Integrative Proximal-Ubiquitomics Profiling for Deubiquitinase and E3 Ligase Substrate Discovery Applied to USP30

Increasing interest in deubiquitinases (DUBs) and ubiquitin E3 ligases as drug targets to modulate critical molecular pathways in disease is driven by the discovery of specific cellular roles of these enzymes. Key to this is the identification of DUB or E3 ligase substrates. While global cellular ubiquitination changes upon perturbation of DUB/E3 ligase activity can be studied using mass spectrometry-based proteomic methods, these datasets include indirect and downstream ubiquitination events. To enrich for direct substrates of DUB/E3 ligase enzymes, we have combined proximity-labelling methodology (APEX2) and subsequent ubiquitination enrichment (based on the K-ε-GG motif) to form a proximal-ubiquitome workflow. We have applied this technology to identify altered ubiquitination events in the proximity of the DUB ubiquitin specific protease 30 (USP30) upon its inhibition. We show ubiquitination events previously linked to USP30 on TOMM20 and FKBP8 and the previously undescribed candidate substrate LETM1, which is deubiquitinated in a USP30-dependent manner.