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An improved SUMmOn-based methodology for the identification of ubiquitin and ubiquitin-like protein conjugation sites identifies novel ubiquitin-like protein chain linkages

2009; Wiley; Volume: 10; Issue: 2 Linguagem: Inglês

10.1002/pmic.200900648

1615-9861

Stanley M. Jeram, Tharan Srikumar, Xiang Dong Zhang, H. Anne Eisenhauer, Richard S. Rogers, Patrick G. A. Pedrioli, Michael J. Matunis, Brian Raught,

Autophagy in Disease and Therapy

PROTEOMICSVolume 10, Issue 2 p. 254-265 Research Article An improved SUMmOn-based methodology for the identification of ubiquitin and ubiquitin-like protein conjugation sites identifies novel ubiquitin-like protein chain linkages Stanley M. Jeram, Stanley M. Jeram Ontario Cancer Institute, and McLaughlin Centre for Molecular Medicine, Toronto, ON, CanadaSearch for more papers by this authorTharan Srikumar, Tharan Srikumar Ontario Cancer Institute, and McLaughlin Centre for Molecular Medicine, Toronto, ON, CanadaSearch for more papers by this authorXiang-Dong Zhang, Xiang-Dong Zhang Bloomberg School of Public Health, Department of Biochemistry and Molecular Biology, The Johns Hopkins University, Baltimore, MD, USASearch for more papers by this authorH. Anne Eisenhauer, H. Anne Eisenhauer Ontario Cancer Institute, and McLaughlin Centre for Molecular Medicine, Toronto, ON, CanadaSearch for more papers by this authorRichard Rogers, Richard Rogers Institute for Systems Biology, Seattle, WA, USASearch for more papers by this authorPatrick G. A. Pedrioli, Patrick G. A. Pedrioli Institute of Biochemistry, ETH Zurich, Zurich, SwitzerlandSearch for more papers by this authorMichael Matunis, Michael Matunis Bloomberg School of Public Health, Department of Biochemistry and Molecular Biology, The Johns Hopkins University, Baltimore, MD, USASearch for more papers by this authorBrian Raught, Corresponding Author Brian Raught [email protected] Ontario Cancer Institute, and McLaughlin Centre for Molecular Medicine, Toronto, ON, CanadaOntario Cancer Institute, and McLaughlin Centre for Molecular Medicine, 101 College St., MaRS TMDT 9-805, Toronto, ON, M5G 1L7, Canada Fax: +1-416-581-7629===Search for more papers by this author Stanley M. Jeram, Stanley M. Jeram Ontario Cancer Institute, and McLaughlin Centre for Molecular Medicine, Toronto, ON, CanadaSearch for more papers by this authorTharan Srikumar, Tharan Srikumar Ontario Cancer Institute, and McLaughlin Centre for Molecular Medicine, Toronto, ON, CanadaSearch for more papers by this authorXiang-Dong Zhang, Xiang-Dong Zhang Bloomberg School of Public Health, Department of Biochemistry and Molecular Biology, The Johns Hopkins University, Baltimore, MD, USASearch for more papers by this authorH. Anne Eisenhauer, H. Anne Eisenhauer Ontario Cancer Institute, and McLaughlin Centre for Molecular Medicine, Toronto, ON, CanadaSearch for more papers by this authorRichard Rogers, Richard Rogers Institute for Systems Biology, Seattle, WA, USASearch for more papers by this authorPatrick G. A. Pedrioli, Patrick G. A. Pedrioli Institute of Biochemistry, ETH Zurich, Zurich, SwitzerlandSearch for more papers by this authorMichael Matunis, Michael Matunis Bloomberg School of Public Health, Department of Biochemistry and Molecular Biology, The Johns Hopkins University, Baltimore, MD, USASearch for more papers by this authorBrian Raught, Corresponding Author Brian Raught [email protected] Ontario Cancer Institute, and McLaughlin Centre for Molecular Medicine, Toronto, ON, CanadaOntario Cancer Institute, and McLaughlin Centre for Molecular Medicine, 101 College St., MaRS TMDT 9-805, Toronto, ON, M5G 1L7, Canada Fax: +1-416-581-7629===Search for more papers by this author First published: 21 January 2010 https://doi.org/10.1002/pmic.200900648Citations: 25Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onEmailFacebookTwitterLinkedInRedditWechat Abstract Ubiquitin (Ub) and the ubiquitin-like proteins (Ubls) comprise a remarkable assortment of polypeptides that are covalently conjugated to target proteins (or other biomolecules) to modulate their intracellular localization, half-life, and/or activity. Identification of Ub/Ubl conjugation sites on a protein of interest can thus be extremely important for understanding how it is regulated. While MS has become a powerful tool for the study of many classes of PTMs, the identification of Ub/Ubl conjugation sites presents a number of unique challenges. Here, we present an improved Ub/Ubl conjugation site identification strategy, utilizing SUMmOn analysis and an additional protease (lysyl endopeptidase C), as a complement to standard approaches. As compared with standard trypsin proteolysis-database search protocols alone, the addition of SUMmOn analysis can (i) identify Ubl conjugation sites that are not detected by standard database searching methods, (ii) better preserve Ub/Ubl conjugate identity, and (iii) increase the number of identifications of Ub/Ubl modifications in lysine-rich protein regions. Using this methodology, we characterize for the first time a number of novel Ubl linkages and conjugation sites, including alternative yeast (K54) and mammalian small ubiquitin-related modifier (SUMO) chain (SUMO-2 K42, SUMO-3 K41) assemblies, as well as previously unreported NEDD8 chain (K27, K33, and K54) topologies. Supporting Information Detailed facts of importance to specialist readers are published as "Supporting Information". Such documents are peer-reviewed, but not copy-edited or typeset. They are made available as submitted by the authors. 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Proteomics 2010, 10, doi:10.1002/pmic.200900627, this issue. Citing Literature Volume10, Issue2No. 2 January 2010Pages 254-265 ReferencesRelatedInformation