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Quantifying Protein–Ligand Interactions by Direct Electrospray Ionization-MS Analysis: Evidence of Nonuniform Response Factors Induced by High Molecular Weight Molecules and Complexes

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journal contribution
posted on 2013-10-01, 00:00 authored by Hong Lin, Elena N. Kitova, John S. Klassen
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.

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