Mass Spectrometry-based bioassay for the screening of soluble orphan receptors

The suitability of a novel mass spectrometric (MS) based flow-injection bio assay format to rapidly detect ligands for soluble orphan receptors in comp lex matrices was demonstrated, using digoxin and antidigoxigenin FAb as the model ligand and the model protein target, respectively. In addition, char acteristic MS and MSn (n = 2 or 3) data, that is, molecular mass as well as mass fingerprints, were obtained during a single run and provided a basis for the identification of the active compounds that were found. The flow-in jection setup used is characterized by the integration of two restricted ac cess columns (RA column) and a pH dissociation step. Bioactive compounds ar e separated both from unbound ligands as well as the targeted proteins, e.g ., as described in the text. This approach allows the isolated bioactive mo lecules to be introduced in the ion trap MS detector in a clean and well-de fined matrix, which minimizes background noise during sample analysis while simultaneously improving bioassay robustness. In this article, we demonstr ate the potential of the bioassay format to detect ligands for soluble orph an receptors. Natural extracts, both spiked and unspiked with the model com pound digoxin, were analyzed, which demonstrated that only those compounds possessing affinity for the model protein target were detected by MS. Using an antidigoxigenin FAb concentration of 2 muM, digoxin could be detected d own to 250 nM in spiked plant extracts. In contrast, however, molecules tha t did not bond to the protein target were efficiently trapped by the RA col umn and did not show up in the MS spectra, thus illustrating the principle and potential of the bioassay format. (C) 2001 Elsevier Science B.V.