应用iTRAQ-HPLC-MS技术筛选奶牛脂肪肝病尿液蛋白标志物

Screening Protein Markers in Urine of Dairy Cows with Fatty Liver Disease Based on ITRAQ-HPLC-MS Technology

应用同位素标记相对和绝对定量(iTRAQ)技术筛选并鉴定脂肪肝病奶牛尿液中的差异表达蛋白,为诊断奶牛脂肪肝病提供新的生物标志物。收集40头奶牛的尿液,分为脂肪肝病组A1、A2和健康对照组B1、B2,每组各10头奶牛。组内每10个样品等量混合后进行试验,将符合要求的样品进行iTRAQ标记后利用高效液相色谱法层析,经串联质谱鉴定。共筛选出110个差异表达蛋白,其中50个表达下调,60个表达上调;通过生物信息学分析得到了4个关键性蛋白:玻连蛋白(Vitronectin,VTN)、脂质运载蛋白(Lipocalin,LCN2)、凝血酶原(Prothrombin,F2)和丛集素(Clusterin,CLU)。本试验成功筛选出脂肪肝病奶牛尿液中的差异表达蛋白,并鉴定出其中4种与奶牛脂肪肝病存在密切关系的关键性调节因子,如能进一步证实为奶牛脂肪肝病的重要生物标志物,将会为奶牛脂肪肝病的早期检测和诊断提供新的方法。

To obtain new protein markers of fatty liver disease in dairy cows,the relative and absolute quantification isotope labeling(iTRAQ)technology was used to screen and identify differentially expressed proteins in urine of dairy cows with fatty liver disease.40cows urines were collected and divided into diseased groups A1,A2and healthy groups B1,B2,respectively including 10cows in each group.Every 10urine samples in the same group was equally mixed into 1sample for the experiment.Subsequently,the samples which the requirements were marked by an iTRAQ kit.Finally,the high-performance liquid chromatography(HPLC)and tandem mass spectrometry were used.In this way,we got a series of data as a result,then the results were conducted by a series of bioinformatics analysis.Totally,110differentially expressed proteins were screened,in which 50were upregulated and 60were downregulated.4key proteins were acquired by bioinformatic analysis:Vitronectin(VTN),Lipocalin(LCN2),Prothrombin(F2)and Clusterin(CLU). Differentially expressed proteins in the urine of dairy cows with fatty liver disease were screenedin this experiment,4key regulatory factors were closely related to fatty liver disease,and should be further confirmed as important biological markers of fatty liver disease in dairy cows,which will provide a new method in early detection and diagnosis of fatty liver disease of dairy cows.