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

N. Jonker, J. Kool, J.G. Krabbe, K. Retra, H. Lingeman, H. Irth

Research output: Contribution to JournalArticleAcademicpeer-review

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 (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 K
Original languageEnglish
Pages (from-to)71-7
JournalJournal of Chromatography A
Volume1205
Issue number1-2
DOIs
Publication statusPublished - 2008

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