Development of ultra-miniaturized weak affinity chromatography coupled to mass spectrometry as a high throughput fragment screening method against wild-type and purified membrane proteins embedded in biomimetic membranes

Background: Membrane proteins, which make up approximately 30 % of the proteome, are important drug targets but present many challenges in drug discovery, including limited production rates, low final yields of pure and functionally folded proteins, and instability in aqueous media. The problems encountered with membrane proteins are even more critical in the Fragment Based Drug Discovery, where the discovery of potential drug candidates is hampered by the limited availability of efficient methods for rapid screening of weak fragmentprotein interactions. Results: In this work, we propose the coupling of miniaturized weak affinity chromatography with mass spectrometry (nano-WAC-MS) as an innovative strategy for the rapid screening of fragments capable of weak binding to a selected membrane protein. An integral membrane protein (AA2AR) was incorporated into biotinylated nanodiscs, which were subsequently immobilized on a miniaturized monolithic streptavidin column (75 mu m i.d., 300 nL volume). The coupling of these miniaturized affinity columns (each consuming less than 1 mu g of protein) to mass spectrometry (MRM mode) has been optimized to maximize the low affinity range and increase throughput so that 150 fragments can be injected in a single analysis, with a DMSO content as high as 10 %, with no influence on the affinity. Hits are identified by comparing their retention with that measured on control columns prepared with empty nanodiscs. Significance: The results of this screening are compared with those obtained by NMR and newly identified hits are confirmed by either competition experiments or frontal affinity experiments. We show that this nanodisc-based strategy, which provides a stable and native-like lipid environment for the protein (columns can be used for several days), should also work with other membrane proteins embedded in nanodiscs.