Direct Method for Discovering and Ranking Multiple Ligands

Direct Method for Discovering and Ranking Multiple Ligands 

6 Frontal Affnity Chromatography - Mass Spectrometry for Ligand Discovery and Characterization 

Deng and Sanyal have suggested that FAC is not applicable to ligands with low on- and off-rates [20]. As will be shown below, this is not true particularly when indirect FAC methods are applied, but it is also misleading in direct assays as described in this section. With flow-rate programming, for example, slow kinetics 

1) These displacements (or roll-ups ) can only occur if there is competition for a common binding site, or a strongly-negative 

Mass spectrometry enables the type of direct analyses described, but it does have its limitations. Online operation forces detection at infusion concentrations, in salty buffer and under complex mixture conditions. General ion suppression results from the buffer and mixture components, and mixture complexity can tax the resolution of even the best mass spectrometers. Increasing compound concentration is not the answer, as this leads to problems of solubility and increased compound consumption. We have found that the online method can work successfully for up to 100 compounds per analysis, but the false negative rate becomes appreciable [21]. As an alternative for ligand discovery purposes, we have developed a FAC-LC/MS system in which FAC effluent is sampled and analyzed by LC/MS [19]. This system offers the ability to concentrate mixture components and introduces another dimension to the data in order to tolerate more complex mixtures (Fig. 6.9). Using this system, we have screened approximately 1000 modified trisaccharide acceptor analogs targeting immobilized N- 

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