Profiling the Activity of Deubiquitinating Enzymes Using Chemically Synthesized Ubiquitin-Based Probes

Deubiquitinating enzymes (DUBs) are of interest as potential new targets for pharmacological intervention. Active-site-directed probes can be used for the accurate profiling of DUB activity as well as the identification of DUBs and DUB inhibitor selectivity. Previously, active-site directed DUB probes have been obtained using intein-based methods that have inherent limitations. Total chemical synthesis of ubiquitin allows for easy incorporation of different tags, such as fluorescent reporters, affinity tags, and cleavable linkers. Here, we describe the total chemical synthesis of a fluorescent active-site directed DUB probe, which facilitates fast, in-gel detection of active DUBs and circumvents the use of Western blot analysis. In addition, an in-gel activity-based DUB profiling assay is described in detail, in which the fluorescent DUB probe is used to visualize active DUBs in cell lysates. Finally, an inhibition assay is described in which the fluorescent probe is used to determine the specificity and potency of a small molecule DUB inhibitor.