Proteomic Profiling of Potential E6AP Substrates via Ubiquitin-based Photo-Crosslinking Assisted Affinity Enrichment

The ubiquitin (Ub) ligase E6AP, encoded by the UBE3A gene, has been causally associated with human diseases including cervical cancer and Angelman syndrome, a neurodevelopmental disorder. Yet, our knowledge about disease-relevant substrates of E6AP is still limited, presumably because at least some of these interactions are rather transient, a phenomenon observed for many enzyme-substrate interactions. Here, we introduce a novel approach to trap such potential transient interactions by combining a stable E6AP-Ub conjugate mimicking the active state of this enzyme with photo-crosslinking (PCL) followed by affinity enrichment coupled to mass spectrometry (AE-MS). To enable PCL, we equipped Ub with diazirine moieties at distinct positions. We validated our PCL assisted AE-MS approach by identification of known (e. g. PSMD4, UCHL5) and potential new (e. g. MSH2) substrates of E6AP. Our findings suggest that PCL assisted AE-MS is indeed suited to identify substrates of E6AP, thereby providing insights into E6AP-associated pathologies, and, potentially, of other enzymes of the Ub-conjugating system.