基于稳定同位素标记衍生化的靶向脂质组学LC-MS分析方法

LC-MS based targeted lipidomics analysis greatly promotes the development of lipidomics. But it also faces the underlying problems of low detection sensitivity, the serious matrix effect, and lack of standards or isotope internal standards. The technique of stable isotope labeling derivatization (SILD) is one of the effective ways to solve the above problems. However, the reported SILD reagents are less, and hard to be synthesized. And also the LC-MS/MS analytical performance of reported SILD derivatives needs to be improved. Therefore, the design and synthesis of novel SILD reagents have become one of the key scientific problems in this field. This project intends to use cheap and commercial available 6-quinoline-acetic acid and rhodamine 6G as raw materials, and H/D haloalkanes as the source of isotopic labels, to prepare three kinds of SILD reagents (light/heavy, isobaric and iTRAQ-like) by using 1-3 steps common organic synthesis reactions. It can be realized that fatty acid, hydroxyl steroids, phosphatidylserines and hemolysis phosphatidylserines can be selectively labeled. On this basis, LC-MS/MS analytical methods of those lipids above mentioned will be developed. Combined with statistical analysis of bioinformatics, the research objects of this project about lipid biomarkers screening, metabolic pathways construction, the structural analysis of lipid isomers, new lipid metabolites findings, and high throughput tests of clinical samples will be studied. The development of this project will remarkably improve the accuracy and comprehensiveness about information collection and searching of lipidomics, and this will accelerate its rapid development.

基于LC-MS的靶向脂质组学分析极大地推进了脂质组学的发展，但也面临灵敏度低、基质效应严重、缺乏标准品或同位素内标的问题。稳定同位素标记衍生化(SILD)技术是解决上述问题的有效办法之一，但现有SILD试剂种类较少，且难于合成，标记物LC-MS/MS分析性能有待提高。因此，设计、合成新型SILD试剂就成为该领域的关键科学问题之一。本项目拟采用廉价易得的6-喹啉-乙酸和罗丹明6G为原料，H/D卤代烷引入同位素标签，设计1-3步常规有机反应，制备轻/重、等重、iTRAQ-like三类SILD试剂，实现脂肪酸、羟基类固醇、磷脂酰丝氨酸、溶血磷脂酰丝氨酸的选择性标记，在此基础上建立LC-MS/MS分析方法，再结合生物信息学统计分析，以实现脂质标志物筛选、代谢通路构建、异构体结构解析、新代谢物发现、临床样本高通量检测等目的。本项目的实施可显著提高脂质组学信息挖掘的准确性和全面性，进而推动其快速发展。

基于LC-MS的靶向脂质组学分析存在灵敏度低、基质效应严重、缺乏标准品或同位素内标的问题。稳定同位素标记衍生化(SILD)技术是解决这些问题的有效办法之一，在本项目资金的支持下，项目组人员首先从理论和实践角度对比探寻SILD母核结构，开展了6-喹啉乙酸、罗丹明类、吡哌酸/沙星类分子的色谱、质谱、光谱、合成反应性的对比实验，优选出罗丹明类作为本项目最主要的SILD试剂母核结构。项目重点开展了基于SILD的羟基类固醇类标志物、神经鞘氨醇酯类标志物、脂肪醛类标志物、磷脂酰丝氨酸、溶血磷脂酰丝氨酸的靶向脂质组学LC-MS分析方法开发，方法在脂质标志物筛选与定量、代谢通路构建、异构体分离分析、新代谢物分析方法、临床样本高通量定性/定量检测等方面有良好应用。同时开展了小分子疾病标志物代谢组学LC-MS分析方法研究，并将本项目开发的上述SILD技术应用到了暴露组学、药物分析领域。此外，受本项目在化学标记生物标志物分析方法及应用工作的启发，项目团队在化学/生物/纳米标记传感分析方面开展了一些工作，为酶生物标志物、小分子疾病标志物和环境标志物的分析方法提供了新思路和新途径。在项目的资助下，发表35篇论文（项目主持人赵先恩作为第一作者或通讯作者的论文33篇），其中入选ESI高被引论文4篇、热点论文1篇，授权发明专利3项，申请发明专利3项。