Hydrogen/Deuterium Exchange Mass Spectrometry for Weak Binders
Yoshitomo Hamuro, Stephen J. Coales
Abstract
This note describes theoretical and experimental considerations to observe perturbation of a protein upon binding to a ligand with weak affinity by hydrogen/deuterium exchange mass spectrometry (HDX-MS). The most popular application of HDX-MS is to determine the binding site of a drug or drug lead in a protein target. However, when the affinity of a ligand is weak, driving the equilibrium to the formation of a complex is difficult, and thus, observing the perturbation upon binding is also challenging. Theoretical consideration indicates that the original concentration of a ligand over the dissociation constant ([L0]/KD) is roughly equal to the maximum protection factor expected for the experiment when the original concentration of a ligand is significantly larger than the original concentration of a protein and the dissociation constant ([L0] ≫ [P0] and [L0] ≫ KD). When HDX-MS analysis of a protein with a ligand of low affinity and low solubility is carried out, it may be challenging to achieve high enough ligand concentration to drive the equilibrium in favor of the complex due to the low solubility. There are two methods to alleviate this issue: (i) spiking a low affinity/low solubility ligand to exchange buffer to lower the required ligand concentration in aqueous protein stock solution and (ii) mixing a 1:1 ratio of aqueous protein–ligand stock solution and deuterated buffer to initiate the exchange reaction instead of the commonly used 1:9 ratio.