Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2014 Oct;28:105-11.
doi: 10.1016/ Epub 2014 Aug 30.

HDX-MS Guided Drug Discovery: Small Molecules and Biopharmaceuticals

Free PMC article

HDX-MS Guided Drug Discovery: Small Molecules and Biopharmaceuticals

David P Marciano et al. Curr Opin Struct Biol. .
Free PMC article


Hydrogen/deuterium exchange coupled with mass spectrometry (HDX-MS or DXMS) has emerged as an important tool for the development of small molecule therapeutics and biopharmaceuticals. Central to these advances have been improvements to automated HDX-MS platforms and software that allow for the rapid acquisition and processing of experimental data. Correlating the HDX-MS profile of large numbers of ligands with their functional outputs has enabled the development of structure activity relationships (SAR) and delineation of ligand classes based on functional selectivity. HDX-MS has also been applied to address many of the unique challenges posed by the continued emergence of biopharmaceuticals. Here we review the latest applications of HDX-MS to drug discovery, recent advances in technology and software, and provide perspective on future outlook.


Figure 1
Figure 1. Schematic of a typical HDX-MS workflow
a. A protein sample in the absence or presence of a ligand (shown in magenta) is incubated at 4°C in D2O containing buffers for various time intervals b. After “on-exchange”, the protein is denatured and the deuterium uptake is quenched under acidic conditions (pH 2.5) at 0° C followed by proteolytic digestion using an on-line pepsin column c. Proteolytic peptides are then separated using a gradient column and subjected to mass determination using a high resolution mass spectrometer d. Average deuterium incorporation for each peptide over time is calculated from their mass shifts (top) and the differential HDX data (apo versus ligand bound) is overlaid onto an available three-dimensional structure (bottom). Regions that are differentially protected are color coded according to the HDX WorkBench software scheme.
Figure 2
Figure 2. Correlating HDX-MS with functional output
Analysis of pharmacologically distinct PPARγ ligands for both cellular transactivation and HDX demonstrates a strong correlation between helix 12 peptide (SLHPLLQEIYKDLY) protection and receptor activity. Thus, HDX can be used as a predictive assay to support lead optimization of functional selective PPARγ modulators (FSPPARMs).

Similar articles

See all similar articles

Cited by 24 articles

See all "Cited by" articles

Publication types

MeSH terms