Screening of protein-ligand interactions using dynamic protein-affinity chromatography solid-phase extraction-liquid chromatography-mass spectrometry

Journal of Chromatography. a
Niels JonkerHubertus Irth

Abstract

A novel methodology is shown enabling the screening of mixtures of compounds for their affinity to a receptor protein. The system presented, dynamic protein-affinity chromatography solid-phase extraction (DPAC-SPE), overcomes the limitations of the existing methods by performing an incubation of the His-tagged protein with a mixture of possible ligands, which are still in their native conditions. This is followed by a fully automated affinity trapping step, coupled on-line to an LC-MS system in order to detect and identify the bound ligands. The system has been optimized using a commercially available on-line SPE system, using the estrogen receptor alpha (ERalpha) as model protein. A representative range of ligands with sub-nanomolar to millimolar affinities has been identified successfully from a mixture. The weakest binder that can be identified is norethindrone (approximately K(d)=0.1-1mM). The same setup also provides the possibilities to measure EC50 curves of both weak and strong binders.

References

Jul 5, 2001·Protein Expression and Purification·S EilerM Ruff
Jul 4, 2006·Nature Reviews. Drug Discovery·Steven A Hofstadler, Kristin A Sannes-Lowery
Aug 21, 2007·Journal of Chromatography. a·Jerome P Ferrance
Jan 29, 2008·Combinatorial Chemistry & High Throughput Screening·Yongsoo Choi, Richard B van Breemen

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