Frontal affinity chromatography–mass spectrometry assay technology for multiple stages of drug discovery: applications of a chromatographic biosensor

Abstract

This article presents new concepts in affinity chromatography/mass spectrometry for the study of molecular interactions. Chromatographic assays involving estrogen receptor-β, sorbitol dehydrogenase, human α-thrombin, cholera toxin B subunit, β-galactosidase, and Griffonia simplicifolia isolectin B 4 were established in microaffinity columns and operated in frontal analysis mode. Methods and formalism are presented for the measurement of dissociation constants, using direct methods in which the mass spectrometric signature of the ligand is used to measure breakthrough time and, hence, binding strength. The direct approach is capable of measuring sub-micromolar K d and higher, on sub-pmol amounts of immobilized protein, as shown in the cholera toxin assay. Indirect assays that demonstrate the advantage of routine, rugged performance were developed. By tracking the effect of a test ligand on a selected probe, or indicator ligand, dissociation constants in the low nanomolar range could be reliably determined for ligands to estrogen receptor-β. Mass spectrometry supports the resolution of complex ligand mixtures, and it is demonstrated in the sorbitol dehydrogenase assay that ligands can be rank ordered across approximately three orders of magnitude in K d , in a single run. A new concept for rapid mixture prescreening is presented, in which an indicator ligand can be used to discriminate between mixtures that contain high levels of weak ligands and those that contain single strong ligands.