天然药物是创新药物研发的重要来源，但受限于技术瓶颈，天然药物靶标发现十分困难，严重限制了基于天然药物的创新药物研发。天然药物与靶蛋白的弱结合性，导致复合物在检测过程中易被破坏，是天然药物靶标发现的核心难点问题。本项目拟建立非变性质谱与解吸附电喷雾电离源串联新技术，突破药物-靶蛋白弱结合带来的检测困难，实现直接检测复合物质谱信号；建立非变性体系的在线酶切辅助靶标鉴定新技术，还原复合物在生物体内的结构状态，依据酶切效率受蛋白碰撞几率及结构的影响原理，实现对药物-靶蛋白结合位点的动态分析；建立化学交联技术进行结合位点的验证及定量检测。项目研究将为天然药物靶标发现特别是弱结合靶蛋白的发现提供共性技术，助推基于天然药物的创新药物研发和新靶标的发现。

Natural product is one of important sources for novel drug development. However, most of their targets remain unknown due to current technical limitations, which obstructs structural transformation and has become a bottleneck for further development. The weak binding between drug and its target protein is the major problem as it would be easily broken during detection. In this proposal, we suggest to develop a novel technique by combining Native-Mass Spectrometry (MS) with Desorption Electrospray Ionization (DESI) for direct detection of drug-protein complexes, which maintains weak bindings connected during detection. In addition, the establishment of online enzymatic digestion-assisted structural analysis is suitable for non-denaturing systems, which mimics the conformational states of complexes in cells or organisms and achieves dynamic analysis of binding sites. Finally, chemical crosslinking provides validation and quantitative analysis of binding sites and conformational changes. Therefore, the development of this proposed techniques would benefit for target discovery of natural drugs, especially for maintaining drug-target protein weak bindings during detection, which promotes the discovery of novel drug innovation and their target discovery.