Mass spectrometry-based ligand binding assays on adenosine A1 and A2A receptors.

A Massink,G Spijksma,J Louvel,A P Ijzerman,D Guo,M Holzheimer,A Hölscher,T Hankemeier

doi:10.1007/s11302-015-9477-0

Abstract

Conventional methods to measure ligand-receptor binding parameters typically require radiolabeled ligands as probes. Despite the robustness of radioligand binding assays, they carry inherent disadvantages in terms of safety precautions, expensive synthesis, special lab requirements, and waste disposal. Mass spectrometry (MS) is a method that can selectively detect ligands without the need of a label. The sensitivity of MS equipment increases progressively, and currently, it is possible to detect low ligand quantities that are usually found in ligand binding assays. We developed a label-free MS ligand binding (MS binding) assay on the adenosine A1 and A2A receptors (A1AR and A2AAR), which are well-characterized members of the class A G protein-coupled receptor (GPCR) family. Radioligand binding assays for both receptors are well established, and ample data is available to compare and evaluate the performance of an MS binding assay. 1,3-Dipropyl-8-cyclopentyl-xanthine (DPCPX) and 4-(2-((7-amino-2-(furan-2-yl)-[1,2,4]triazolo[1,5-a]-[1,3,5]triazin-5-yl)amino)ethyl)phenol (ZM-241,385) are high-affinity ligands selective for the A1AR and A2AAR, respectively. To proof the feasibility of MS binding on the A1AR and A2AAR, we first developed an MS detection method for unlabeled DPCPX and ZM-241,385. To serve as internal standards, both compounds were also deuterium-labeled. Subsequently, we investigated whether the two unlabeled compounds could substitute for their radiolabeled counterparts as marker ligands in binding experiments, including saturation, displacement, dissociation, and competition association assays. Furthermore, we investigated the accuracy of these assays if the use of internal standards was excluded. The results demonstrate the feasibility of the MS binding assay, even in the absence of a deuterium-labeled internal standard, and provide great promise for the further development of label-free assays based on MS for other GPCRs.Electronic supplementary materialThe online version of this article (doi:10.1007/s11302-015-9477-0) contains supplementary material, which is available to authorized users.

Highlights

Conventional methods to measure ligand-receptor binding parameters typically require labeled probes such as radiolabeled [1] or fluorescently labeled ligands [2]
The results demonstrate the feasibility of the mass spectrometry (MS) binding assay, even in the absence of a deuterium-labeled internal standard, and provide great promise for the further development of label-free assays based on MS for other G protein-coupled receptor (GPCR)
We developed and validated MS binding assays for the adenosine A1 and A2A receptors

Summary

Introduction

Conventional methods to measure ligand-receptor binding parameters typically require labeled probes such as radiolabeled [1] or fluorescently labeled ligands [2]. The development of the mass spectrometry (MS) binding assay by the group of Wanner permits to measure binding of an unlabeled ligand to its target [4]. Instead of the radiolabeled ligand in radioligand binding assays, an unlabeled marker. The amount of marker ligand bound to the target receptor is detected by mass spectrometry. The marker ligand still has to fulfill the same requirements as radioligands: high affinity and selectivity for the target and low non-specific binding [5]. It is practical to choose a ligand for MS binding applications that has already been validated as a good radioligand. This ensures a straightforward validation of an MS binding assay by comparing it to existing radioligand binding assays

Methods

Results

Discussion

Conclusion