Screening of protein-ligand interactions using dynamic protein-affinity chromatography solid-phase extraction-liquid chromatography-mass spectrometry.
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.
Jonker
Niels
N
VU University Amsterdam, Faculty of Sciences, Department of Chemistry, BioMolecular Analysis Group, De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands. N.Jonker@few.vu.nl
Kool
Jeroen
J
Krabbe
Johannes G
JG
Retra
Kim
K
Lingeman
Henk
H
Irth
Hubertus
H
D001667:Binding, Competitive; D002846:Chromatography, Affinity / Q000379:methods*; D047628:Estrogen Receptor alpha / Q000737:chemistry*; D008024:Ligands; D013058:Mass Spectrometry / Q000379:methods*; D011485:Protein Binding; D011506:Proteins / Q000737:chemistry*; D052616:Solid Phase Extraction / Q000379:methods*
D047628:Estrogen Receptor alpha; D008024:Ligands; D011506:Proteins