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 rob...

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Bibliographic Details
Published in:Analytical and bioanalytical chemistry 2020-09, Vol.412 (22), p.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: Article
Language:English
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
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Description
Summary: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
ISSN:1618-2642
1618-2650
DOI:10.1007/s00216-020-02772-9