A cell-permeable Ub-Dha probe for profiling E1-E2-E3 enzymes in live cells

Activity-based ubiquitin probes (Ub-ABPs) have recently been developed as effective tools for studying the capabilities of E1-E2-E3 enzymes, but most of them can only be used in cell lysates. Here, we report the first cell-penetrating Ub-Dha probes based on thiazolidine-protected cysteines, which enable successful delivery into cells confirmed by a fluorophore at the N-terminus of Ub and live-cell fluorescence microscopy. A total of 18 E1-E2-E3 enzymes in live cells were labelled and enriched in combination with label-free quantification (LFQ) mass spectrometry. This work provided a new cell-penetrating Ub tool for studying the activity and function of Ub-related enzymes. We report the first cell-penetrating Ub-Dha probes based on thiazolidine-protected cysteines, which were successfully used to label and enrich the E1-E2-E3 enzymes in live cells in combination with label-free quantification (LFQ) mass spectrometry.