Improved accuracy of low affinity protein-ligand equilibrium dissociation constants directly determined by electrospray ionization mass spectrometry.

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.
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Submitted on : Wednesday, August 29, 2012 - 2:00:07 PM
Last modification on : Friday, June 7, 2019 - 5:50:04 PM

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Lucie Jaquillard, Fabienne Saab, Françoise Schoentgen, Martine Cadene. Improved accuracy of low affinity protein-ligand equilibrium dissociation constants directly determined by electrospray ionization mass spectrometry.. Journal of The American Society for Mass Spectrometry, Springer Verlag (Germany), 2012, 23 (5), pp.908-22. ⟨10.1007/s13361-011-0305-7⟩. ⟨hal-00726223⟩

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