FIGURE 2.6 FAC/ESI mass chromatograms of a six-component oligosaccharide library, which passed through an affinity column, directed against a Salmonella polysaccharide. Selected ion chromatograms of the indicated masses correspond to unretained components (m/z 576.6, 681.4, 697.3) and to components with increasing degrees of binding (m/z 347.0, 509.1 and 1157.3, respectively). (Reprinted from Schriemer et al. , used with permission. Copyright 1998 by Wiley-VCH Verlag GmbH, Weinheim, Germany.)
chromatograms for each of the components. An example of FAC/ESI-MS is illustrated in Figure 2.6 for the resolution of polysaccharides with a mi-croaffinity column containing an immobilized monoclonal antibody directed against a Salmonella polysaccharide. The three nonactive oligosaccharides (m/z 576.2,681.4, 697.3) break through in the void volume, while the successive ion chromatograms corresponding to oligosaccharides with m/z 347.0, 509.1 and 1157.3 indicate weak, moderate, and fairly strong binding compounds, assuming competitive binding to the same binding site. In general, however, these results can also be interpreted as due to multiple binding sites with different binding affinities for the different components. Advantages of the FAC/ESI-MS approach for screening include the following: large numbers of compounds can be easily screened; the amounts of drugs and substrates used are relatively low; the affinity column can be reused repeatedly, further reducing the consumption of substrates; relative binding strengths can be ranked, since they are proportional to the breakthrough times; throughput can be increased with parallel affinity columns and multisprayers; isobars can be distinguished with MS/MS detection methods; and dissociation constants (Kd) can be obtained from concentration studies. Mixtures of up to 100 components have been analyzed by FAC/ESI-MS, however, as the size of the mixture increases, charge suppression can occur, which reduces or prevents the ionization of components in the mixture.
An extension of the FAC/ESI-MS method, described in the preceding paragraph, is theprescreening of a compound library for unknown ligands. This can be accomplished by use of an "indicator" compound, viz., a known competitive ligand preequilibrated with the FAC affinity column and added to a mixture prior to FAC analysis . Under these conditions, the breakthrough curve for the known indicator is shifted toward the breakthrough curve for the void volume, if a competitive binder is present in the mixture. The shifting of the indicator breakthrough curve is evidence that the mixture contains components that are binding specifically to the substrate, since they compete with the indicator. This approach is used to rapidly prescreen a library for new ligands by use of a weaker indicator ligand that has a short breakthrough time as a means for indicating the presence of a stronger binder, which may have a much longer breakthrough time under normal FAC conditions. Such a mixture would have to be deconvoluted with normal FAC/ESI-MS conditions to identify the unknown binder. However, the savings in time by use of the prescreening methodology is justified, since most mixtures are not likely to contain strong binders.
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