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Activity-Based Ubiquitin-α-Amidomethyl Acrylate Probe for Profiling of Ubiquitinating Enzymes and Deubiquitinases

ABSTRACT: Activity-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.

ORGANISM(S): Homo Sapiens

SUBMITTER: Xu Li

PROVIDER: PXD080401 | iProX | Tue Jun 30 00:00:00 BST 2026

REPOSITORIES: iProX

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