An E2-ubiquitin thioester-driven approach to identify substrates modified with ubiquitin and ubiquitin-like molecules

Covalent modifications of proteins with ubiquitin and ubiquitin-like molecules are instrumental to many biological processes. However, identifying the E3 ligase responsible for these modifications remains a major bottleneck in ubiquitin research. Here, we present an E2-thioester-driven identification (E2 similar to dID) method for the targeted identification of substrates of specific E2 and E3 enzyme pairs. E2 similar to dID exploits the central position of E2-conjugating enzymes in the ubiquitination cascade and provides in vitro generated biotinylated E2 similar to ubiquitin thioester conjugates as the sole source for ubiquitination in extracts. This enables purification and mass spectrometry-based identification of modified proteins under stringent conditions independently of the biological source of the extract. We demonstrate the sensitivity and specificity of E2-dID by identifying and validating substrates of APC/C in human cells. Finally, we perform E2 similar to dID with SUMO in S. cerevisiae, showing that this approach can be easily adapted to other ubiquitin-like modifiers and experimental models.