基于靶向蛋白质组学技术的尿蛋白疾病标志物验证筛选平台的建立及其在IgA肾病研究中的应用

Urine is an important source of biomarkers, especially for renal diseases. A large number of biomarker candidates have been identified by proteomic studies and now need to be validated by high throughput and highly precise assays. In another hand, because most of the high-abundant urinary proteins are come from the plasma proteome and many proteins secreted from the kidney are of low abundance, the identification of disease-specific biomarkers that are generated by the kidney injury site is very difficult. Targeted proteomic approaches provide high throughput, precise and sensitive techniques for low-abundant protein quantification. A crucial step is to select proper proteins for the targeted analysis. In the previous works of our group, a urinary protein biomarker dataset (http://122.70.220.102/biomarker) was set up. And, kidney-originated urinary proteins (proteins could serve as disease-specific biomarkers) were identified by analyzing the urine from perfused isolated rat kidneys. Based on these databases, we plan to build an experimental platform to screen and validate urinary biomarkers by targeted prtoemic approaches. A database will also be set up to facilitate further studies. This platform will also be applied to study IgA nephropathy in this project.

尿液是重要的疾病标志物、尤其是肾脏病标志物的来源。目前已经通过蛋白质组学的研究发现了很多候选标志物，需要高通量、高精度的方法进行验证。另一方面，由于尿蛋白质组中，大部分的高丰度蛋白为血浆蛋白，而由肾脏分泌到尿液中的蛋白多为低丰度蛋白，鉴定由损伤部位直接产生的、疾病特异的标志物非常困难。靶向蛋白质组学为高通量、高灵敏度和高精确度地进行低丰度蛋白的定量提供了技术保障。选取适当的蛋白进行靶向分析是研究的关键。在本课题组的前期工作中，建立了尿液中的蛋白标志物数据库（http://122.70.220.102/biomarker）；并通过肾脏灌流的方法鉴定了肾脏分泌到尿液中的蛋白质组，这些蛋白极有可能作为疾病特异性的标志物。本研究拟在这两个数据库的基础上，建立一个利用靶向蛋白质组学技术验证和筛选疾病标志物的实验平台和数据库。课题还将应用此平台进行IgA肾病标志物的研究。

尿液是重要的生物标志物来源。IgA肾病是中国最为常见的原发性肾小球肾炎。目前只有肾活检结果是其诊断的金标准。蛋白质组学技术、尤其是靶向蛋白组学方法的发展为其新生物标志物的筛选和验证提供了高效可靠的工具。本项目取得的主要成果分为以下四个方面：1）建立和优化了蛋白质组分析和多反应检测(Multiple Reaction Monitoring, MRM)实验设计的生物信息学方法；2）通过蛋白质组实验和文献编审建立和完善了尿蛋白质组数据库，可用于辅助生物标志物的可信度和特异性的分析以及蛋白靶向多肽的确定；3）建立了生物标志物筛选和验证的生物信息学和质谱实验流程；4）通过实验发现了IgA肾病的新的潜在生物标志物。