Journal
ANALYTICAL AND BIOANALYTICAL CHEMISTRY
Volume 412, Issue 22, Pages 5525-5535Publisher
SPRINGER HEIDELBERG
DOI: 10.1007/s00216-020-02772-9
Keywords
MPEP; mGlu5 receptor; MS binding assays; Radioligand binding assays; Saturation assay
Funding
- FEDER/Ministerio de Ciencia, Innovacion y Universidades - Agencia Estatal de Investigacion [CTQ2017-89222-R, PCI2018-093047]
- Catalan government [2017SGR1604]
- Neuron-ERANET
- Agence Nationale de la Recherche [ANR-17-NEU3-0001]
- CNRS [PICS 08212]
- Labex EpiGenMed (program Investissements d'avenir) [ANR-10LABX-12-01]
- Agence Nationale de la Recherche (ANR) [ANR-17-NEU3-0001] Funding Source: Agence Nationale de la Recherche (ANR)
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Mass spectrometry (MS) binding assays are a label-free alternative to radioligand or fluorescence binding assays, so the readout is based on direct mass spectrometric detection of the test ligand. The study presented here describes the development and validation of a highly sensitive, rapid, and robust MS binding assay for the quantification of the binding of the metabotropic glutamate 5 (mGlu5) negative allosteric modulator (NAM), MPEP (2-methyl-6-phenylethynylpyridine) at the mGlu5 allosteric binding site. The LC-ESI-MS/MS (liquid chromatography-electrospray ionization-tandem mass spectrometric) analytical method was established and validated with a deuterated analogue of MPEP as an internal standard. The developed MS binding assay described here allowed for the determination of MS binding affinity estimates that were in agreement with affinity estimates obtained from a tritiated MPEP radioligand saturation binding assay, indicating the suitability of this methodology for determining affinity estimates for compounds that target mGlu5 allosteric binding sites. Graphical abstract
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