title: 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.
authors.affiliation: Centre de Biophysique Moléculaire, CNRS UPR 4301, affiliated with Université d'Orléans, rue Charles-Sadron, 45071 Orléans, Cedex 2, France.
authors.forename: Lucie
authors.initials: L
authors.lastname: Jaquillard
authors.forename: Fabienne
authors.initials: F
authors.lastname: Saab
authors.forename: Françoise
authors.initials: F
authors.lastname: Schoentgen
authors.forename: Martine
authors.initials: M
authors.lastname: Cadene
mesh_terms: D000818:Animals; D002417:Cattle; D002645:Chickens; D005740:Gases / Q000737:chemistry; D006801:Humans; D007700:Kinetics; D008024:Ligands; D008958:Models, Molecular; D009711:Nucleotides / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
chemical_list: D005740:Gases; D008024:Ligands; D009711:Nucleotides; D011506:Proteins