Identification of E3 ligase substrates and PROTAC-induced ubiquitylation sites using proximity-based identification of ubiquitin sites (PrIUS).

The ubiquitin system regulates virtually all cellular processes, yet the vast majority of ubiquitylation sites identified in the human proteome cannot be attributed to specific E3 ligases. This knowledge gap hampers our understanding of ubiquitin signaling and the development of therapeutics leveraging the ubiquitylation system. Here we present Proximity-based Identification of Ubiquitin Sites (PrIUS), a versatile mass spectrometry-based approach combining proximity-dependent biotinylation (BioID) with ubiquitin remnant (diGly) enrichment. PrIUS enables simultaneous identification of E3 ligase interactors and direct mapping of substrate ubiquitylation sites within a single workflow. Using the E3 ligase NEDD4L, we demonstrated that E3 interactomes are inherently rich for diGly-modified peptides that remain largely undetectable without PTM-specific enrichment. PrIUS identified hundreds of high-confidence ubiquitylation sites, successfully distinguished substrates from non-substrate interactors (including adapters and E2 enzymes), and identified non-degradative ubiquitylation events. We also demonstrate utility for characterizing targeted protein degrader mechanisms by precisely mapping PROTAC-induced ubiquitylation sites. PrIUS thus provides a useful approach for elucidating E3-substrate relationships and characterizing mechanisms for emerging degrader therapeutics at amino acid resolution.