Abstract: Provided are multiplexed methods for characterizing binding of a test compound to different receptor target molecules using mass spectroscopy techniques. The methods employ receptor molecules that have different functions or found in different tissues, such as cerebral cortex, cerebellum, ventricular and hepatic membrane preparations. The methods enable determination of undesirable off-target binding of a test compound. The methods comprise incubation of a heterologous mixture of different receptor target molecules with ligands (known binders), and a test compound. Various wells contain different amounts of molecules for use in construction of concentration curves. Next, unbound ligands are separated from the well contents. Next, ligands that were bound to the receptors are separated. An LC/ESI-MS/MS method may be used to reduce irrelevant mass spectroscopy peaks.

Abstract: The present invention relates to a reliable and effective in vitro assay based on cAMP levels modulated by Gi-coupled receptors expressed in a selected cell for screening and identifying pharmaceutically effective compounds that specifically interact with and modulate the activity of a cellular receptor.