Residue-Level Determination of Small MoleculeProtein Affinities by HydrogenDeuterium Exchange Mass Spectrometry

TY - JOUR

T1 - Residue-Level Determination of Small MoleculeProtein Affinities by HydrogenDeuterium Exchange Mass Spectrometry

AU - Lin, De

AU - Godoy Magalhaes, Luma

AU - McMillan, Joel

AU - Eadsforth, Thomas

AU - Stewart, Greg

AU - Cartmill, Kieran

AU - Postis, Vincent L G

AU - Masson, Glenn R

N1 - Publisher Copyright: © 2026 The Authors. Published by American Chemical Society.

PY - 2026/3/31

Y1 - 2026/3/31

N2 - 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 (K 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.

AB - 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 (K 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.

KW - HDX-MS

KW - HDX-MS/MS

KW - hydrogen Isotopes

KW - ions

KW - ligands

KW - mass spectrometry

KW - peptides and proteins

U2 - 10.1021/jasms.6c00020

DO - 10.1021/jasms.6c00020

M3 - Article

C2 - 41915381

SN - 1044-0305

VL - 37

SP - 1391

EP - 1401

JO - Journal of The American Society for Mass Spectrometry

JF - Journal of The American Society for Mass Spectrometry

IS - 6

ER -