Influence of Ammonium Acetate Concentration on Receptor–Ligand Binding Affinities Measured by Native Nano ESI-MS: A Systematic Study

Native electrospray ionization (ESI) mass spectrometry (MS) is a powerful technique for analyzing biomolecules in their native state. However, ESI-MS is incompatible with nonvolatile solution additives. Therefore, biomolecules have to be electrosprayed from a solution that differs from their purification or storage buffer, often aqueous ammonium acetate (AmAc). In this study, the effect of the ionic strength on the dissociation constants of six different noncovalent complexes, that cover interactions present in many biological systems, was investigated. Complexes were electrosprayed from 10 mM, SO mM, 100 mM, 300 mM, and 500 mM aqueous AmAc. For all systems, it was shown that the binding affinity is significantly influenced by the ionic strength of the solution. The determined dissociation constant (K-d) was affected more than 50% when increasing the AmAc concentration. The results are interpreted in terms of altered ionic interactions induced by the solution. This work emphasizes the modulating effect of the ions on noncovalent interactions and the importance of carefully choosing the AmAc concentration for quantifying the receptor ligand binding strengths.