View All PostersP2037
Dynamic Protein Affinity Chromatography SPE-LC-MS Finding New Receptor Ligands in Complex MixturesPresenter Niels Jonker, Vrije Universiteit, Netherlands
Additional Authors: Kim Retra, Hans Krabbe, Jeroen Kool, Hubertus IrthConventional, microtitreplate-based High-Throughput Screening (HTS) assays have the drawback that no information is obtained on the chemical nature of the active ligand(s). In the present paper we describe an analytical screening methodology that provides information on both binding affinity and chemical structure of potential ligands in a single analysis. For this purpose, we have combined the solution-phase incubation of the protein target and the screening sample with the metal affinity chromatography isolation of the protein-ligand complex and the subsequent LC-MS determination of the dissociated active ligand(s). The screening set-up has been implemented and automated on a Spark Symbiosis Pharma workstation. Rather than covalently immobilizing the (His-tagged) protein target on a solid support as in conventional affinity chromatography, the current method relies on the isolation of the protein-ligand complex using a nickel(II) loaded immobilized metal affinity chromatography (IMAC) column. This dynamic protein affinity chromatography (DPAC) method allows the efficient separation of bound ligands from inactive compounds and overcomes the drawbacks of covalently immobilizing a high amount of target proteins. After dissociation of the protein-ligand complex, the Ni(II)-IMAC column is regenerated allowing the use of fresh protein for each screening measurement. We demonstrate a proof-of-principle for this screening concept using the His-tagged steroid binding domain of the estrogen receptor a as protein target and a variety of low to high affinity estrogens as model ligands. The present methodology has been fully characterized in terms of detection limits, within-day and between-day reproducibility, and can be in operation continuously and unattended for 72-hour sequences. The identification of active ligands is performed on the basis of MS spectrum matching using both MS and MS-MS spectra obtained with a quadrupole time-of-flight (QTOF) mass spectrometer.