Competitive MS Binding Assays for Dopamine D₂Receptors Employing Spiperone as a Native Marker

Abstract

A competitive MS binding assay employing spiperone as a native marker and a porcine striatal membrane fraction as a source for dopamine D2 receptors in a nonvolatile buffer has been established. Binding of the test compounds to the target was monitored by mass-spectrometric quantification of the nonbound marker, spiperone, in the supernatant of the binding samples obtained by centrifugation. A solid-phase extraction procedure was used for separating spiperone from ESI-MS-incompatible supernatant matrix components. Subsequently, the marker was reliably quantified by LC-ESI-MS-MS by using haloperidol as an internal standard. The affinities of the test compounds, the dopamine receptor antagonists (+)-butaclamol, chlorpromazine and (S)-sulpiride obtained from the competitive MS binding assay were verified by corresponding radioligand binding experiments with [3H]spiperone. The results of this study demonstrate that competitive MS binding assays represent a universally applicable alternative to conventional radioligand binding assays.

MS‐Binding Assays: Kinetic, Saturation, and Competitive Experiments Based on Quantitation of Bound Marker as Exemplified by the GABA Transporter mGAT1

“…The mixture was heated at reflux for 5 min and after cooling to room temperature, it was poured into ice-cold HCl (1.5 m, 31 mL). (14).…”

“…We introduced the concept of competitive MS-binding assays [13,14] in which a native marker, that is, a nonlabeled ligand, and mass spectrometric detection is used to characterize test compounds effectively in a competitive binding assay. The feasibility of this approach has been exemplified with native dopamine D 1 and D 2 receptors from porcine striatal cell membranes as targets.…”

“…This technique is implemented in all areas of research, including drug discovery and development. [8][9][10][11][12][13][14] By exploiting the characteristic mass over charge ratio (m/z) for each compound and the corresponding m/z value for daughter ions after fragmentation in tandem MS techniques, mass spectrometry is a true alternative to scintillation counting and fluorescence measurements. With MS, the quantitation of ligands in binding assays is possible without special labeling.…”

Label-Free, LC-MS-Based Assays to Quantitate Small-Molecule Antagonist Binding to the Mammalian BLT1 Receptor

“…28 In addition, the application of the coupled LC-MS methodology to study low-molecular-weight ligand binding to membrane-associated receptors in a labelfree manner was pioneered and extensively developed in the Wanner laboratory. [29][30][31] The relative simplicity of this label-free approach to studying a wide array of biochemical binding processes held great appeal to us. We now report studies employing a modified, highly flexible, LC-MSbased method to quantitate small-molecule antagonist binding to membrane-associated BLT1 receptors in both equilibrium direct saturation binding and competition binding formats.…”

Recent developments in protein–ligand affinity mass spectrometry

“…A method similar to that used in the work by Annis et al [68] and Derks et al [69] used a native marker ligand for, e.g., the dopamine D2 receptor, thereby allowing the analysis of ligand binding to GPCRs after a rapid solid-phase extraction MS step [70, 71]. A followup technology used MALDI as an ionization source.…”