Quantifying protein-ligand interactions by direct electrospray ionization-MS analysis: evidence of nonuniform response factors induced by high molecular weight molecules and complexes.

The deleterious effects of high molecular weight (MW) solute (polymers and noncovalent assemblies) on protein-ligand (PL) affinity measurements carried out using the direct electrospray ionization mass spectrometry (ESI-MS) assay are described. The presence of high MW solute, that do not interact with the protein (P) or ligand (L) of interest, is shown to result in a decrease in the abundance (Ab) ratio (R) of ligand-bound to free protein ions (i.e., Ab(PL)/Ab(P)) measured for protein-carbohydrate complexes. This effect, which can reduce the apparent association constant by more than 60%, is found to be more pronounced as the differences in the surface properties of P and PL become more significant. It is proposed that the decrease in R reflects a reduction in the number of available surface sites in the ESI droplets upon introduction of large solute and increased competition between P and the more hydrophilic PL for these sites. That a similar decrease in R is observed upon introduction of surfactants to solution provides qualitative support for this hypothesis.