BioMolecular Analysis, Department of Chemistry and Pharmaceutical Sciences, Faculty of Sciences, VU University Amsterdam, De Boelelaan 1083, 1081HV Amsterdam, The Netherlands.
J Chromatogr B Analyt Technol Biomed Life Sci. 2011 Jun 15;879(20):1781-8. doi: 10.1016/j.jchromb.2011.04.023. Epub 2011 Apr 28.
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.
建立了一种基于磁性珠的亲和选择方法，用于筛选混合物和纯化合物文库中的乙酰胆碱结合蛋白（AChBP）配体。该方法如下工作：在溶液中使 His 标记的 AChBP 与潜在配体孵育后，加入钴（II）涂层的顺磁珠，然后通过注射将形成的珠-AChBP-配体复合物从溶液中取出，并在外置可调磁铁中捕获在 LC 管中。然后将非配体洗涤到废物中，然后用变性溶液冲洗将配体洗脱到 SPE 小柱上。最后，通过 SPE-LC-MS 分析鉴定配体。当前方法的优点是在溶液中孵育，然后固定化 AChBP 配体捕获，并能够将磁性珠系统用作可移动/在线可传输的亲和 SPE 材料。该系统经过优化，然后成功地用于鉴定作为纯化合物注射的 AChBP 配体，以及用于混合物中配体的捕捞。用 AChBP 作为靶蛋白获得的结果可靠地区分了 pK(i) 值至少为 6.26 至 8.46 的配体和非配体。
