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

质谱（MS）结合测定是放射性配体或荧光结合测定的无标记替代方法，因此读数基于测试配体的直接质谱检测。此处介绍的研究描述了用于定量代谢型谷氨酸5（mGlu5）负变构调节剂（NAM），MPEP（2-甲基-6-苯基乙炔基吡啶）位于mGlu5变构结合位点。建立了LC-ESI-MS / MS（液相色谱-电喷雾电离串联质谱）分析方法，并以MPEP的氘代类似物作为内标进行了验证。

图形概要