Detection and identification of huwentoxin-IV interacting proteins by biotin-avidin chemistry combined with mass spectrometry

Background : Numerous spider toxins are of interest as tools for neurophysiological research or as lead molecules for the development of pharmaceuticals and insecticides. Direct detection and identification of the interacting proteins of a spider toxin are helpful for its action-mechanism analysis and practical application. The present study employed a combinative strategy for the analysis of interacting proteins of huwentoxin-IV (HWTX-IV), a peptidic neurotoxin from the venom of the spiderSelenocosmia huwena.Results : HWTX-IV was first lightly labeled with biotin under the optimized mild experimental conditions and the toxin labeled with a single biotin group (monobiotinylated HWTX-IV) was demonstrated by electrophysiological experiments to retain its original bioactivity and was used in combination with far-western blotting to detect its interacting proteins. Comparative experiments indicated that some membrane proteins from rat neuromuscular junction preparations bind to monobiotinylated HWTX-IV after being transferred onto a PVDF membrane from the SDS-gel. With capillary high performance liquid chromatography-tandem mass spectrometry, several membrane proteins with which HWTX-IV potentially interacted were identified from the preparations and then bioinformatically analyzed.Conclusions : This work has provided not only a new insight into the action mechanism of HWTX-IV but also a reference technology for the relevant researches.

背景：众多蜘蛛毒素作为神经生理学研究工具，或是开发药物与杀虫剂的先导分子，均受到广泛关注。直接检测并鉴定蜘蛛毒素的相互作用蛋白，有助于解析其作用机制并推动实际应用。本研究针对虎纹捕鸟蛛（Selenocosmia huwena）毒液中的肽类神经毒素虎纹毒素-IV（huwentoxin-IV, HWTX-IV），采用组合式研究策略对其相互作用蛋白进行分析。
结果：首先在优化后的温和实验条件下对HWTX-IV进行轻度生物素标记；经电生理实验验证，仅结合单个生物素基团的标记产物（单生物素化HWTX-IV）保留了原有的生物活性，随后将其与远西方印迹（far-western blotting）技术结合，用于检测其相互作用蛋白。对照实验显示，大鼠神经肌肉接头制剂中的部分膜蛋白经SDS凝胶电泳分离并转印至PVDF膜后，可与单生物素化HWTX-IV结合。通过毛细管高效液相色谱-串联质谱（capillary high performance liquid chromatography-tandem mass spectrometry），从该制剂中鉴定出数种HWTX-IV的潜在相互作用膜蛋白，并对其进行了生物信息学分析。
结论：本研究不仅为解析HWTX-IV的作用机制提供了全新视角，也为相关领域研究提供了可借鉴的技术方法。