Online magnetic bead based dynamic protein affinity selection coupled to LC-MS for the screening of acetylcholine binding protein ligands

L. Pochet, F. Heus, N. Jonker, H. Lingeman, W.M.A. Niessen, J. Kool, A.B. Smit

Résultats de recherche: Contribution à un journal/une revueArticle

Résumé

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 values ranging from at least 6.26 to 8.46 and non-binders.
langueAnglais
Pages1781-1788
Nombre de pages8
journalJournal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences
Volume879
Numéro20
Les DOIs
étatPublié - 15 juin 2011

Empreinte digitale

Acetylcholine
Carrier Proteins
Screening
Ligands
Proteins
Binders
Tubing
Cobalt
Magnets
Libraries
Injections

Citer ceci

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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 values ranging from at least 6.26 to 8.46 and non-binders.",
author = "L. Pochet and F. Heus and N. Jonker and H. Lingeman and W.M.A. Niessen and J. Kool and A.B. Smit",
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Online magnetic bead based dynamic protein affinity selection coupled to LC-MS for the screening of acetylcholine binding protein ligands. / Pochet, L.; Heus, F.; Jonker, N.; Lingeman, H.; Niessen, W.M.A.; Kool, J.; Smit, A.B.

Dans: Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences, Vol 879, Numéro 20, 15.06.2011, p. 1781-1788.

Résultats de recherche: Contribution à un journal/une revueArticle

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AU - Heus,F.

AU - Jonker,N.

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AU - Niessen,W.M.A.

AU - Kool,J.

AU - Smit,A.B.

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