The growing interest in deubiquitinases (DUBs) as drug targets for modulating critical molecular pathways in disease is fuelled by the discovery of their specific cellular roles. A crucial aspect of this fact is the identification of DUB substrates. While mass spectrometry-based proteomic methods can be used to study global changes in cellular ubiquitination following DUB activity perturbation, these datasets often include indirect and downstream ubiquitination events. To enrich for the direct substrates of DUB enzymes, we have developed a proximal-ubiquitome workflow that combines proximity labeling methodology (ascorbate peroxidase-2 [APEX2]) with subsequent ubiquitination enrichment based on the K-ε-GG motif. We applied this technology to identify altered ubiquitination events in the vicinity of the DUB ubiquitin-specific protease 30(USP30)upon its inhibition. Our findings reveal ubiquitination events previously associated with USP30 on
TOMM20 and FKBP8, as well as the candidate substrate LETM1, which is deubiquitinated in a USP30-dependent manner. This 'proxy-ubiquitomics' workflow may potentially be useful also for the discovery of ubiquitin E3 ligase / PROTAC / molecular glue and DUB / DUBTAC substrates.