应用iTRAQ-LC-MS/MS方法筛选大鼠DAI后脑组织差异表达蛋白质

目的应用相对和绝对定量同位素标记结合液相色谱-串联质谱法(isobaric tag for relative and absolute quantification-liquid chromatograph-mass spectrometer/mass spectrometer,iTRAQ-LC-MS/MS)筛选SD大鼠弥漫性轴索损伤(diffuse axonal injury,DAI)后脑组织差异表达的蛋白质,寻找诊断DAI的潜在生物标志物。方法参照Marmarou法建立DAI动物模型,分为空白对照组(n=4)、假打击组(n=4)和打击致死组(n=4),采用iTRAQ-LC-MS/MS技术对大鼠脑组织蛋白质进行检测,并对检测结果进行生物信息学分析及验证,筛选出差异表达的蛋白质。结果定量检测出2016种蛋白质,生物信息学分析显示其在细胞中分布广泛、功能多样,并且参与了多种生物过程,16种蛋白质在打击致死组具有差异表达,包括1种表达上调蛋白质和15种下调蛋白质;Western印迹法验证了iTRAQ-LC-MS/MS的结果。结论 iTRAQ-LC-MS/MS的检测技术筛选出大鼠DAI后脑组织中多个差异表达的蛋白质,不仅为深入探讨DAI后轴索损伤的发病机制提供了研究方向,也为DAI的诊断提供了潜在生物标志物。

Screening of Differential Expression Proteins in Rat Brain Tissues after DAI by iTRAQ-LC-MS/MS

Objective To screen for the differential expression proteins in brain tissues of SD rat after diffuse axonal injury (DAI) by isobaric tag for relative and absolute quantification-liquid chromatographmass spectrometer/mass spectrometer (iTRAQ-LC-MS/MS),and to explore potential biomarkers available for the diagnosis of DAI.Methods Animal models of DAI were established with the Marmarou method as reference,and the subjects were divided into blank control group (n=4),sham strike group (n=4) and fatal strike group (n=4),respectively.The proteins in rat brain tissues were detected by iTRAQ-LC-MS/MS,and bioinformatics analysis and verification were performed on the results and screened for the differential expression proteins.Results A total of 2 016 proteins were identified and quantified.The bioinformatics analysis revealed that the proteins had wide distribution and function,and participated in different biological processes.There were 16 proteins showed differential expression in fatal strike group,including one up-regulated expression protein and 15 down-regulated expression proteins.The results of iTRAQLC-MS/MS were confirmed by Western blotting method.Conclusion Multiple differential expression proteins in rat brain tissues after DAI can be screened by iTRAQ-LC-MS/MS.This not only indicates a research direction for exploring the pathogenesis of DAI,but also provides potential biomarkers available for the diagnosis of DAI.