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

• Published in: Analytical and bioanalytical chemistry Vol. 412; no. 22; pp. 5525 - 5535
• Main Authors: Ricart-Ortega, Maria, Berizzi, Alice E., Catena, Juanlo, Malhaire, Fanny, Muñoz, Lourdes, Serra, Carmen, Lebon, Guillaume, Goudet, Cyril, Llebaria, Amadeu
• Format: Journal Article
• Language: English
• Published: Berlin/Heidelberg Springer Berlin Heidelberg 01.09.2020; Springer; Springer Nature B.V; Springer Verlag
• Subjects: Affinity; Allosteric properties; Allosteric Site; Analytical Chemistry; Assaying; Binding sites; Biochemistry; Biochemistry, Molecular Biology; Characterization and Evaluation of Materials; Chemistry; Chemistry and Materials Science; Chromatography, High Pressure Liquid - methods; Deuteration; Estimates; Fluorescence; Food Science; Glutamic acid receptors (metabotropic); HEK293 Cells; Humans; Ionization; Ions; Laboratory Medicine; Life Sciences; Ligands; Limit of Detection; Liquid chromatography; Mass spectrometry; Mass spectroscopy; Monitoring/Environmental Analysis; Protein Binding; Radioligand Assay; Receptor, Metabotropic Glutamate 5 - metabolism; Reproducibility of Results; Research Paper; Scientific equipment and supplies industry; Scientific imaging; Spectrometry, Mass, Electrospray Ionization - methods; Spectroscopy; Tandem Mass Spectrometry - methods; United States; Spain; MS binding assays; Saturation assay; MPEP; mGlu5 receptor; Radioligand binding assays
• ISSN: 1618-2642; 1618-2650
• DOI: 10.1007/s00216-020-02772-9

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