MS-Binding Assays: Kinetic, Saturation, and Competitive Experiments Based on Quantitation of Bound Marker as Exemplified by the GABA Transporter mGAT1

• Published in: ChemMedChem 2006-02, Vol.1 (2), p.208-217
• Main Authors: Zepperitz, Christine, Höfner, Georg, Wanner, Klaus T.
• Format: Article
• Language: English
• Subjects: Chromatography, Liquid; drug screening; GABA Plasma Membrane Transport Proteins - metabolism; GABA transporters; Kinetics; ligand-binding assays; liquid chromatography; Magnetic Resonance Spectroscopy; mass spectrometry; Radioligand Assay; Reproducibility of Results; Spectrometry, Mass, Electrospray Ionization
• ISSN: 1860-7179; 1860-7187
• DOI: 10.1002/cmdc.200500038

A new kind of binding assay is described in which the amount of a nonlabeled marker bound to the target is quantified by LC–ESI‐MS–MS. This new approach was successfully implemented with nonlabeled NO 711 as marker and the GABA transporter subtype mGAT1 as target. The native marker bound to the target was liberated from the receptor protein by methanol denaturation after filtration. A reliable and sensitive LC–ESI‐MS–MS method for the quantitation of NO 711 was developed, and data from mass spectrometric detection were analyzed by nonlinear regression. Kinetic MS‐binding experiments yielded values for k+1 and k−1, while in saturation MS‐binding experiments, Kd and Bmax values were determined. In competitive MS‐binding experiments, Ki values were obtained for various test compounds covering a broad range of affinities for mGAT1. All experiments were performed in 96‐well plate format with a filter plate for the separation step which improved the efficiency and throughput of the procedure. The method was validated by classical radioligand‐binding experiments with the labeled marker [3H2]NO 711 in parallel. The results obtained from MS‐binding experiments were found to be in good agreement with the results of the radioligand‐binding assays. The new kind of MS‐binding assay presented herein is further adapted to the conventional radioligand‐binding assay in that the amount of bound marker is securely quantified. This promises easy implementation in accordance with conventional binding assays without the major drawbacks that are inherent in radioligand or fluorescence binding assays. Therefore, MS‐binding assays are a true alternative to classical radioligand‐binding assays. No need to label: A new kind of MS‐binding assay allows the amount of a nonlabeled marker bound to the target to be determined by LC–ESI‐MS–MS instead of by quantitation of nonbound marker, as done in previous studies. The new MS‐binding assay is methodologically similar to classical radioligand‐binding studies, but is free of the major drawbacks inherent in the latter.