Unveiling BCL-xL-specific PROTAC efficiency and dissociation pathways using native mass spectrometry

the ubiquitin-proteasome system, thereby reducing off-target effects associated with small molecule inhibitors. However, rational design and optimization of PROTACs remain challenging due to the need to balance simultaneous interactions with both an E3 ubiquitin ligase and the target protein. Here we employ native mass spectrometry (MS) as a rapid, label-free platform to screen and characterize the formation and stability of ternary complexes between BCL-xL, VHL E3 ligase complex (VCB), and various targeting PROTACs. Native MS enables direct detection of binary BCL-xL·PROTAC and ternary BCL-xL·PROTAC·VCB complexes and provides semi-quantitative insights into PROTAC affinity and cooperativity with both binding partners. Furthermore, we explore the dissociation pathways of these complexes in the gas phase using collision-induced dissociation (CID) and ultraviolet photodissociation (UVPD), revealing distinct fragmentation and subunit release patterns that reflect the structural organization and gas-phase stability of the complexes. Variable-temperature ESI-MS (vT-ESI) further allows assessment of thermal stabilities of the complexes in solution. Together, our study demonstrates the power of native MS to both screen and mechanistically characterize PROTAC-induced ternary complex formation.