摘要
目的:应用同重同位素相对与绝对定量(iTRAQ)结合质谱技术筛选冷应激大鼠血浆差异表达蛋白。方法:将30只健康SPF级雄性Wistar大鼠随机分为冷应激组A和常温饲养组B,将A、B两组分别随机分为3组,即A1、A2、A3组和B1、B2、B3组(n=5)。常温饲养温度为(24.0±0.1)℃,冷应激温度为(4.0±0.1)℃。两组实验大鼠经不同温度处理12 h后,用肝素钠抗凝负压管腹主动脉采血,分离血浆,提取血浆蛋白、定量、酶解、iTRAQ标记、强阳离子交换色谱(SCX分离)和质谱分析。结果:共鉴定出1 085个蛋白,筛出39个差异表达蛋白,其中29个表达上调蛋白,10个表达下调蛋白。经生物信息学分析,筛出3个与动物冷应激相关的重要差异表达蛋白,分别为:组织相容性蛋白HA-1、Ras相关蛋白Rap1b、整合蛋白β-1。结论:本实验成功筛出冷应激大鼠血浆差异表达蛋白,iTRAQ技术为筛选大鼠冷应激蛋白诊断标志物提供了一个良好的平台,为深入研究动物冷应激发生机制奠定了良好基础。
Objective： Isobaric tags for relative and absolute quantitation （iTRAQ） combined with mass spectrometry were used to screen differentially expressed plasma proteins in cold stress rats. Methods： Thirty health SPF Wistar rats were randomly divided into cold stress group A and control group B, then A and B were randomly divided into 3 groups（ n = 5） ： A1 ,A2,A3 and B1 ,B2,B3.The temperature of room rais- ing was（24.0 ± 0.1 ） ℃, and the cold stress temperature was（4.0 ± 0.1 ） ℃. The rats were treated with different temperatures until 12 h. The abdominal aortic blood was collected with heparin anticoagulation suction tube. Then, the plasma was separated for protein extraction, quanti- tative, enzymolysis, iTRAQ labeling, SCX fractionation and mass spectrometry analysis. Results： Totally, 1 085 proteins were identified in the test, 39 differentially expressed proteins were screened, including 29 up-regulated proteins and 10 down-regulated proteins. Three important differentially expressed proteins related to cold stress were screened by bioinformatics analysis （Minor histocompatibility protein HA-l, Ras-re- lated protein Rap- 1 b, Integrin beta- 1 ）. Conclusion： In the experiment, the differentially expressed plasma proteins were successfully screened in cold stress rats. iTRAQ technology provided a good platform to screen protein diagnostic markers on cold stress rats, and laid a good founda- tion for further study on animal cold stress mechanism.
出处
《中国应用生理学杂志》
CAS
CSCD
2015年第5期392-395,400,共5页
Chinese Journal of Applied Physiology
基金
国家自然科学基金项目(31372398)
黑龙江省自然科学基金面上项目(C2015041)
