Native mass spectrometry and gas-phase fragmentation provide rapid and in-depth topological characterization of a PROTAC ternary complex

Proteolysis-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 by recruiting an E3 ligase. Here, we show that charge reduction, native mass spectrometry, and 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 POI dissociation. Collision-induced dissociation (CID) causes elimination of the nonperipheral PROTAC, producing an intact VCB-POI complex not seen in solution but consistent with PROTAC-induced protein-protein interactions. In addition, we 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 and PROTAC-protein contacts and may report on PROTAC-induced protein-protein interactions, a characteristic correlated with PROTAC selectivity and efficacy. •Native MS yields structural analysis of a PROTAC-induced ternary complex•Multiple activation methods probe unique subunit dissociation pathways•Conformational analysis identifies two gas-phase PROTAC complex topologies•Subsequent subunit dissociation interrogates relevant protein-protein interactions Song et al. demonstrate the utility of native mass spectrometry, ion mobility spectrometry, and multiple activation methods to characterize PROTAC-induced complex topologies and conformation-specific protein-protein interactions.