超短肽的液相色谱-质谱谱库建立及在食管癌预警生物标志物筛选中的应用研究

Esophageal cancer (EC) is highly malignant that early diagnosis and early treatment are the most efficient therapeutic schedules for curing EC in spite of no clinically available biomarkers for EC prediction. Of note, overshort peptides (OSPs) have proved to reveal good capabilities in early prediction of diseases. However, the large-scale profiling of OSPs is being limited by the lack of OSP spectrum database which is essential to the selection of OSP-biomarkers. To figure out these issues, we propose to establish the OSP spectrum database based on our previous related studies. In this study, a series of works are conducted for the database construction, including optimization of OSP preparation and analysis, OSP prediction with QSRR and interactive models, OSP identification via comparison of common MSMS fragments, and summarization of information including LC-MS methods, retention times, MS and MS2 spectra. With the employment of the spectrum database, we further conduct the selection of OSP-biomarkers for EC early prediction, in which metabolic profiles of OSPs are acquired from sera of EC patients and their control subjects. Furthermore, by using multi- and univariate analyses, the OSPs presenting early prediction capabilities are selected and validated based on different sample sets. The absolute concentrations and cutoff values of predictive OSPs are further determined, providing researchers with valuable information for EC early prediction. Therefore, the OSP-biomarkers for EC prediction are of great clinical value.

食管癌是一种恶性程度较高的肿瘤，早发现、早治疗是其最有效的治疗手段，但临床上尚无用于食管癌早期预警的生物标志物。超短肽作为生物标志物展现了较好的早期预警效能。但高覆盖超短肽谱库的缺乏限制了其大规模分析，构建全面、高覆盖的超短肽谱库是开展超短肽标志物筛选的重要前提。针对上述问题，拟在实验室前期谱库构建研究基础上，构建超短肽谱库。首先通过优化预处理及LC-MS方法建立超短肽分析体系，利用改良的QSRR模型、交互模型及共有特征对比等方法对超短肽进行预测及鉴定，并对已鉴定超短肽的色谱质谱信息进行整理，构建超短肽谱库。在此基础上，以食管癌及其对照样本为分析对象，利用构建的分析体系及谱库采集食管癌血清超短肽代谢轮廓，并基于不同的样本集利用多变量及单变量分析完成预警超短肽的筛选及验证，并对其进行定量及阈值划定，为食管癌的早期预警提供价值信息。可用于食管癌早期预警的超短肽标志物具有重要的临床价值。

超短肽在炎症及免疫调控、食品风味调节、疾病生物标志物等方面展现出其特有价值。目前仍然没有专门用于复杂生物体系超短肽大规模表征的方法。食管鳞癌（ESCC）作为我国高发的恶性肿瘤具有恶性程度高、预后差的特点，有研究表明ESCC的发生及预后与其炎性分子的表达调控密切相关，而二肽基肽酶对肿瘤中炎性分子相关信号通路有激活作用，但其分子机制不清晰。本研究拟基于液相色谱质谱联用（LC-MS）结合QSRR保留时间预测建立复杂生物样本中超短肽的高覆盖注释方法。在此基础上，对ESCC血清、组织及细胞中的超短肽代谢轮廓进行系统表征，并对后者对炎性分子的影响进行探究。.主要研究结果如下：.1.基于LC-MS结合QSRR建立超短肽高覆盖筛选方法及谱库。通过对训练集及QSRR算法的优化证实网格二肽/氨基酸作为训练集结合支持向量机回归具有最好的二肽保留时间预测效果，进一步利用网格二肽及迭代QSRR策略从4种生物样本的LC-autoMSMS数据中注释198种二肽及149种三肽；将所建方法及谱库应用于3种肝病的血清超短肽代谢轮廓差异分析，发现脯氨酸二肽随着肝病严重程度增加而升高。.2.基于所建LC-MS方法及谱库开展了ESCC二肽代谢轮廓分析，最终从ESCC血清及组织分别鉴定90种及102种二肽，其中大部分在ESCC血清及组织中呈现升高趋势，以脯氨酸、天冬酰胺、甘氨酸二肽升高最为显著。ESCC组织中，脯氨酸二肽体现出最为显著的二肽-二肽基肽酶表达相关性，进一步基于ESCC细胞系证实天冬酰胺-脯氨酸可显著上调炎性相关转录因子NF-κB。该结果为ESCC的炎性调控提供新的靶点。.综上所述，本研究建立了高覆盖、高灵敏的超短肽注释方法，在此基础上系统地表征了ESCC二肽代谢轮廓的变化，筛选了具有潜在功能的活性二肽，并初探功能二肽对炎性分子的潜在作用。