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

Abstract

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.