快速全二维液相色谱-质谱联用系统的构建及其用于转基因水稻的代谢组学研究

Comprehensive two-dimensional liquid chromatography (LC×LC) is a powerful tool for analysis of complex biological samples. However, the very long timescale (typically several hours to tens of hours) of LC×LC is clearly its chief drawback. In the project, the carbon phase coated on the high surface area of alumina (C/Al2O3) and silica particles (C/Al/SiO2) will be developed, which can provide higher retentivity and comparable selectivity than C/ZrO2. Based on the novel high temperature columns, a fast comprehensive two-dimensional liquid chromatography/mass spectrometry system (LC×LC-MS) will be carried out by utilizing high temperature and high pressure to decrease the time needed to do the second dimension.The system can provide a high peak capacity of two dimensional separations in a relatively short analysis time..In addition, LC×LC generates rich information and complex peak patterns that require automated processing for peak alignment. A novel peak alignment algorithm will be developed using a distance and spectrum correlation optimization method. This algorithm can detect and merge multiple peak entries of the same metabolite into one peak entry in each input peak list.A progressive retention time map searching method will be used to align metabolite peaks in all samples together based on optimization of the Euclidean distance and mass spectrum similarity. .The rice introduced cryIAc and sck genes can improve the insect resistance. Metabolic profiles of the wild and transgenic rice will be obtained by the above LC×LC-MS system. The significantly distinguishable metabolites between transgenic and wild rice grains will be picked out and identified. The unintended effects related to gene modification can be assessed.

针对目前全二维液相色谱存在的分析时间长、峰容量损失和稀释效应等制约因素，本项目拟通过化学气相沉积法将碳沉积在多孔Al2O3和Al/SiO2微球表面，研制高比表面、高柱效的新型耐高温色谱柱。将其作为第二维柱，通过高温和高压的结合实现第二维的快速分析，从而增加第一维峰的调制次数，提高总峰容量和缩短分析时间，构建高通量、高分辨、高灵敏的快速全二维液相色谱-质谱联用系统。结合欧氏距离和谱图相关性优化算法，开发适用于全二维液相色谱-质谱系统的峰匹配和谱图对比软件，解决复杂二维代谢数据处理难题。以抗虫性转基因水稻为研究对象，利用建立的全二维系统对转基因水稻和野生型水稻进行代谢组学分析，建立水稻的二维代谢指纹谱，找到差异代谢物，监测转基因水稻是否引入了非预期的代谢反应。

近年来在线二维液相色谱方法发展迅速，已成为复杂样品分离分析的强有力工具。然而，由于其运行时间长，峰容量损失和稀释效应等制约因素限制了它的实际应用。针对上述难题，本项目从新型液相色谱柱的研制、基于不同模式的多维液相色谱-质谱系统的构建及在植物代谢组学中应用等多个方面开展研究工作。首先研制了一系列基于离子液体表面修饰的新型液相色谱柱，包括咪唑两性离子的核-壳型亲水作用色谱柱、葡糖胺基离子液体的混合模式色谱柱、双阳离子液体色谱柱和三阳离子液体色谱柱。这些色谱柱对核苷、有机酸、水溶性维生素和黄酮类物质显示更有效的保留和更好的选择性。其次构建了不同模式的多维液相色谱-质谱系统，包括停留模式的二维液相色谱-质谱系统用于血样中脂质轮廓的分析，两维分别选择亲水作用色谱（HILIC）和反相色谱（RPLC）分离机理，根据脂质分子的极性头基和非极性尾基的不同进行分离。代谢组和脂质组同时分离的二维液相色谱-质谱系统，使用捕集柱实现复杂样品中代谢组和脂质组组份的分离和转移，结合后续分别针对代谢组和脂质组的平行柱分离，实现一次进样同时有效的分离代谢组和脂质组代谢物。在线三维液相色谱-质谱系统，通过引入第一维液相色谱，将复杂样品按其组分性质差异分成若干馏分，降低了复杂样品体系的复杂度，结合后续经典全二维液相色谱分离系统，实现了三维液相色谱系统卓越的分离能力。最后将建立的分析系统成功用于人参中三萜类皂甙代谢轮廓以及转基因水稻代谢组学研究中，取得了良好的结果。已发表SCI收录论文16篇，申请发明专利6项（授权1项），培养博士生2人。

内容获取失败，请点击重试