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Accueil > Publications > Recherche par années > Années 2010 > 2012

Jaquillard, L., Saab, F., Schoentgen, F. and Cadene, M.

Improved accuracy of low-affinity protein-ligand equilibrium dissociation constants determined by ESI-MS

Journal of The American Society for Mass Spectrometry 23 (5) 908-922

par Frapart - publié le , mis à jour le

Abstract :

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.