Analytical Sciences, Talk
AS-025
Native ESI-MS: Buffer Concentration Effect on Protein-Ligand Binding Affinities
Agni Gavriilidou1, Basri G├╝lbakan1, Renato Zenobi1
1ETH Zurich
The characterization of protein-ligand interactions is important for the understanding of biochemical reactions and pathways as well as for a subsequent design of new therapeutics for treatment of different human diseases, thus it is of crucial importance to study the effect of buffer concentration on the protein-ligand affinities.

A powerful technique for the investigation of noncovalent interactions is native electrospray ionization mass spectrometry (ESI-MS). Ammonium acetate is the most frequent buffer choice for studying non-covalent protein-ligand interactions by native ESI-MS analysis. The buffer concentration used in native ESI-MS titration measurements is generally in the low to high mM range. Previous studies have shown that by varying the buffer concentration the relative abundances of the protein-ligand complex to the free protein can vary (Benkestock et al. 2004; Kapur et al. 2001). However, to date it was not shown to which extent the concentration of the buffer affects the apparent affinity of the ligand to a receptor .
A well studied system (lysozyme-NaG3) was chosen in order to show to which extent the concentration of ammonium acetate buffer used for nanoESI-MS affects the value of the dissociation constant (KD). Five different ammonium acetate buffer concentrations were used (10 mM, 50 mM, 100 mM, 300 mM and 500 mM) for this study. It can be seen that the KD is decreasing with increasing the ammonium acetate concentration (Figure 1).
A general trend is observed that by increasing the ammonium acetate concentration the KD values are decreasing. The affinity of NaG3 to lysozyme is altered by a factor of up to 50% with increasing the buffer concentration. .

To date it has not been shown to which extent the buffer concentration affects the value of the dissociation constant (KD). Different model systems (protein/aptamers-ligands) will be tested in order to investigate this effect.