临床标志物的MS-MRM定量新方法研究

A high efficient validation strategy may significantly promote the translation from scientific discovery to clinic application. LC-MS technology shows promise as a tool to rapidly develop clinical assays for the emerging biomarkers, and to detect them with high sensitivity, high specificity and high throughput. As a result, FDA（Food and Drug Administration ,FDA） released draft guidelines and make the principles clear for the MS-based Lab developed test（LDT）in 2014, which makes an encouraging development of clinical mass spectrometry technologies. Scientist have discovered a lot of candidate protein biomarkers of liver cancer from the specimen of the liver tissue, the cell lines and the bio fluid samples of patients, and most of them are lack of further verification and validation. Therefore, in this project, we will make efforts to develop a MRM-based strategy for the clinical quantitation and verification of the promise biomarker panels. By making a step-wise and strict selection of high confident protein biomarker panels; by developing a high efficient and high reproducible methodology and corresponding quantity control for serum protein digestion; by designing and synthesizing a series of isotope labeled peptides for the standard curves, and by setting up a standard MRM quantitation method, we will develop a clinical applicable MRM assay for the test of candidate liver cancer biomarker. We will further apply the MRM-assay to the precise quantitation of targeted proteins in clinical serum samples, and afford reference data and technology for the further testing in large scale of samples.

发展适用的目标分子验证技术,是蛋白质组学临床转化医学的前沿。质谱检测技术以其方法开发高效、快速，检测灵敏度高、特异性强和高通量等特点，成为很有临床检验应用前景的定量技术。近年来FDA针对基于质谱的临床实验室自建项目（LDT）相关标准的建立，更进一步规范和促进了临床质谱检验技术的发展。多年来科学家已通过组织、细胞和体液研究，发现了一系列潜在的肝癌诊断蛋白质标志物，急需研究其临床检验价值。本课题拟针对这些标志物进行逐级筛选，挑选出具有高特异性和高灵敏度的血清诊断标志物群。针对该血清标志物群及其相关特异性肽段，研究高效稳定的酶解和质控方案，设计并合成序列同源的同位素标记定量标准曲线标品，探索实用的LC-MRM定量检测方法，并整合为符合临床检测标准的试剂盒以及定量研究方案，用于临床正常人、肝脏良性疾病和不同类型肝癌患者血清标志物的精准定量检测，并为在更大规模的血清验证提供技术手段和参考。

发展适用的目标分子验证技术,是蛋白质组学临床转化医学的前沿。质谱检测技术以其方法开发高效、快速，检测灵敏度高、特异性强和高通量等特点，成为很有临床检验应用前景的定量技术。近年来FDA针对基于质谱的临床实验室自建项目（LDT）相关标准的建立，更进一步规范和促进了临床质谱检验技术的发展。多年来科学家已通过组织、细胞和体液研究，发现了一系列潜在的肝癌诊断蛋白质标志物，急需研究其临床检验价值。本课题拟针对这些标志物进行逐级筛选，挑选出具有高特异性和高灵敏度的血清诊断标志物群。由此本项目开发了一系列技术以完成以全面快速肝癌血清标志物筛选为目标的工作流程，该流程集合血清样品快速前处理的技术、针对特定疾病的血清标志物数据库构建技术、基于质谱数据深度识别的谱图预测技术和大规模标志物靶向蛋白质监测的方法开发技术(DeepDIA and DeepPRM)、大规模血清样品质谱分析的质控和归一化方法，以及基于机器学习的方法结合定量数据，筛选高效的血清标志物组合的一系列技术等。我们将这套流程用于快速分析316例包含肝癌多个发展进程中患者的血清，我们也将发展的方法用于共计316例正常人、肝炎、肝硬化和肝癌患者血清样品（发现集246例样品,n = 246, within 肝癌（HCC，n =70), 肝硬化(LC ，n = 42), HBV引起的肝炎患者(HBV， n = 42), HCV引起的肝炎患者(HCV， n = 42), and 健康对照组(NC， n = 50）分析中，并在另外两个验证集Cohort II （n = 35, within HCC (n = 5), LC (n = 5), HBV (n = 5), HCV (n = 5), and NC (n = 5)）, and cohort III （n = 35, within HCC (n = 5), LC (n = 5), HBV (n = 5), HCV (n = 5), and NC (n = 5)）中进行验证，找到了13个蛋白用于区分肝癌患者和正常及良性疾病患者，准确率达到93.3%，找到了10个蛋白用于区分HBV引起的肝炎和肝硬化患者。