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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.

2025-11-13 biophysics
10.1101/2025.11.13.688232 bioRxiv
Show 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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