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dc.contributor.authorBakos, Gen_US
dc.contributor.authorYu, Len_US
dc.contributor.authorGak, IAen_US
dc.contributor.authorRoumeliotis, TIen_US
dc.contributor.authorLiakopoulos, Den_US
dc.contributor.authorChoudhary, JSen_US
dc.contributor.authorMansfeld, Jen_US
dc.coverage.spatialEnglanden_US
dc.date.accessioned2019-02-20T07:48:43Z
dc.date.issued2018-11-14en_US
dc.identifierhttps://www.ncbi.nlm.nih.gov/pubmed/30429481en_US
dc.identifier10.1038/s41467-018-07251-5en_US
dc.identifier.citationNat Commun, 2018, 9 (1), pp. 4776 - ?en_US
dc.identifier.urihttps://repository.icr.ac.uk/handle/internal/3062
dc.identifier.eissn2041-1723en_US
dc.identifier.doi10.1038/s41467-018-07251-5en_US
dc.description.abstractCovalent modifications of proteins with ubiquitin and ubiquitin-like molecules are instrumental to many biological processes. However, identifying the E3 ligase responsible for these modifications remains a major bottleneck in ubiquitin research. Here, we present an E2-thioester-driven identification (E2~dID) method for the targeted identification of substrates of specific E2 and E3 enzyme pairs. E2~dID exploits the central position of E2-conjugating enzymes in the ubiquitination cascade and provides in vitro generated biotinylated E2~ubiquitin thioester conjugates as the sole source for ubiquitination in extracts. This enables purification and mass spectrometry-based identification of modified proteins under stringent conditions independently of the biological source of the extract. We demonstrate the sensitivity and specificity of E2-dID by identifying and validating substrates of APC/C in human cells. Finally, we perform E2~dID with SUMO in S. cerevisiae, showing that this approach can be easily adapted to other ubiquitin-like modifiers and experimental models.en_US
dc.format.extent4776 - ?en_US
dc.languageengen_US
dc.language.isoengen_US
dc.rights.urihttp://creativecommons.org/licenses/by/4.0/en_US
dc.subjectAnaphase-Promoting Complex-Cyclosomeen_US
dc.subjectCell Lineen_US
dc.subjectHeLa Cellsen_US
dc.subjectHumansen_US
dc.subjectSUMO-1 Proteinen_US
dc.subjectSaccharomyces cerevisiaeen_US
dc.subjectSaccharomyces cerevisiae Proteinsen_US
dc.subjectUbiquitinen_US
dc.subjectUbiquitin-Activating Enzymesen_US
dc.subjectUbiquitin-Conjugating Enzymesen_US
dc.subjectUbiquitin-Protein Ligasesen_US
dc.subjectUbiquitinsen_US
dc.titleAn E2-ubiquitin thioester-driven approach to identify substrates modified with ubiquitin and ubiquitin-like molecules.en_US
dc.typeJournal Article
dcterms.dateAccepted2018-10-10en_US
rioxxterms.versionofrecord10.1038/s41467-018-07251-5en_US
rioxxterms.licenseref.startdate2018-11-14en_US
rioxxterms.typeJournal Article/Reviewen_US
dc.relation.isPartOfNat Communen_US
pubs.issue1en_US
pubs.notesNot knownen_US
pubs.organisational-group/ICR
pubs.publication-statusPublished onlineen_US
pubs.volume9en_US
pubs.embargo.termsNot knownen_US
dc.contributor.icrauthorChoudhary, Jyotien_US


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