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

2008; Elsevier BV; Volume: 1205; Issue: 1-2 Linguagem: Inglês

10.1016/j.chroma.2008.07.089

1873-3778

Niels Jonker, Jeroen Kool, Johannes G. Krabbe, Kim Retra, H. Lingeman, Hubertus Irth,

Monoclonal and Polyclonal Antibodies Research

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 (ERα) 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 Kd = 0.1–1 mM). The same setup also provides the possibilities to measure EC50 curves of both weak and strong binders.