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bioRxiv · 10.1101/2025.11.13.688232

Spatially Resolved Determination of Small Molecule Protein Affinities by Hydrogen Deuterium Exchange Mass Spectrometry

Lin, D.·Magalhaes, L. G.·McMillan, J.·Eadsforth, T. C.·Stewart, G.·Cartmill, K.·Postis, V.·Masson, G.
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Abstract

Hydrogen Deuterium Exchange Mass Spectrometry (HDX-MS) is an established tool in drug discovery, used to characterize target engagement and conformational dynamics, frequently used in both biopharmaceutical and small molecule development. Conventional HDX-MS experiments are performed at saturating ligand concentrations to generate a binding "footprint", where decreased solvent exchange reflects a local structural stabilization and/or reduced solvent accessibility upon binding. Here, we present an extended HDX-MS and HDX-MS/MS titration workflow with electron capture dissociation (ECD) fragmentation capable of estimating apparent dissociation constants (KDapp) at global, peptide and single amino acid resolution by fitting uptake-concentration relationships under EX2 exchange and Langmuir binding assumptions. The ability to determine affinity constants in a spatially resolved manner combined with the automation available in HDX-MS sample handling and data analysis enables quantitative mapping of ligand-protein interactions and provides a scalable approach for structure-activity relationship studies in drug discovery.

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Lin, D., Magalhaes, L. G., McMillan, J., Eadsforth, T. C., Stewart, G., Cartmill, K., Postis, V., Masson, G.. 2025-11-13. Spatially Resolved Determination of Small Molecule Protein Affinities by Hydrogen Deuterium Exchange Mass Spectrometry. https://doi.org/10.1101/2025.11.13.688232

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