基于高通量双重同位素电荷标记的真菌甾醇代谢质谱分析新方法研究

Steroid metabolism of fungus is one of the antifungal targets and resistant mechanism. This project aims to establish a systematic strategy for mass spectrometric analysis of steroid metabolism and related enzyme, we develop a new double isotopic charge labeling method based on isoquinoline/pyridine for selective labeling steroids in fungus, N-cationic tag is directly attached onto the target compounds, the detection limits and compatibility in further mass spectrometric analysis is significantly improved. The difference of steroid metabolism network in multiple fungus and drug treated fungus is high-throughput identified and quantified by comparing d0/d5 pairs and d0/d7 pair with matrix-assisted laser desorption/ionization Fourier transform mass spectrometry and nano-liquid chromatography-mass spectrometry. The isotopic labeling method will avoid the use of isotope-labeled internal standards and sample manipulation. The related metabolic enzyme is also analyzed and verified; the relationship among fungus steroid metabolism, metabolic enzyme and the resistance of antifungal drug is further systematical studied. This is the first double isotope labeling method used for quantitative analysis of alcoholic small molecules in the multiple groups. This project offers a systematic tool for investigating the potential drug, antifungal targets and the mechanism of resistance.

真菌细胞甾醇代谢是抗真菌药作用靶点和产生耐药性的机制之一。本项目旨在建立真菌甾醇代谢及相关酶的系统性质谱分析策略：开发新型异喹啉/吡啶双重同位素电荷标记方法，标记真菌细胞中甾醇，产生新的正电荷中心，提高质谱检测灵敏度和兼容性，结合基质辅助激光解吸离子化－傅立叶变换质谱和纳流液相-串联质谱，通过寻找相差5Da 和7Da的质谱峰对，实现高通量定性、定量分析多组耐药真菌细胞及在药物作用下甾醇代谢网络的差异，定量分析无需同位素内标和繁琐的分离纯化步骤。并对甾醇代谢酶变化进行分析和验证，进一步系统性研究真菌甾醇代谢、代谢酶及抗真菌药物耐药的相关性。该项目首次开发出双重同位素标记方法相对定量多组样本中醇类小分子，为筛选潜在抗真菌药物、作用靶点及耐药性机理研究提供系统性分析方法。

真菌细胞甾醇代谢是抗真菌药作用靶点和产生耐药性的机制之一。本项目建立了真菌甾醇代谢及相关酶的系统性质谱分析策略：开发新型异喹啉/吡啶双重同位素电荷标记方法和香豆素类新基质，标记真菌细胞中甾醇，产生新的正电荷中心，提高质谱检测灵敏度和兼容性，结合基质辅助激光解吸离子化－傅立叶变换质谱和纳流液相-串联质谱，通过寻找相差5Da 和7Da的质谱峰对，实现高通量定性、定量分析多组耐药真菌细胞及在药物作用下甾醇代谢网络的差异，定量分析无需同位素内标和繁琐的分离纯化步骤。并对甾醇代谢酶变化进行分析和验证，进一步系统性研究真菌甾醇代谢、代谢酶及抗真菌药物耐药的相关性。该项目首次开发出双重同位素标记方法相对定量多组样本中醇类小分子，为筛选潜在抗真菌药物、作用靶点及耐药性机理研究提供系统性分析方法。进一步结合质谱成像技术（Mass Spectrometry Imaging, MSI），在基质中加入内标，对药物及其代谢物的空间分布进行分子水平成像和相对定量分析，实现高时间分辨率监测药物的摄入和代谢变化。并与传统的高效液相色谱-串联质谱（HPLC-MS/MS）分析方法比较，比较两者相对定量结果的相关性。