Residue-Level Determination of Small-Molecule–Protein Affinities by Hydrogen–Deuterium Exchange Mass Spectrometry

Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS) is an established tool in drug discovery, used to characterize target engagement and conformational dynamics, and frequently used in both biopharmaceutical and small molecule drug discovery. Conventional HDX-MS experiments are performed at saturating ligand concentrations to generate a binding "footprint", where decreased solvent exchange reflects a local structural stabilization 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 (<i>K</i>_D^app) 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.