Coupling the Torpedo Microplate-Receptor Binding Assay with Mass Spectrometry to Detect Cyclic Imine Neurotoxins
Coupling the Torpedo Microplate-Receptor Binding Assay with Mass Spectrometry to Detect Cyclic Imine Neurotoxins
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DOI：
10.1021/ac3027564
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发表时间：
2012-12-04
影响因子：
7.4
通讯作者：
Molgo, Jordi
中科院分区：
文献类型：
--
作者：
Araoz, Romulo;Ramos, Suzanne;Molgo, Jordi
环亚胺神经毒素是一类新出现的甲藻神经毒素家族，是烟碱型乙酰胆碱受体的强效拮抗剂。我们根据烟碱型乙酰胆碱受体的竞争性激动剂/拮抗剂的作用机制，建立了一种靶向性功能检测方法，用于检测海洋亚胺类神经毒素。方法开发的关键步骤是将富含烟碱型乙酰胆碱受体的鱼雷电细胞膜固定在微板孔表面，并使用生物素化的α-银环蛇毒素作为示踪剂。环亚胺神经毒素以浓度依赖的方式竞争性地抑制生物素化的α-银环蛇毒素与鱼雷-烟碱乙酰胆碱受体的结合。微孔板-受体结合试验可以直接在贝类样品中快速检测环亚胺神经毒素的纳摩尔浓度。尽管受体结合试验对于检测以烟碱型乙酰胆碱受体为靶标的神经毒素具有高度的敏感性和特异性，但不能识别特定的分析物。为了解决微板-受体结合分析的低选择性问题，用甲醇洗脱了与包被的鱼雷烟碱受体紧密结合的环亚胺神经毒素，并用质谱仪鉴定了洗脱配体的化学性质。将鱼雷电细胞膜固定在微孔板表面是一种高通量的方法，可用于直接在贝类基质中检测针对烟碱型乙酰胆碱受体的神经毒素，具有很高的灵敏度和重复性。
Cyclic imine neurotoxins constitute an emergent family of neurotoxins of dinoflagellate origin that are potent antagonists of nicotinic acetylcholine receptors. We developed a target-directed functional method based on the mechanism of action of competitive agonists/antagonists of nicotinic acetylcholine receptors for the detection of marine cydic imine neurotoxins. The key step for method development was the immobilization of Torpedo electrocyte membranes rich in nicotinic acetylcholine receptors on the surface of microplate wells and the use of biotinylated-alpha-bungarotoxin as tracer. Cyclic imine neurotoxins competitively inhibit biotinylated-alpha-bungarotoxin binding to Torpedo-nicotinic acetylcholine receptors in a concentration-dependent manner. The microplate-receptor binding assay allowed rapid detection of nanomolar concentrations of cyclic imine neurotoxins directly in shellfish samples. Although highly sensitive and specific for the detection of neurotoxins targeting nicotinic acetylcholine receptors as a class, the receptor binding assay cannot identify a given analyte. To address the low selectivity of the microplate-receptor binding assay, the cyclic imine neurotoxins tightly bound to the coated Torpedo nicotinic receptor were eluted with methanol, and the chemical nature of the eluted ligands was identified by mass spectrometry. The immobilization of Torpedo electrocyte membranes on the surface of microplate wells proved to be a high-throughput format for the survey of neurotoxins targeting nicotinic acetylcholine receptors directly in shellfish matrixes with high sensitivity and reproducibility.