To demonstrate proper functioning of the MS-based bioassay and illustrate the potential for orphan target screening, a model system using anti-digoxigenin antibodies was chosen, and digoxin samples were injected under several bioassay conditions. The presence of digoxin onto the second RA column was evaluated by monitoring the reconstructed ion currents of two m/z values, 651.1 and 798.2, that were characteristic for digoxin under the conditions applied. Figure 5.16 shows the results of digoxin injections (1 mM) using three different bioassay conditions. First, digoxin was injected into a bioassay system in which the anti-digoxigenin FAb as well as the dissociation solution were replaced by buffer (10 mM ammonium acetate, pH 6.7). The ion current traces for m/z 651 and 798 are comparable to those obtained for blank injections, thus indicating that digoxin is efficiently trapped onto the first RA column. The ion current peaks, which can be observed in the total, as well as extracted ion current profiles are caused by the introduction of residual ions and solvent (DMSO), which were not completely removed from the RA column during flushing. During a second step, anti-digoxigenin FAb was added to the biochemical assay, while the dissociation solution was still replaced by buffer. Again, the digoxin injections showed hardly any increase in ion current, indicating that the affinity complexes formed passed both the first and second RA columns. Finally, digoxin was injected into a complete bioassay system containing both the affinity protein as well as the dissociation solution. The ion current traces of m/z 651 and 798 both clearly show a significant increase in intensity compared with the previous digoxin injections. From these experiments it can be concluded that the affinity complexes between digoxin and anti-digoxigenin FAb were indeed formed, passed the first RA column, and finally were dissociated by the pH shock applied. As a result, digoxin molecules were trapped onto the second RA column and were detected by full-scan MS analysis after column desorption. Under these semi-optimized conditions, digoxin could be detected down to 250 nM, whereas the relative standard deviation of 1 mM digoxin injections equaled 13.0% (n = 5).
5.5 Future Prospects 211
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