基于同位素编码探针的LC-MS代谢轮廓分析新方法及其在肺癌标志物筛选中的应用

Liquid chromatography-mass spectrometry (LC-MS) has become a powerful tool for clinical research, environmental science, and so on. However, its feasibility and reliabilities is seriously challenged in metabolic profile analysis. In the non-targeted analysis, it is very difficult to extract the comprehensive features from high resolution mass spectrometry data. In the targeted analysis, it often seriously suffers from matrix effect, reducing the typical advantages of mass spectrometric detection in terms of selectivity and specificity. The development of simple and effective way to overcome these problems is of great significance to the LC-MS based metabolomics studies. In this project, we will develop series of novel isotope-coded probes (also kown as isotope-coded derivatization reagents) for amines and carboxylic acids. These probes are designed by incorporating 14N/15N coding or multiple isotopic coding into the aromatic fused imidazole structure. With the aid of these new probes, the isotope-assisted non-targeted and targeted LC-MS metabolic profile analysis method will be developed. The performance of LC-MS metabolic file analysis can be drastically improved by making full use of the advantages of isotope-coded probes during the data acquisition and preprocessing, which is favorable to providing the high quality of the data for the subsequent multivariate statistical analysis. For non-targeted metabolomics, the isotope-assisted LC-HRMS approaches enable global data extraction and feature annotation for true metabolites and can help to improve the probability and reliability for discovering biomarkers. For targeted metabolomics, the isotope-assisted LC-MS/MS approaches enable isotope internal standard quantification for the global target analytes, which can efficiently overcome the matrix effects and enhance the accuracy. These proposed methods will be applied in the research of lung cancer related metabolic samples, and it is expected that more biomarkers would be discovered or verification for early diagnosis, curative effect evaluation and prognosis evaluation. These results will provide a solid theoretical basis for improving the level of diagnosis and treatment of lung cancer.

LC-MS在诸多领域显示了强大能力，然而，在代谢轮廓分析方面仍存在缺陷。在非靶向分析方面，全面可靠地提取分析特征非常困难；在靶向分析方面，常受到基质效应的困扰。本项目以芳环并咪唑结构为母体，制备适用于胺基和羧基代谢物的14N/15N编码（及多重编码）分子探针，借助新型探针建立靶向和非靶向LC-MS代谢轮廓分析方法。在数据采集和预处理环节，充分利用同位素编码探针的辅助作用，提高LC-MS分析方法的性能，为后续多元统计分析提供高质量的数据。在非靶向分析过程中，发挥同位素辅助特征提取的作用，提取更多真正有意义的分析特征，提高发现生物标志物的概率；在靶向代谢轮廓分析过程中，发挥同位素内标定量的作用，克服基质效应的影响，提高方法的准确度和分析通量。将新方法应用于肺癌相关的胺类代谢谱和羧酸代谢谱的研究，发现或验证更多用于早期诊断、疗效评价和预后评估的代谢标志物，为提高肺癌的诊治水平提供理论基础。

本研究工作基本按计划进行，制备了一系列同位素编码标记试剂，发展了基于同位素编码标记的代谢组学分析方法。借助新型探针建立靶向和非靶向的分析方法，对具有特定官能团的代谢物进行化学标记，实现选择性绘制代谢轮廓。利用同位素编码标记实现更为准确的特征提取和内标定量，提高代谢组分析的覆盖度和准确性，为标志物的筛选和检测提供有力的工具，将本项目建立的方法示范性地应用于肺癌标志物的研究工作。（1）以芳环并咪唑结构为母体，制备并表征了具有质谱增敏功能的H/D和14N/15N编码分子探针16种，可用于选择性绘制胺基、羰基和羧基等代谢谱。（2）利用本项目开发的同位素编码标记试剂，建立的非靶向代谢谱分析方法，用于血清中胺基、羧酸和羰基代谢谱的研究。主要创新点集中在数据采集和预处理环节，充分利用同位素编码探针的辅助作用，提高液相色谱-质谱（LC-MS）分析方法的性能，提取更多真正有意义的分析特征，为后续多元统计提供高质量的数据；（3）建立了基于同位素编码标记的高通量靶向分析方法。发挥同位素内标校正的作用，克服基质效应的影响，以模式分析物为代表，探索了“同位素全内标法”、“同系物同位素双重内标法”、“多重编码标准加入法”等多种定量模式，通过缩短运行时间与并行进样提高靶向分析的通量。（4）发展了一系列样品前处理新技术。针对项目实施中遇到的衍生效率低的问题，发展了基于金属有机骨架（MOF）的分散固相萃取方法，先后制备了MIL-101(Cr)、MOF-5(Zn)和ZIF-8(Zn)等MOF材料用于富集水相介质中的低含量分析物，取得了满意的效果。（5）将建立的代谢谱分析方法示范性地应用于肺癌相关的胺类谱、羰基谱和羧酸谱的研究，结果表明本项目建立的方法能够准确有效提取分析特征，结合多元统计分析，可实现判别分析和差异特征筛选，可为诸多领域的代谢组学研究提供可靠的技术支撑。