新型靶向线粒体蛋白质活性探针的合成及应用

Mitochondria are energy-producing organelles performed a remarkably wide range of cellular functions, and mitochondrial dysfunction is linked to a diverse set of human diseases. The annotation of mitochondrial functions always attracts researchers’ attentions. Conclusive identification of mitochondrial proteins is essential to characterize new biological processes in mitochondria. However, it is still difficult and challenging, including tedious fractionation procedures and high possibility of false positive during isolating mitochondria from living cells and limited coverage using chemical biology tools. Herein, in this proposal, we are going to develop a series of smart probes composed of (Aza)quinone-methide function, mitochondria-targeting modalities, light control, reactions inside mitochondria and bioorthogonal activity-based probe profiling. These probes are designed that penetrate biomembranes and spontaneously concentrate in mitochondria, where proximal protein captures are triggered by light or active components in mitochondria. These probes combined with mass spectrometry will facilitate identification of mitochondrial proteins in live cultured cells and tissues. The successful completion of this project would provide not only the exploration of new mitochondrial proteins, but also new insight in the functions of mitochondria.

线粒体是细胞能量工厂，与许多疾病的发生发展相关，一直以来都是研究的热点。因此，完善线粒体蛋白“目录”是深入研究线粒体的生物过程、疾病发病机理的必要基础和前提条件。然而，现有的线粒体蛋白质研究方法有着分离提取线粒体技术操作繁琐，假阳性高和化学生物方法提取量少等局限性。本项目拟构建线粒体蛋白特异性靶向分子探针，以具有触发式自降解释放及固载特性的（氮杂）亚甲基苯醌机制为核心，结合线粒体定位基团、光控手段、线粒体内部活性物质相关的化学反应及正交活性蛋白谱技术，设计合成一系列免分离、高效率的智能响应活性探针，用于活性细胞、组织内原位提取鉴定线粒体蛋白质，促进新的线粒体蛋白质的发现，进一步地完备线粒体蛋白质组库，为线粒体功能研究的突破提供更准确更科学的信息。

线粒体是真核细胞内一种重要的细胞器，是生物氧化过程的主要场所。研究表明，线粒体蛋白质水平的异常是功能紊乱的主要原因之一。但现有的线粒体蛋白质研究方法主要依赖于细胞器分离提取技术，操作繁琐，并且假阳性较高。本项目以构建简单、准确、高效地研究线粒体蛋白质组新工具为目标，从多功能探针设计思路出发，将线粒体靶向物质、光控或酶控触发基团及化学蛋白质组学结合，设计合成了特异性靶向线粒体蛋白质的活性探针，简化了线粒体分离步骤，并可用于活性细胞中原位分离提取捕获的蛋白质。我们利用蛋白质质谱鉴定技术分析了捕获的线粒体蛋白质。本项目经过三年的努力，基本完成了课题任务，达到了预期目标。本项目相关研究结果为线粒体功能研究提供了更丰富的信息。