Development and validation of a mass spectrometry binding assay for mGlu5 receptor


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.

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We thank Prof. Jean-Philippe Pin (IGF, University of Montpellier, CNRS, INSERM, Montpellier) for scientific support and contributions to this work.


This work was financially supported by FEDER/Ministerio de Ciencia, Innovación y Universidades - Agencia Estatal de Investigación (CTQ2017-89222-R and PCI2018-093047), by the Catalan government (2017SGR1604), by Neuron-ERANET, by the Agence Nationale de la Recherche (ANR-17-NEU3-0001 under the frame of Neuron Cofund) and by the Programme International de Collaboration Scientifique (PICS 08212) of the CNRS. AEB was supported by the Labex EpiGenMed (program « Investissements d’avenir », ANR-10-LABX-12-01).

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Ricart-Ortega, M., Berizzi, A.E., Catena, J. et al. Development and validation of a mass spectrometry binding assay for mGlu5 receptor. Anal Bioanal Chem (2020).

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  • MPEP
  • mGlu5 receptor
  • MS binding assays
  • Radioligand binding assays
  • Saturation assay