Gas phase affinity selection-native mass spectrometry for automated ligand screening

Significance of the topic

Ligand screening of protein targets is central to drug discovery and structural biology because it identifies chemical entities that modulate biomolecular function. Native mass spectrometry (native MS) preserves non-covalent protein-ligand complexes and yields stoichiometric and affinity information that complements orthogonal biophysical methods. Automating native-MS workflows and increasing throughput while retaining sensitivity for weak binders addresses a practical bottleneck in early hit discovery and fragment screening.

Objectives and overview of the study

This application note describes an integrated liquid chromatography–native mass spectrometry (LC-nMS) workflow that automates online buffer exchange, multiplexed ligand binding, and direct native MS analysis to support high-throughput ligand screening. Key aims were to (1) compare pre-column (on-column) versus post-column ligand binding for preservation of weak interactions, (2) demonstrate automated apparent Kd determination, and (3) introduce a Gas Phase Affinity Selection MS approach using MSn to increase throughput and enable ligand identification/structural elucidation.

Methodology

Experimental design used carbonic anhydrase (CA) as a model receptor and a small set of sulfonamide-class ligands with published Kd values spanning submicromolar to low tens of micromolar affinities. The workflow steps included:

• Preparation of CA in 200 mM ammonium acetate and ligand stocks in DMSO.
• Online buffer exchange of CA into native-compatible volatile buffer using a NativePac OBE-1 SEC column.
• Fraction collection into multiwell plates prefilled with ligands (post-column binding) or pre-mixing protein and ligand prior to SEC (pre-column binding).
• Direct infusion to native MS via a 15 µm EASY-Spray capillary emitter for intact complex analysis, and MSn experiments for ligand release and fragmentation.

The approach also tested increased ligand multiplicity per well and a gas-phase selection strategy to isolate complexes in the quadrupole, dissociate ligands via HCD/CID, and identify them by high-resolution Orbitrap detection and IT-based MSn fragmentation.

Used instrumentation

• Thermo Scientific Orbitrap Ascend Structural Biology Tribrid mass spectrometer (native MS and MSn capability).
• Thermo Scientific Q Exactive UHMR Hybrid Quadrupole-Orbitrap (used in parallel experiments).
• Thermo Scientific Vanquish LC system with fraction collector, dual pumps and autosampler for online buffer exchange and plate-based fractionation.
• NativePac OBE-1 SEC column for online desalting/buffer exchange.
• EASY-Spray capillary emitter (15 µm bullet type) and EASY-Spray source for nano/low-flow native infusion.
• Software: BioPharma Finder 5.0 for spectral deconvolution and GraphPad Prism for Kd fitting.

Main results and discussion

Pre-column (on-column) binding showed significant loss of weaker binders due to dilution and on-column dissociation during SEC, leading to detection biased toward the strongest ligand only. In contrast, post-column binding—where desalted protein fractions are collected into wells already containing ligands—preserved weaker complexes (L2, L3) and produced apparent Kd rankings consistent with literature values (L1 > L2 > L3 > L4).

Automated titrations using fixed protein concentration and varying ligand concentrations (molar ratios 1:0.5 to 1:10) allowed extraction of apparent Kd values from fractional occupancy of protein-ligand complexes observed in the native spectra. These apparent Kd values agreed with published affinities for the CA sulfonamide ligands, demonstrating quantitative potential of the workflow.

The Gas Phase Affinity Selection MS strategy increased throughput by allowing multiple ligands per well while still enabling unambiguous identification. The method isolates the intact protein–ligand complex in the quadrupole, uses higher-energy collisional dissociation (HCD/FHCD) to release ligands to lower m/z, and records high-resolution Orbitrap MS (and IT-based MSn) spectra of released ligands for identification and structural elucidation. Practical findings included the need to record ligand release in both positive and negative ion modes because some released ligands (or their adducted forms) ionize preferentially in negative mode. The approach successfully identified an unexpected impurity (a chlorinated dibenzenedisulfonamide) via MS3 fragmentation and mass-shift analysis.

Key performance metrics: operational throughput of ~300 samples per day (one ligand per well workflow) and robust detection of complexes across a wide Kd range, with improved sensitivity to weak binders using post-column binding.

Benefits and practical applications

• Preservation of native complexes enables direct measurement of stoichiometry and relative occupancy without denaturation steps.
• Post-column binding reduces loss of weak binders that can dissociate during chromatographic desalting, improving hit identification for fragment and weak-affinity screens.
• Automated fraction collection and plate-based workflows integrate with high-throughput sample handling and decrease manual labor.
• Gas Phase Affinity Selection MS with MSn supports multiplexed screenings and structural elucidation of bound ligands, including detection of impurities or unexpected adducts.
• Compatible with follow-up structural studies and can prioritize compounds for orthogonal assays (SPR, ITC, biochemical assays).

Limitations and practical considerations

• Apparent Kd values derived from native MS reflect gas-phase–to–solution correlations and may require orthogonal validation for absolute thermodynamics.
• Adducts, cofactors, and similar m/z among ligands can complicate assignment—careful MSn and ion mode selection (positive/negative) are often required.
• Column and buffer conditions must be carefully optimized to minimize artefacts and maintain native-like conformations.

Future trends and potential applications

• Scaling multiplexing strategies together with advanced deconvolution algorithms and machine learning to increase ligand-per-well complexity while maintaining confident identifications.
• Integration of time-resolved native MS or SLOMO-like approaches to extract kinetic parameters (kon/koff) in addition to equilibrium affinities.
• Higher-throughput native-MS platforms coupled with automated sample handling could be applied to fragment libraries, covalent fragment mapping, and targeted protein degrader ternary complex screens.
• Improved ion sources, front-end desalting, and MS instrumentation (higher m/z/upper mass range) will enhance detection of large complexes and multi-ligand stoichiometries.

Conclusion

The presented LC-nMS workflow combining online buffer exchange, plate-based post-column ligand binding, and native MS provides an automated, higher-throughput platform for ligand screening that preserves weak interactions and enables apparent Kd determination. The Gas Phase Affinity Selection MS extension further increases throughput and uses MSn to identify and structurally characterize bound ligands, including unexpected impurities. This pipeline offers a practical route to accelerate early discovery screening while maintaining access to structural information through MSn.

Reference

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