To demonstrate proper functioning of the MS-based bioassay for anti-digoxigenin antibodies using complex sample matrices, higher plant extracts were diluted ten times with 10 mM TRIS buffer (pH 7.0), spiked with digoxin (250 nM), and subsequently analyzed. Figure 5.17 shows an example of such a spiked extract. First, an unspiked aliquot of the extract was injected into the MS-based bioassay of which the first RA column was removed (no target present). Consequently, moderately polar to hydrophobic compounds present in the sample were trapped onto the second RA column and were eluted towards the MS detector. The principle of the MS-based bioassay was then demonstrated by reinserting the RA column again, followed by the injection of a spiked aliquot of the same plant extract in the presence of protein target. As can be seen in Fig. 5.17, the first RA column efficiently traps the non-bioactive molecules, which were present in the plant extract. The bioactive compound digoxin, however, is successfully isolated from the plant extract and can clearly be observed in the MS spectrum (m/z 859.2 and 902.0). By injecting spiked and unspiked aliquots of natural extracts, the ability of the bioassay format to rapidly detect ligands for protein targets, such as soluble orphan receptors, is demonstrated.
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