iProXProteomicsXu LiHomo Sapienshttp://www.iprox.org/page/project.html?id=IPX0018073000lixuqd@qdu.edu.cnQingdao UniversityiProXfalseActivity-Based Ubiquitin-α-Amidomethyl Acrylate Probe for Profiling of Ubiquitinating Enzymes and DeubiquitinasesActivity-based ubiquitin probes have emerged as powerful tools for dissecting protein ubiquitination. In this work, we discovered that an α-amidomethyl substituted acrylate electrophile exhibits markedly enhanced reactivity. Based on this finding, we designed and synthesized a new activity-based probe, Ubiquitin-α-Amidomethyl Acrylate (Ub-AMA). In vitro biochemical assays demonstrated that Ub-AMA exhibits broad crosslinking capability across the entire ubiquitin regulatory enzyme system—including E1, E2, and E3 enzymes as well as DUBs. Compared to the classic Ub-VME probe, Ub-AMA achieves significantly higher labeling efficiency for E2 enzymes. Finally, proteomic profiling in cell lysates confirmed that Ub-AMA can capture E1, E2, and E3 enzymes as well as DUBs in a complex biological environment. In summary, the Ub-AMA probe developed here provides a new tool for in depth analysis of the dynamic regulatory mechanisms of ubiquitination. Moreover, this work offers valuable insights for the design of other activity based probes based on α,β-unsaturated carbonyl scaffolds.Tue Jun 30 00:00:00 GMT+01:00 2026PXD0804019606