Competitive MS binding assays for dopamine D2 receptors employing spiperone as a native marker.
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.
Niessen
Karin V
KV
Zentrales Institut des Sanitätsdienstes der Bundeswehr München, Ingolstädter Landstrasse 102, 85748 Garching-Hochbrück, Germany.
Höfner
Georg
G
Wanner
Klaus T
KT
D000818:Animals; D002849:Chromatography, Gas; D003342:Corpus Striatum / Q000737:chemistry / Q000378:metabolism; D018492:Dopamine Antagonists / Q000378:metabolism*; D065127:Dopamine D2 Receptor Antagonists; D008566:Membranes / Q000737:chemistry / Q000378:metabolism; D017448:Receptors, Dopamine D2 / Q000378:metabolism*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods; D013134:Spiperone / Q000378:metabolism*; D013379:Substrate Specificity; D013552:Swine
D018492:Dopamine Antagonists; D065127:Dopamine D2 Receptor Antagonists; D017448:Receptors, Dopamine D2; D013134:Spiperone