二维液相色谱质谱代谢组学新方法及其在淋巴瘤研究中的应用

There are a large number of metabolites with complicated physical and chemical properties and wide concentration distributions, which present a great challenge for their rapid and comprehensive analysis. Two-dimensional liquid chromatography (2DLC) - mass spectrometry method can greatly enlarge the coverage of metabolite analysis, but the analytical time is too long and also there is a dilution effect. This project is to construct a rapid alternated 2DLC system for the simultaneous analysis of polar, medium polar metabolome and lipidome. During the construction of the two-dimensional system, firstly, the matching of column systems should be solved, with the mixed-mode columns preferred for the retention and separation of polar, medium polar compounds. Then, by choosing the optimal trap column, the lipid analysis column and the mixed-mode column is integrated. Further, the efficient and comprehensive serum sample preparing method is to be developed for acquisition of serum metabolic spectra employing the 2DLC - mass spectrometry system. Finally, the research on serum metabolic disturbance and diagnosis biomarker for NK/T cell lymphoma will be finally exerted through integrating the metabolome and lipidome data. The execution of this project will not only provide a solution for the high-coverage and high-throughput metabolomics analysis, but also put forward a new strategy for future method development.

代谢物种类繁多、理化性质复杂、浓度分布范围宽等特点对其快速全面分析造成了巨大挑战。二维液相色谱（2DLC）质谱联用方法理可以大幅增加代谢物分析覆盖面，但存在分析时间过长和稀释效应问题。本项目拟构建快速交替2DLC质谱系统，首次实现经一次进样同时分析极性，中等极性代谢组和非极性脂类组。二维系统构建中，首先需要解决分离柱系统匹配的问题，拟侧重选择混合模式色谱柱以用于极性及中等极性代谢物的保留和分离；继而选取最佳捕集柱，整合脂质分析柱与混合模式柱；进一步开发高效全面的血清预处理方法，利用该二维系统进行血清代谢谱的构建。最终通过整合血清代谢组与脂质组数据，研究NK/T细胞淋巴瘤代谢紊乱和临床诊断标志物。本项目的实施，不仅能满足代谢组学方法高代谢物覆盖度和高分析通量的要求，同时也为方法开发中调和分析覆盖度和分析通量的矛盾提供了新思路。

针对代谢组学分离检测方法需要兼顾代谢物分析覆盖度、灵敏度和分析通量的问题, 我们建立了一种新型快速交替2DLC质谱分离检测系统，可用于极性、中等极性代谢组和疏水性脂质组同时分离分析。基于该系统建立的2DLC-MS方法，我们获取了全面的血清代谢谱。进一步优化柱系统建立的新方法，可实现全链长酰基CoA同时检测。这些2DLC-MS新方法的建立不仅可以实现单次进样中检测覆盖度和分析通量的大幅提升，同时也降低了对样本用量的需求，可用于珍贵样本的分析。.将上述交替2DLC-MS方法应用于食管癌患者血清代谢紊乱研究，发现食管癌患者血清中存在脂质代谢失调这一代谢特征及Chok酶高表达与食管癌病人预后密切相关。进一步联合全面代谢组和脂质组学研究，揭示了NK/T细胞淋巴瘤患者血清中发生了全面的代谢紊乱，并筛选了具有潜在诊断能力的3-Hydroxypyridine 和TCDCA的组合标志物，SM(d20:0/20:2)和ChE(18:2)组合脂质标志物，这些标志物可辅助临床诊断，同时为NK/T细胞淋巴瘤的发病机理研究提供线索。本项目相关工作结果在国际会议上交流了1次，已发表SCI收录论文4篇，申请发明专利3项。