Ubiquitin signalling relies on E3 ligases, the architects of the system, to build ubiquitin chains that specify protein fate, for example proteasomal turnover. Progress in E3 biology has been hindered by overlapping lists, inconsistent naming, and uneven annotation across datasets. To reset and unite the E3 landscape we assembled the E3-ome, a harmonised compendium of 670 human E3 ligases with unified nomenclature and expert curation. This community-driven resource integrates published and unpublished observations, experimental datasets, bioinformatics analyses, and literature review, enabling hypothesis driven testing across screening, degrader design, and biological discovery.
Using the E3-ome as a map, we built a reporter based screen to identify E3 ligases that enhance targeted protein degradation, and paired it with a quantitative, branch aware proteomics workflow. By using a combination of Ub clipping and AQUA mass spectrometry, we resolved PROTAC-induced branched K48, K29 architectures on a model substrate in cells, and uncovered an E3 that cooperates with CUL4 and CUL2 PROTACs to boost neo-substrate degradation. These data link ligase identity to chain architecture and degradation output, and support branched chain regulation as a tunable axis for selective protein degradation. Together, the E3-ome and our branch aware workflow provide a foundation for community use, offering a unified map and a measurement toolkit to decode and steer ubiquitin signalling.