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

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Abstract

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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Acknowledgments

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

Funding

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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Authors and Affiliations

  1. MCS, Laboratory of Medicinal Chemistry & Synthesis, Department of Biological Chemistry, Institute for Advanced Chemistry of Catalonia (IQAC-CSIC), Jordi Girona 18, 08034, Barcelona, Spain

    Maria Ricart-Ortega, Juanlo Catena, Lourdes Muñoz, Carmen Serra & Amadeu Llebaria

  2. IGF, CNRS, INSERM, University of Montpellier, 34094, Montpellier, France

    Maria Ricart-Ortega, Alice E. Berizzi, Fanny Malhaire, Guillaume Lebon & Cyril Goudet

  3. SIMchem, Service of Synthesis of High Added Value Molecules, Institute for Advanced Chemistry of Catalonia (IQAC-CSIC), Jordi Girona 18, 08034, Barcelona, Spain

    Lourdes Muñoz, Carmen Serra & Amadeu Llebaria

Authors
  1. Maria Ricart-Ortega
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  2. Alice E. Berizzi
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Correspondence to Cyril Goudet or Amadeu Llebaria.

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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 412, 5525–5535 (2020). https://doi.org/10.1007/s00216-020-02772-9

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  • DOI: https://doi.org/10.1007/s00216-020-02772-9

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