We synthesized a novel linker (1) with biotin, alkyne and amino groups for the identification of target proteins using a small molecule that contains an azide group (azide probe). The alkyne in the linker bound the azide probe via an azide-alkyne Huisgen cycloaddition. A protein cross-linker effectively bound the conjugate of the linker and an azide probe with a target protein. The covalently bound complex was detected by western blotting. Linker 1 was applied to a model system using an abscisic acid receptor, RCAR/PYR/PYL (PYL). Cross-linked complexes of linker 1, the azide probes and the target proteins were successfully visualized by western blotting. This method of target protein identification was more effective than a previously developed method that uses a second linker with biotin, alkyne, and benzophenone (linker 2) that acts to photo-crosslink target proteins. The system developed in this study is a method for identifying the target proteins of small bioactive molecules and is different from photo-affinity labelling.

我们使用具有叠氮基团的小分子（叠氮化物探针）合成了具有生物素，炔烃和氨基的新型接头（1），用于鉴定目标蛋白。连接子中的炔通过叠氮化物-炔烃Huisgen环加成结合叠氮化物探针。蛋白质交联剂有效地将接头和叠氮化物探针的缀合物与靶蛋白结合在一起。通过蛋白质印迹检测共价结合的复合物。将连接子1应用于使用脱落酸受体RCAR / PYR / PYL（PYL）的模型系统。接头1的交联复合物，叠氮化物探针和目标蛋白已成功通过Western blotting可视化。这种目标蛋白鉴定方法比以前开发的方法更有效，该方法使用了带有生物素，炔烃和二苯甲酮的第二种连接子（连接子2），可对目标蛋白进行光交联。在这项研究中开发的系统是一种识别小生物活性分子目标蛋白的方法，它不同于光亲和标记。