Accelerating the Throughput of Affinity Mass Spectrometry-Based Ligand Screening toward a G Protein-Coupled Receptor

Affinity mass spectrometry (MS) enables rapid screening of compound mixtures for ligands bound to a specific protein target, yet its current throughput is limited to individually assay pools of 400–2000 compounds. Typical affinity MS screens implemented in pharmaceutical industry laboratories identify putative ligands based on qualitative analysis of compound binding to the target whereas no quantitative information is acquired to discriminate high- and low-affinity ligands in the screening phase. Furthermore, these screens require purification of a stabilized form of the protein target, which poses a great challenge for membrane receptor targets. Here, we describe a new, potentially general affinity MS strategy that allows screening of 20,000 compounds in one pool for highly efficient ligand discovery toward a G protein-coupled receptor (GPCR) target. Quantitative measurement of compound binding to the receptor enables high-affinity ligand selection using both the purified receptor and receptor-embedded cell membranes. This high-throughput, label-free and quantitative affinity MS screen resulted in discovery of three new antagonists of the A2A adenosine receptor.