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

The ubiquitin (Ub) ligase E6AP, which is encoded by the UBE3A gene, has been associated with several human diseases including cervical cancer and Angelman syndrome, a neurodevelopmental disorder. Yet, our knowledge about disease-relevant substrates of E6AP is still limited. The formation of a thioester complex between Ub and the catalytic Cys residue of E6AP represents an essential intermediate step in E6AP-mediated ubiquitination. As potential substrates have to come into close proximity of the thioester bond to be ubiquitinated, we reasoned that a stable E6AP-Ub conjugate should represent a suitable affinity matrix for the identification of E6AP substrates. In addition, we employed a Ub variant equipped with a diazirine (Ub-DEA), as the resulting E6AP-Ub-DEA conjugate enables to covalently trap substrate proteins via photo-crosslinking (PCL). We validated the applicability of our approach in PCL-assisted affinity enrichment coupled to mass spectrometry (PCL-AE-MS) experiments. The results obtained indicate that PCL-AE-MS is indeed suited to identify substrates of E6AP and, presumably, of other enzymes of the Ub-conjugating system forming thioester complexes with Ub.