Journal
CELL CHEMICAL BIOLOGY
Volume 28, Issue 10, Pages 1528-+Publisher
CELL PRESS
DOI: 10.1016/j.chembiol.2021.05.005
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Funding
- National Institutes of Health [P41GM103422, R24GM136766]
- Bristol Myers Squibb Company
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By using charge reduction, native mass spectrometry, and gas-phase activation methods, we demonstrated an integrated approach for in-depth analysis of PROTAC-linked ternary complexes. This approach may help in screening for ternary complex formation and PROTAC-protein contacts, potentially reporting on PROTAC-induced protein-protein interactions known to be correlated with PROTAC selectivity and efficacy.
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.
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