基于SPME-配体置换反应识别有机污染物的潜在靶蛋白及毒性效应研究

有机污染物与蛋白的结合是在生物体内发挥毒性作用的关键环节，识别靶蛋白对研究有机污染物的毒性至关重要。传统的亲和纯化等识别手段需修饰小分子配体或固定蛋白受体，这将影响配体的空间位阻或受体的构象，不仅操作复杂，且会导致亲和作用的偏差。免标记的结构蛋白组学可实现高通量的靶蛋白筛查，但也面临分析成本高、数据解析难度大等不足。本项目拟从小分子配体的检测出发，选择与蛋白具有高亲和力的小分子作为竞争性配体，置换与受体蛋白结合的有机污染物，通过固相微萃取（SPME）技术监测置换前后有机污染物的游离浓度。通过逐一添加竞争性配体，实现复杂生物样品中有机污染物的靶向蛋白识别，利用表面等离子共振分析和分子对接模拟从分子层面揭示其结合机制，结合代谢组学分析阐释有机污染物的毒性效应。本研究不仅可提供一种快速识别配体-受体相互作用的免标记方法，也将有助于实现有机污染物潜在靶蛋白的快速自动化筛查,揭示其潜在毒性作用。

The binding of organic pollutants and proteins is a key point to exert toxic effects in organisms. Identification of target proteins is very important to study the toxicity of organic pollutants. Traditional identification methods such as affinity purification require modification of ligands or immobilization of receptors. This would affect the steric hindrance of ligands or the conformation of receptors, which is complicated and could bias the affinity result. Label-free proteomics can achieve high-throughput screening, but it also faces the disadvantages of high analysis cost and difficulty in data analysis. Based on the detection of small molecular ligands, this project plans to select small molecules with high affinity to proteins as competitive ligands, displace organic pollutants bound to the receptor protein, and detect the free organic pollutants before and after displacement reaction through solid phase microextraction (SPME), to identify potential target proteins of organic pollutants in complex biological samples by adding competitive ligands one by one. For unknown organic pollutants-protein receptors, surface plasmon resonance and molecular docking are used to explore their affinity mechanism, combined with non-targeted metabolomics to reveal the toxic effects of organic pollutants. This project not only provides a new label-free method for rapid identification of ligand-receptor interactions, but also helps to achieve rapid automated screening of target proteins, and reveals the toxic mechanism of organic pollutants.