title: Online magnetic bead based dynamic protein affinity selection coupled to LC-MS for the screening of acetylcholine binding protein ligands.
abstract: A magnetic beads based affinity-selection methodology towards the screening of acetylcholine binding protein (AChBP) binders in mixtures and pure compound libraries was developed. The methodology works as follows: after in solution incubation of His-tagged AChBP with potential ligands, and subsequent addition of cobalt (II)-coated paramagnetic beads, the formed bead-AChBP-ligand complexes are fetched out of solution by injection and trapping in LC tubing with an external adjustable magnet. Non binders are then washed to the waste followed by elution of ligands to a SPE cartridge by flushing with denaturing solution. Finally, SPE-LC-MS analysis is performed to identify the ligands. The advantage of the current methodology is the in solution incubation followed by immobilized AChBP ligand trapping and the capability of using the magnetic beads system as mobile/online transportable affinity SPE material. The system was optimized and then successfully demonstrated for the identification of AChBP ligands injected as pure compounds and for the fishing of ligands in mixtures. The results obtained with AChBP as target protein demonstrated reliable discrimination between binders with pK(i) values ranging from at least 6.26 to 8.46 and non-binders.
authors.affiliation: BioMolecular Analysis, Department of Chemistry and Pharmaceutical Sciences, Faculty of Sciences, VU University Amsterdam, De Boelelaan 1083, 1081HV Amsterdam, The Netherlands. lionel.pochet@fundp.ac.be
authors.forename: Lionel
authors.initials: L
authors.lastname: Pochet
authors.forename: Ferry
authors.initials: F
authors.lastname: Heus
authors.forename: Niels
authors.initials: N
authors.lastname: Jonker
authors.forename: Henk
authors.initials: H
authors.lastname: Lingeman
authors.forename: August B
authors.initials: AB
authors.lastname: Smit
authors.forename: Wilfried M A
authors.initials: WM
authors.lastname: Niessen
authors.forename: Jeroen
authors.initials: J
authors.lastname: Kool
mesh_terms: D002352:Carrier Proteins / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D002853:Chromatography, Liquid / Q000379:methods*; D055808:Drug Discovery / Q000379:methods*; D004867:Equipment Design; D008280:Magnetics; D013058:Mass Spectrometry; D008863:Microspheres; D004364:Pharmaceutical Preparations / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D054852:Small Molecule Libraries / Q000737:chemistry / Q000378:metabolism; D052616:Solid Phase Extraction / Q000379:methods*
chemical_list: C428944:AChBP protein, Lymnaea; D002352:Carrier Proteins; D004364:Pharmaceutical Preparations; D054852:Small Molecule Libraries