Journal
JOURNAL OF THE AMERICAN SOCIETY FOR MASS SPECTROMETRY
Volume 23, Issue 5, Pages 908-922Publisher
SPRINGER
DOI: 10.1007/s13361-011-0305-7
Keywords
Accuracy; Equilibrium dissociation constant; KD; Gas phase dissociation; Aggregation; ESI; Model; Low affinity; Interaction; Protein-ligand; Mass spectrometry
Funding
- ANR
- Ligue Nationale contre le Cancer
- Canceropole Grand-Ouest
- CNRS
- Region Centre
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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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