A guide to mapping ubiquitin and ubiquitin-like E3 ligases to their substrates

Ubiquitination is a post-translational modification that plays a key role in the maintenance of protein homeostasis. Ubiquitin is covalently attached to the target proteins through a three-step enzymatic cascade in which substrate specificity is conferred by the E3 ligases. However, to match more than 600 E3s with their specific substrates is one of the major challenges in the field. The dynamic and reversible nature of ubiquitination requires the development of techniques to systematically address this question. Here we provide a comprehensive overview of the current methodologies used to reveal targets of E3 ligases, discussing their strengths and limitations. This is particularly relevant in light of emerging pharmacological strategies for targeted protein degradation.