Site-Specific Hydrogen–Deuterium Exchange Mass Spectrometry for Deep Explorations of Dynamic Shifts in Protein–Drug Interactions

Abstract

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 its ability to quickly identify binding sites and to provide a 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 five 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.