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Updated: Feb 24, 2026
Author Spotlight: Evaluating Biophysical Assays for Characterizing PROTACS Ternary Complexes
Published on: January 12, 2024
Unveiling BCL-xL-specific PROTAC efficiency and dissociation pathways using native mass spectrometry
Mohamed I Gadallah1,2, Kailyn L Nonhof1, Digant Nayak3
1Department of Chemistry, The University of Texas at Austin Austin TX 78712 USA jbrodbelt@cm.utexas.edu.
Abstract:
Overexpression of anti-apoptotic proteins such as BCL-xL is a hallmark of various cancers and a major driver of resistance to conventional chemotherapies. While small-molecule BCL-xL inhibitors have shown promising outcomes, their clinical use is hindered by dose-limiting toxicities, especially thrombocytopenia. Proteolysis-targeting chimeras (PROTACs) offer a promising alternative by promoting selective degradation of target proteins via 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.
Insights
This study uses native mass spectrometry (MS) to rapidly screen and characterize proteolysis-targeting chimeras (PROTACs) that degrade anti-apoptotic BCL-xL protein. Native MS effectively analyzes PROTACs, revealing insights into ternary complex formation and stability for cancer therapy development.
Area of Science:
- Biochemistry
- Chemical Biology
- Mass Spectrometry
Background:
- Overexpression of anti-apoptotic proteins like BCL-xL drives cancer and chemotherapy resistance.
- Small-molecule BCL-xL inhibitors face toxicity issues, particularly thrombocytopenia.
- Proteolysis-targeting chimeras (PROTACs) offer an alternative by degrading target proteins via the ubiquitin-proteasome system.
Purpose of the Study:
- To utilize native mass spectrometry (MS) as a platform for screening and characterizing PROTACs targeting BCL-xL.
- To investigate the formation, stability, and dissociation pathways of ternary complexes involving BCL-xL, PROTACs, and the VHL E3 ligase complex (VCB).
- To assess the utility of native MS techniques, including collision-induced dissociation (CID), ultraviolet photodissociation (UVPD), and variable-temperature electrospray ionization MS (vT-ESI-MS), for PROTAC analysis.
Main Methods:
- Native mass spectrometry (MS) was employed for label-free screening and characterization.
- Direct detection of binary (BCL-xL·PROTAC) and ternary (BCL-xL·PROTAC·VCB) complexes.
- Analysis of complex dissociation pathways using collision-induced dissociation (CID) and ultraviolet photodissociation (UVPD).
- Assessment of thermal stability in solution using variable-temperature ESI-MS (vT-ESI-MS).
Main Results:
- Native MS successfully detected and semi-quantified PROTAC binding affinities and cooperativity in both binary and ternary complexes.
- CID and UVPD revealed distinct fragmentation patterns, providing insights into the structural organization and gas-phase stability of the complexes.
- vT-ESI-MS allowed for the evaluation of the thermal stabilities of these complexes in solution.
- The study confirmed the effectiveness of native MS in analyzing PROTAC-induced ternary complex formation.
Conclusions:
- Native MS is a powerful, rapid, and label-free tool for screening and mechanistic characterization of PROTACs.
- This approach provides valuable insights into ternary complex formation and stability, crucial for PROTAC design and optimization.
- Native MS facilitates the development of novel PROTAC-based therapeutics for cancers driven by proteins like BCL-xL.
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