Activity-based E2 conjugating enzyme (E2)-ubiquitin (Ub) probes have recently emerged as effective tools for studying the molecular mechanism of E3 ligase (E3)-catalyzed ubiquitination. However,the preparation of existing activitybased E2-Ub 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 ahydrazide-based native chemical ligation,whichenabledtheconstruction of astructurally new activity-based E2-Ub probe to covalentlycapture the catalytic site of Cys-dependent E3s.Chemical cross-linking coupled with mass spectrometry (CXMS) demonstrated the utility of this new probe in structural analysis of the intermediates formed during Nedd4 and Parkin-mediated transthiolation.Thisstudyexemplifiestheutilityofchemicalprotein synthesis for the development of protein probes for biological studies.