Updated publication reference for PubMed record(s): 29263404. E3 ubiquitin (UB) ligases, the ending module of the E1-E2-E3 cascades, transmit diverse signals in the cell by attaching UB to cellular proteins. Identifying E3 substrates holds the key to elucidate the roles of E3s in cell regulation. We constructed an orthogonal UB transfer (OUT) cascade to identify the substrates of E6AP, a HECT E3 also known as Ube3a that is implicated in neurodevelopmental disorders and cancer. We used yeast cell surface display to engineer E6AP to exclusively transfer an affinity-tagged UB variant (xUB) to its substrate proteins. Proteomic identification of xUB-conjugated proteins in HEK293 cells afforded 131 potential E6AP targets. Among them we verified MAPK1, CDK1 and CDK4, and PRMT5 are directly ubiquitinated by E6AP in vitro and in the cell. Our work establishes OUT as an efficient platform to profile E6AP substrates and would guide the engineering of OUT cascades with other E3s to interrogate their biological functions.