Native Mass Spectrometry and Gas-Phase Fragmentation Provide Rapid and In-Depth Topological Characterization of a PROTAC Ternary Complex

SummaryProteolysis targeting chimeras (PROTACs) represent a new direction in small-molecule therapeutics whereby a heterobifunctional linker to a protein-of-interest (POI) induces its ubiquitination-based proteolysis via recruiting an E3 ligase. We show here that native mass spectrometry and associated gas-phase activation methods combine for an in-depth analysis of a PROTAC-linked ternary complex. Electron-capture dissociation (ECD) of the intact POI-PROTAC-VCB complex (a trimeric subunit of an E3 ubiquitin ligase) promotes significant PROTAC ejection in competition with minor POI dissociation. Collision-induced dissociation (CID) causes elimination of the non-peripheral PROTAC, producing an intact VCB-POI complex not seen in solution but consistent with PROTAC-induced protein-protein interactions. We further used ion mobility spectrometry (IMS) and collisional activation to identify the source of this unexpected dissociation. Together, the evidence shows that this integrated approach can be used to screen for ternary complex formation, PROTAC-protein contacts, and PROTAC-induced protein-protein interactions, a characteristic correlated with PROTAC selectivity and efficacy.