Automated online protein-ligand binding and its detection using native mass spectrometry

Significance of the Topic

Native mass spectrometry (nMS) preserves noncovalent interactions and enables direct detection of protein–ligand and protein–protein complexes. This capability is critical for characterizing targets lacking classical small-molecule pockets, including so-called "undruggable" proteins. Integrating liquid chromatography (LC) with native MS streamlines buffer exchange, increases throughput, and permits online monitoring of binding events, addressing limitations of manual sample prep and direct infusion.

Study Objectives and Overview

The study presents an automated, online workflow combining an integrated LC system with native MS to detect protein–ligand interactions and ternary complexes formed by molecular glues. Initial validation uses carbonic anhydrase (CA) with four ligands spanning low- to high-affinity ranges. The method is then applied to screen molecular glues that stabilize CRBN-DDB1 interactions with a target protein.

Methodology and Instrumentation

• Workflow Steps:
  
  1. Prefill 96-well plate with ligands or molecular glues.
  2. Set fraction collector at binding temperature.
  3. Autosampler → Valve 1 → Online buffer exchange (OBE) → UV detection → Fraction collection.
  4. Transfer fractions back to autosampler → Valve 2 → Electrospray ionization into MS.
• Instrument Configuration:
  
  • Thermo Scientific Vanquish LC with dual pumps, UV detector, fraction collector, dual-injection autosampler.
  • Thermo Scientific Q-Exactive UHMR and Orbitrap Ascend Structural Biology Edition MS.
  • Mass range up to m/z 80,000; native top-down and direct MS capabilities.

Main Results and Discussion

• Carbonic Anhydrase Binding:
  
  • Four ligands (L1–L4) showed apparent Kd values of 13.2 to 0.27 µM.
  • Post-column (on-line) binding preserved weak interactions better than pre-column, consistent with published affinities.
  • Ranking of ligand affinities aligned with known data (L4 strongest, L1 weakest).
• Molecular Glue Screening:
  
  • Five glues (A1–E1) evaluated for stabilizing CRBN-DDB1–target complexes.
  • High-throughput fraction collection and direct infusion allowed detection of ternary complex formation.
  • Strong glues (e.g., B1, C1) produced high fractional complex abundance; weak or control compounds showed minimal binding.

Benefits and Practical Applications

• High-throughput screening of ligands and molecular glues for drug discovery campaigns.
• Rapid buffer exchange minimizes dissociation of weak complexes.
• Quantitative ranking of binding affinities directly from native MS data.

Future Trends and Potential Applications

• Integration with data-driven workflows and machine-learning analysis to predict binding outcomes.
• Expansion to membrane proteins, large multiprotein assemblies, and post-translationally modified targets.
• Further miniaturization and multiplexing for ultra-high throughput screening.

Conclusion

The integrated LC–native MS workflow enables automated, online detection of protein–ligand and ternary molecular glue complexes with high sensitivity and throughput. It provides reliable affinity measurements and a scalable platform for drug discovery targeting challenging proteins.

Reference

No formal reference list was provided in the source document.