a8c06bbf18a98aa66298a14d9f1d9b619715a3</citedby><cites>FETCH-LOGICAL-c585t-756aeaf75e30f2b326c013ea78a8c06bbf18a98aa66298a14d9f1d9b619715a3</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktohtml>$$Uhttps://www.sciencedirect.com/science/article/pii/S1097276511006757$$EHTML$$P50$$Gelsevier$$Hfree_for_read</linktohtml><link.rule.ids>230,314,776,780,881,3537,27901,27902,65306</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/21906983$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Kim, Woong</creatorcontrib><creatorcontrib>Bennett, Eric J.</creatorcontrib><creatorcontrib>Huttlin, Edward L.</creatorcontrib><creatorcontrib>Guo, Ailan</creatorcontrib><creatorcontrib>Li, Jing</creatorcontrib><creatorcontrib>Possemato, Anthony</creatorcontrib><creatorcontrib>Sowa, Mathew E.</creatorcontrib><creatorcontrib>Rad, Ramin</creatorcontrib><creatorcontrib>Rush, John</creatorcontrib><creatorcontrib>Comb, Michael J.</creatorcontrib><creatorcontrib>Harper, J. Wade</creatorcontrib><creatorcontrib>Gygi, Steven P.</creatorcontrib><title>Systematic and Quantitative Assessment of the Ubiquitin-Modified Proteome</title><title>Molecular cell</title><addtitle>Mol Cell</addtitle><description>Despite the diverse biological pathways known to be regulated by ubiquitylation, global identification of substrates that are targeted for ubiquitylation has remained a challenge. To globally characterize the human ubiquitin-modified proteome (ubiquitinome), we utilized a monoclonal antibody that recognizes diglycine (diGly)-containing isopeptides following trypsin digestion. We identify ∼19,000 diGly-modified lysine residues within ∼5000 proteins. Using quantitative proteomics we monitored temporal changes in diGly site abundance in response to both proteasomal and translational inhibition, indicating both a dependence on ongoing translation to observe alterations in site abundance and distinct dynamics of individual modified lysines in response to proteasome inhibition. Further, we demonstrate that quantitative diGly proteomics can be utilized to identify substrates for cullin-RING ubiquitin ligases. Interrogation of the ubiquitinome allows for not only a quantitative assessment of alterations in protein homeostasis fidelity, but also identification of substrates for individual ubiquitin pathway enzymes.
[Display omitted]
► α-diGly was used to identify/quantify 19,000 ubiquitylation sites in 5,000 proteins ► Kinetics of ubiquitylation allows for classification of distinct substrate types ► Ubiquitinome formation largely requires ongoing protein synthesis ► diGly proteomics can be used to identify cullin-RING ligase substrates</description><subject>Cells, Cultured</subject><subject>Cullin Proteins - metabolism</subject><subject>digestion</subject><subject>Glycylglycine - genetics</subject><subject>HCT116 Cells</subject><subject>homeostasis</subject><subject>Humans</subject><subject>inhibition</subject><subject>lysine</subject><subject>Lysine - genetics</subject><subject>monoclonal antibodies</subject><subject>proteasome endopeptidase complex</subject><subject>proteome</subject><subject>Proteome - metabolism</subject><subject>Proteomics</subject><subject>temporal variation</subject><subject>translation (genetics)</subject><subject>trypsin</subject><subject>ubiquitin</subject><subject>Ubiquitin - metabolism</subject><subject>ubiquitin-protein ligase</subject><subject>Ubiquitination</subject><issn>1097-2765</issn><issn>1097-4164</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2011</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNp9kc1u1TAQhSMEoqXwBgiyK5uEGSd27A1SVfFTqQhQ27XlOJPWV0nc2s6V-va4upcCm67Glr85czynKN4i1AgoPm7q2U-WppoBYg2yBsafFYcIqqtaFO3z_Zl1gh8Ur2LcAGDLpXpZHDBUIJRsDouzi_uYaDbJ2dIsQ_lrNUtyKd-3VJ7ESDHOtKTSj2W6ofKqd3erS26pvvvBjY6G8mfwifxMr4sXo5kivdnXo-Lyy-fL02_V-Y-vZ6cn55Xlkqeq48KQGTtODYysb5iwgA2ZThppQfT9iNIoaYwQLBdsBzXioHqBqkNumqPi0072du1nGmw2F8ykb4ObTbjX3jj9_8vibvS13-qGAbSsywLHe4Hg71aKSc8u5j1OZiG_Rq0ARCObFjP54UkyCwKwBjuR0XaH2uBjDDQ-GkLQD3Hpjd7FpR_i0iB1jiu3vfv3M49Nf_LJwPsdMBqvzXVwUV9dZAWRJyNvJf-7D8o73zoKOlpHi6XBBbJJD9497eE3fkGyjg</recordid><startdate>20111021</startdate><enddate>20111021</enddate><creator>Kim, Woong</creator><creator>Bennett, Eric J.</creator><creator>Huttlin, Edward L.</creator><creator>Guo, Ailan</creator><creator>Li, Jing</creator><creator>Possemato, Anthony</creator><creator>Sowa, Mathew E.</creator><creator>Rad, Ramin</creator><creator>Rush, John</creator><creator>Comb, Michael J.</creator><creator>Harper, J. Wade</creator><creator>Gygi, Steven P.</creator><general>Elsevier Inc</general><scope>6I.</scope><scope>AAFTH</scope><scope>FBQ</scope><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7S9</scope><scope>L.6</scope><scope>7X8</scope><scope>5PM</scope></search><sort><creationdate>20111021</creationdate><title>Systematic and Quantitative Assessment of the Ubiquitin-Modified Proteome</title><author>Kim, Woong ; Bennett, Eric J. ; Huttlin, Edward L. ; Guo, Ailan ; Li, Jing ; Possemato, Anthony ; Sowa, Mathew E. ; Rad, Ramin ; Rush, John ; Comb, Michael J. ; Harper, J. Wade ; Gygi, Steven P.</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c585t-756aeaf75e30f2b326c013ea78a8c06bbf18a98aa66298a14d9f1d9b619715a3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2011</creationdate><topic>Cells, Cultured</topic><topic>Cullin Proteins - metabolism</topic><topic>digestion</topic><topic>Glycylglycine - genetics</topic><topic>HCT116 Cells</topic><topic>homeostasis</topic><topic>Humans</topic><topic>inhibition</topic><topic>lysine</topic><topic>Lysine - genetics</topic><topic>monoclonal antibodies</topic><topic>proteasome endopeptidase complex</topic><topic>proteome</topic><topic>Proteome - metabolism</topic><topic>Proteomics</topic><topic>temporal variation</topic><topic>translation (genetics)</topic><topic>trypsin</topic><topic>ubiquitin</topic><topic>Ubiquitin - metabolism</topic><topic>ubiquitin-protein ligase</topic><topic>Ubiquitination</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Kim, Woong</creatorcontrib><creatorcontrib>Bennett, Eric J.</creatorcontrib><creatorcontrib>Huttlin, Edward L.</creatorcontrib><creatorcontrib>Guo, Ailan</creatorcontrib><creatorcontrib>Li, Jing</creatorcontrib><creatorcontrib>Possemato, Anthony</creatorcontrib><creatorcontrib>Sowa, Mathew E.</creatorcontrib><creatorcontrib>Rad, Ramin</creatorcontrib><creatorcontrib>Rush, John</creatorcontrib><creatorcontrib>Comb, Michael J.</creatorcontrib><creatorcontrib>Harper, J. Wade</creatorcontrib><creatorcontrib>Gygi, Steven P.</creatorcontrib><collection>ScienceDirect Open Access Titles</collection><collection>Elsevier:ScienceDirect:Open Access</collection><collection>AGRIS</collection><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>AGRICOLA</collection><collection>AGRICOLA - Academic</collection><collection>MEDLINE - Academic</collection><collection>PubMed Central (Full Participant titles)</collection><jtitle>Molecular cell</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Kim, Woong</au><au>Bennett, Eric J.</au><au>Huttlin, Edward L.</au><au>Guo, Ailan</au><au>Li, Jing</au><au>Possemato, Anthony</au><au>Sowa, Mathew E.</au><au>Rad, Ramin</au><au>Rush, John</au><au>Comb, Michael J.</au><au>Harper, J. Wade</au><au>Gygi, Steven P.</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Systematic and Quantitative Assessment of the Ubiquitin-Modified Proteome</atitle><jtitle>Molecular cell</jtitle><addtitle>Mol Cell</addtitle><date>2011-10-21</date><risdate>2011</risdate><volume>44</volume><issue>2</issue><spage>325</spage><epage>340</epage><pages>325-340</pages><issn>1097-2765</issn><eissn>1097-4164</eissn><abstract>Despite the diverse biological pathways known to be regulated by ubiquitylation, global identification of substrates that are targeted for ubiquitylation has remained a challenge. To globally characterize the human ubiquitin-modified proteome (ubiquitinome), we utilized a monoclonal antibody that recognizes diglycine (diGly)-containing isopeptides following trypsin digestion. We identify ∼19,000 diGly-modified lysine residues within ∼5000 proteins. Using quantitative proteomics we monitored temporal changes in diGly site abundance in response to both proteasomal and translational inhibition, indicating both a dependence on ongoing translation to observe alterations in site abundance and distinct dynamics of individual modified lysines in response to proteasome inhibition. Further, we demonstrate that quantitative diGly proteomics can be utilized to identify substrates for cullin-RING ubiquitin ligases. Interrogation of the ubiquitinome allows for not only a quantitative assessment of alterations in protein homeostasis fidelity, but also identification of substrates for individual ubiquitin pathway enzymes.
[Display omitted]
► α-diGly was used to identify/quantify 19,000 ubiquitylation sites in 5,000 proteins ► Kinetics of ubiquitylation allows for classification of distinct substrate types ► Ubiquitinome formation largely requires ongoing protein synthesis ► diGly proteomics can be used to identify cullin-RING ligase substrates</abstract><cop>United States</cop><pub>Elsevier Inc</pub><pmid>21906983</pmid><doi>10.1016/j.molcel.2011.08.025</doi><tpages>16</tpages><oa>free_for_read</oa></addata></record> |
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| subjects | Cells, Cultured Cullin Proteins - metabolism digestion Glycylglycine - genetics HCT116 Cells homeostasis Humans inhibition lysine Lysine - genetics monoclonal antibodies proteasome endopeptidase complex proteome Proteome - metabolism Proteomics temporal variation translation (genetics) trypsin ubiquitin Ubiquitin - metabolism ubiquitin-protein ligase Ubiquitination |
| title | Systematic and Quantitative Assessment of the Ubiquitin-Modified Proteome |
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