Improved Accuracy of Low Affinity Protein–Ligand Equilibrium Dissociation Constants Directly Determined by Electrospray Ionization Mass Spectrometry

There is continued interest in the determination by ESI-MS of equilibrium dissociation constants (K D ) that accurately reflect the affinity of a protein–ligand complex in solution. Issues in the measurement of K D are compounded in the case of low affinity complexes. Here we present a K D measurement method and corresponding mathematical model dealing with both gas-phase dissociation (GPD) and aggregation. To this end, a rational mathematical correction of GPD (f sat ) is combined with the development of an experimental protocol to deal with gas-phase aggregation. A guide to apply the method to noncovalent protein–ligand systems according to their kinetic behavior is provided. The approach is validated by comparing the K D values determined by this method with in-solution K D literature values. The influence of the type of molecular interactions and instrumental setup on f sat is examined as a first step towards a fine dissection of factors affecting GPD. The method can be reliably applied to a wide array of low affinity systems without the need for a reference ligand or protein.