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

• Published in: Journal of the American Society for Mass Spectrometry 2012-05, Vol.23 (5), p.908-922
• Main Authors: Jaquillard, Lucie, Saab, Fabienne, Schoentgen, Françoise, Cadene, Martine
• Format: Article
• Language: English
• Subjects: Affinity; Agglomeration; Analytical Chemistry; Animals; Biochemistry, Molecular Biology; Bioinformatics; Biological and medical sciences; Biotechnology; Cattle; Chemistry; Chemistry and Materials Science; Chickens; Constants; Coordination compounds; Dissection; Fundamental and applied biological sciences. Psychology; Gases; Gases - chemistry; Humans; Interactions. Associations; Intermolecular phenomena; Ionization; Kinetics; Life Sciences; Ligands; Mass spectrometry; Mathematical analysis; Models, Molecular; Molecular biophysics; Molecular interactions; Nucleotides; Nucleotides - chemistry; Nucleotides - metabolism; Organic Chemistry; Protein Binding; Proteins; Proteins - chemistry; Proteins - metabolism; Proteomics; Research Article; Spectrometry, Mass, Electrospray Ionization; Spectrometry, Mass, Electrospray Ionization - methods
• ISSN: 1044-0305; 1879-1123
• DOI: 10.1007/s13361-011-0305-7

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.