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

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 effectively crosslinked with deubiquitinases, E1 and E3s. In addition, compared to the classic Ub-VME and Ub-PA probes, Ub-AMA achieves improved 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.