Biophysical techniques that enable the screening and identification of weak affinity fragments against a target protein are at the heart of Fragment Based Drug Design approaches. In the case of membrane proteins, the crucial criteria for fragment screening are low protein consumption, unbiased conformational states and rapidity because of the difficulties in obtaining sufficient amounts of stable and functionally folded proteins. Here we show for the first time that lipidnanodisc systems (membrane-mimicking environment) and ultra-miniaturized affinity chromatography can be combined to identify specific small molecule ligands that bind to an integral membrane protein. The approach is exemplified using the AA2AR GPCR. Home-made affinity nano-columns modified with nanodiscs-embedded AA2AR (only about 1 µg of protein per column) are fully characterized by frontal chromatographic experiments. This method allows (i) to distinguish specific and unspecific ligand/receptor interactions, (ii) to assess dissociation constants, (iii) to identify the binding pocket of uncharacterized ligands using a reference compound with competition experiments. Weak affinity ligands with Kd in the low to high micromolar range be detected. At last, the applicability of this method is demonstrated with 6 fragments recently identified as ligands or non-ligands of AA2AR.

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Quil‐A is a purified extract of saponins with strong immunoadjuvant activity. While specific molecules have been identified and tested in clinical trials, Quil‐A is mostly used as a totum of the Quillaja Saponaria bark extract. Quality control of the extract stability is usually based on the monitoring of specific saponins, whereas the comparison of samples with an initial chromatogram seems more appropriate. A reference fingerprint based on comprehensive two‐dimensional liquid chromatography offers a rapid detection of nonconform samples. To fulfill quality control constraints, off‐line configuration using basic instrumentation was promoted. Hence, reversed‐phase liquid chromatography × reversed‐phase liquid chromatography and hydrophilic interaction chromatography × reversed‐phase liquid chromatography methods with ultraviolet and single‐quadrupole mass spectrometry detection were kinetically optimized. The reversed‐phase liquid chromatography × reversed‐phase liquid chromatography method used a pH switch between dimensions to maximize orthogonality. Despite diagonalization, it led to a high peak capacity of 831 in 2 h. On the other hand, the combination of hydrophilic interaction chromatography and reversed‐phase liquid chromatography offered a larger orthogonality but a lower, yet satisfactory peak capacity of 673. The advantages of both methods were illustrated on degraded samples, where the reversed‐phase liquid chromatography × reversed‐phase liquid chromatography contour plot highlighted the loss of fatty acid chains, while the hydrophilic interaction chromatography × reversed‐phase liquid chromatography method was found useful to evidence enzymatic loss of sugar moieties.