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However, the preparation of existing activity\u2010based E2\u2010Ub probes depends on recombination technology and bioconjugation chemistry, limiting their structural diversity. Herein we describe an expedient total chemical synthesis of an E2 enzyme variant through a hydrazide\u2010based native chemical ligation, which enabled the construction of a structurally new activity\u2010based E2\u2010Ub probe to covalently capture the catalytic site of Cys\u2010dependent E3s. Chemical cross\u2010linking coupled with mass spectrometry (CXMS) demonstrated the utility of this new probe in structural analysis of the intermediates formed during Nedd4 and Parkin\u2010mediated transthiolation. This study exemplifies the utility of chemical protein synthesis for the development of protein probes for biological studies.<\/jats:p>","DOI":"10.1002\/anie.202105870","type":"journal-article","created":{"date-parts":[[2021,5,22]],"date-time":"2021-05-22T12:30:17Z","timestamp":1621686617000},"page":"17171-17177","update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":86,"title":["Chemical Synthesis of Activity\u2010Based E2\u2010Ubiquitin Probes for the Structural Analysis of E3 Ligase\u2010Catalyzed Transthiolation"],"prefix":"10.1002","volume":"60","author":[{"given":"Lu\u2010Jun","family":"Liang","sequence":"first","affiliation":[{"name":"Tsinghua-Peking Center for Life Sciences Ministry of Education Key Laboratory of Bioorganic Phosphorus Chemistry and Chemical Biology Center for Synthetic and Systems Biology Department of Chemistry Tsinghua University  Beijing 100084 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