Characterization of PROTAC specificity and endogenous protein interactomes using ProtacID

Here we describe ProtacID, a flexible BioID (proximity-dependent biotinylation)-based approach to identify PROTAC-proximal proteins in living cells. ProtacID analysis of VHL- and CRBN-recruiting PROTACs targeting a number of different proteins (localized to chromatin or cellular membranes, and tested across six different human cell lines) demonstrates how this technique can be used to validate PROTAC degradation targets and identify non-productive (i.e. non-degraded) PROTAC-interacting proteins, addressing a critical need in the field of PROTAC development. We also demonstrate that ProtacID can be used to characterize native, endogenous multiprotein complexes without the use of antibodies, or modification of the protein of interest with epitope tags or biotin ligase tagging. PROTAC development has surged in popularity, however our ability to characterize PROTAC specificity in living cells has lagged behind. Here, the authors develop ProtacID, a flexible proximity-dependent biotinylation (BioID)-based approach to identify PROTAC-protein interactions in living cells.