Site Specific Hydrogen Deuterium Exchange Mass Spectrometry Enables Deep Explorations of Dynamic Allosteric Networks in Protein-Drug Interactions

Hydrogen Deuterium eXchange Mass Spectrometry (HDX-MS) is a dynamics-sensitive structural method that has rapidly achieved widespread adoption in the biopharmaceuticals industry, owing to it's ability to quickly identify binding sites and to provide molecular mechanism of action for drug candidates.However, a central limitation of conventional HDX-MS is that it has substantially lower spatial resolution than other structural techniques, typically providing information averaged over 'segments' of 5 amino acids or more.Here we demonstrate a sensitive, broadly applicable method for single amino acid resolution, i.e., site specific, HDX-MS measurements.Using a set of five therapeutic candidates targeting the highly druggable cancer target WDR5, we explore the greatly enhanced analytical power that arises from site specificity, including binding mode characterization, affinity ranking and the detection of features that are 'silent' in conventional peptide-level HDX-MS experiments.