Recommendations for performing, interpreting and reporting hydrogen deuterium exchange mass spectrometry (HDX-MS) experiments.

Hydrogen deuterium exchange mass spectrometry (HDX-MS) is a powerful biophysical technique being increasingly applied to a wide variety of problems. As the HDX-MS community continues to grow, adoption of best practices in data collection, analysis, presentation and interpretation will greatly enhance the accessibility of this technique to nonspecialists. Here we provide recommendations arising from community discussions emerging out of the first International Conference on Hydrogen-Exchange Mass Spectrometry (IC-HDX; 2017). It is meant to represent both a consensus viewpoint and an opportunity to stimulate further additions and refinements as the field advances

Identification of Differential Protein Binding Affinities in an Atropisomeric Pharmaceutical Compound by Noncovalent Mass Spectrometry, Equilibrium Dialysis, and Nuclear Magnetic Resonance

Atropisomerism of pharmaceutical compounds is a challenging area for drug discovery programs (Angew. Chem., Int. Ed. 2009, 48, 6398−6401). Strategies for dealing with these compounds include raising the energy barrier to atropisomerization in order to develop the drug as a single isomer (Tetrahedron 2004, 60, 4337−4347) or reducing the barrier to rotation and developing a mixture of rapidly interconverting isomers (Chirality 1996, 8, 364−371). Commonly, however, the atropisomers will be differentiated in terms of their affinity for a given protein target, and it is therefore important to rapidly identify the most active component prior to further compound development. We present equilibrium dialysis and saturation transfer difference NMR (STD-NMR) as techniques for assessing relative affinities of an atropisomeric mixture against antiapoptotic protein targets Bcl-2 and Bcl-x_L. These techniques require no prior separation of the mixture of compounds and are therefore rapid and simple approaches. We also explore the use of noncovalent mass spectrometry for determining <i>K</i>_D values of individual atropisomers separated from the equilibrium mixture and compare the results to solution-phase measurements. Results from equilibrium dialysis, STD-NMR, and noncovalent mass spectrometry are all in excellent agreement and provide complementary information on differential binding, amplification of the strongest binders, and <i>K</i>_D values

Automated Protein–Ligand Interaction Screening by Mass Spectrometry

Identifying protein–ligand binding interactions is a key step during early-stage drug discovery. Existing screening techniques are often associated with drawbacks such as low throughput, high sample consumption, and dynamic range limitations. The increasing use of fragment-based drug discovery (FBDD) demands that these techniques also detect very weak interactions (mM <i>K</i>_D values). This paper presents the development and validation of a fully automated screen by mass spectrometry, capable of detecting fragment binding into the millimolar <i>K</i>_D range. Low sample consumption, high throughput, and wide dynamic range make this a highly attractive, orthogonal approach. The method was applied to screen 157 compounds in 6 h against the anti-apoptotic protein target Bcl-x_L. Mass spectrometry results were validated using STD-NMR, HSQC-NMR, and ITC experiments. Agreement between techniques suggests that mass spectrometry offers a powerful, complementary approach for screening