Multi-dimensional high-throughput molecular glue screening via gas phase affinity selection native mass spectrometry and cryo-EM analysis

Significance of the topic

Targeted protein degradation via molecular glues (MGs) is an emerging modality that expands drug discovery into targets previously considered undruggable by stabilizing weak interactions between a target protein and an E3 ubiquitin ligase. Rapid, high-confidence identification and structural validation of MGs is critical to accelerate hit discovery and progression. Native mass spectrometry (native MS) combined with cryo-electron microscopy (cryo-EM) offers a route to detect intact ternary complexes and obtain structural context, but conventional workflows suffer from low throughput and loss of weak binders during chromatography and sample handling. This study demonstrates an integrated, high-throughput platform that couples online buffer exchange native MS, gas-phase affinity selection, and cryo-EM to detect, identify and structurally validate MG-mediated ternary complexes, using WEE1 binding to the CRBN-DDB1 E3 ligase as a model system.

Objectives and overview of the study

Ulterior aims of the work were to (1) increase native-MS screening throughput for MG discovery, (2) enable direct identification of bound small molecules to E3–MG–target complexes despite weak affinities, and (3) provide orthogonal structural characterization by cryo-EM to support MS-based assignments. The study compares single-compound and multiplexed screening strategies, implements a gas-phase affinity selection and fragmentation workflow to pinpoint bound ligands, and demonstrates the approach on a 96-compound screen for WEE1 engagement with CRBN-DDB1.

Methods and experimental workflow

Key experimental elements and workflow steps:

• Protein and compound preparation: CRBN-DDB1 and WEE1 stocks were mixed at a 1:1 volume ratio (approximately 1:2 molar ratio). Typical concentrations used: CRBN-DDB1 ~3.3 μM, WEE1 ~6.6 μM. Compounds were supplied as 10 mM stocks and diluted into 200 mM ammonium acetate (AmAc) to working concentrations (~5 μM per compound).
• Online buffer exchange (OBE): Rapid buffer exchange to native-friendly AmAc was used to minimize deleterious effects of non-volatile components and permit native MS of intact complexes. Reported OBE time was ~0.7 min per run.
• Screening formats: Single-compound-per-well (96 wells → 96 injections) and multiplexed (4 compounds/well → 24 injections) formats were tested. Incubation regimes evaluated included 25 °C for 1 hour and a rapid 25 °C for 5 minutes; results were comparable, indicating fast complex formation for many hits.
• Gas-phase affinity selection MS workflow: Full injection into the Orbitrap mass analyzer followed by quadrupole (Q1) isolation of the target complex; dissociation of the complex in a higher-energy collision dissociation stage (FHCD); detection of freed ligands and dissociated complex in the Orbitrap; and subsequent fragmentation of released ligands in the ion trap (IT) for structural information.
• Instrumentation used: Thermo Scientific Orbitrap Ascend Structural Biology Tribrid mass spectrometer supporting native MS, top-down workflows, and multiple fragmentation modalities (HCD/CID/ETD/UVPD/PTCR), and ProSight Native software for data analysis. Autosampler, fraction collector and easy-spray interfaces were integrated for sample handling and injection automation.

Used instrumentation

• Thermo Scientific Orbitrap Ascend Structural Biology Tribrid mass spectrometer (native MS-capable; quadrupole isolation to m/z ~8,000; detection to m/z 16,000).
• Automated online buffer exchange system with autosampler and fraction collector to enable rapid OBE and injection cycles.
• ProSight Native software for deconvolution and native MS data processing.
• Cryo-electron microscopy (cryo-EM) employed as an orthogonal method for structural characterization of ligand-bound complexes.

Main results and discussion

Principal findings and observations:

• Throughput: The integrated LC-native MS workflow (OBE ~0.7 min, MS analysis ~1.5 min per run) enables very high throughput screening; the authors estimate capacity on the order of thousands of compounds per day (reported >2,500 compounds/day under optimized conditions).
• Hit identification: In a 96-compound single-per-well screen for compounds that promote WEE1 binding to CRBN-DDB1, 16 putative MG hits were identified. Hits were reproducible between longer (1 h) and rapid (5 min) incubation conditions, consistent with rapid ternary-complex formation for many compounds.
• Multiplexing trade-offs: Multiplexing (4 compounds per well) reduces required injections (24 vs 96) and thereby increases throughput, but compound competition and ambiguity in assigning the bound ligand can arise. To avoid stoichiometric competition and ensure excess of each compound over protein, the authors primarily used one compound per well for confident assignment in the reported dataset.
• Ambiguity and identification strategy: Mass similarity among compounds, adduct formation and cofactors can create ambiguity in deconvoluted mass shifts. The gas-phase affinity selection workflow addresses this by isolating the intact ternary complex in Q1, inducing ligand release by collisional dissociation in FHCD, detecting freed ligands in the Orbitrap, and then fragmenting those ligands in the ion trap to obtain structural information for unambiguous identification.
• Cryo-EM corroboration: Selected MS-identified ligand–E3–target complexes were characterized by cryo-EM to validate binding modes and support MG discovery decisions.

Benefits and practical applications of the method

Advantages demonstrated by this integrated approach include:

• Direct detection of intact E3–ligand–target ternary complexes by native MS, avoiding false negatives from on-column dissociation seen in traditional affinity-selection LC–MS.
• High throughput enabled by rapid OBE and short MS run times, permitting screening at scales relevant to discovery-phase chemistry triage.
• Ability to identify weak binders through post-column and gas-phase workflows that preserve or reveal otherwise labile interactions.
• Structural validation via cryo-EM provides orthogonal evidence for binding and can inform structure-guided optimization.
• Gas-phase MS/MS of released ligands allows identification of unknown or ambiguous binders without relying solely on retention time or intact-mass shifts.

Limitations and considerations

Key limitations and practical caveats noted or implied by the study:

• Non-volatile components in screening matrices prevent direct native MS and necessitate rapid buffer exchange; incomplete removal or insufficient exchange may destabilize weak interactions.
• Multiplexing increases throughput but can create ligand competition and complicate identification when masses are similar or adducted; dedicated single-compound wells improve confidence.
• Gas-phase ligand release is a powerful tool but may not perfectly recapitulate solution-phase affinities; orthogonal validation (e.g., cryo-EM, biochemical assays, cellular degradation assays) remains necessary.
• Identification relies on sufficient mass difference and informative MS/MS fragments; isomeric ligands or identical nominal masses remain challenging.

Future trends and applications

Potential developments and broader applications that follow from this work:

• Automation and scale-up: Further integration with robotics and plate-handling systems could push throughput higher and make native-MS MG screens routine in discovery pipelines.
• Improved data analysis: Advanced deconvolution, database-driven MS/MS matching, and machine-learning approaches could reduce ambiguity in ligand assignment and support robust multiplexing.
• Broader target scope: Extension of the platform to additional E3 ligases and diverse target classes will increase its impact across targeted protein degradation programs.
• Library design: Creating MS-friendly screening libraries (avoiding highly non-volatile salts or labile chemotypes) and orthogonal labeling strategies could enhance detection of weak binders.
• Integration with functional assays: Coupling rapid MS-based identification with downstream biochemical and cellular degradation readouts will accelerate hit-to-lead progression.

Conclusion

This study presents a practical, higher-throughput native-MS platform for molecular glue discovery that combines online buffer exchange, gas-phase affinity selection and fragmentation, and cryo-EM validation. The approach addresses key obstacles in identifying weak ternary-complex-forming ligands, demonstrates rapid and reproducible hit identification (16 hits from a 96-compound screen), and supports structural follow-up. While multiplexing offers throughput gains, single-compound wells currently provide clearer assignments; ongoing improvements in instrumentation, workflows and data analysis are expected to further increase screening efficiency and decrease ambiguity.

Reference

Weijing Liu, Kheewoong Baek, Albert Konijnenberg, Wenfei Song, Christopher Mullen, Yuan Xiong, Ken Durbin, Shane Bechler, Eric Fischer, Rosa Viner, Thomas Moehring. Multi-dimensional high-throughput molecular glue screening via gas phase affinity selection native mass spectrometry and cryo-EM analysis. Thermo Fisher Scientific / Dana-Farber Cancer Institute, 2025. PO397-2025-EN