Identifying E3 Ligase Substrates With Quantitative Degradation Proteomics

Abstract Controlled protein degradation by the ubiquitin‐proteasome pathway is critical for almost all cellular processes. E3 ubiquitin ligases are responsible for targeting proteins for ubiquitylation and subsequent proteasomal degradation with spatial and temporal precision. While studies have revealed various E3‐substrate pairs involved in distinct biological processes, the complete substrate profiles of individual E3 ligases are largely unknown. Here we report a new approach to identify substrates of an E3 ligase for proteasomal degradation using unnatural amino acid incorporation pulse‐chase proteomics (degradomics). Applying this approach, we determine the steady‐state substrates of the C ‐ t erminal to L is H (CTLH) E3 ligase, a multi‐component complex with poorly defined substrates. By comparing the proteome degradation profiles of active and inactive CTLH‐expressing cells, we successfully identify previously known and new potential substrates of CTLH ligase. Altogether, degradomics can comprehensively identify degradation substrates of an E3 ligase, which can be adapted for other E3 ligases in various cellular contexts.