小鼠饲养过程中蛋白质组的整体重稳定性同位素标记

发布时间：2018-06-13 23:18

本文选题：稳定同位素代谢标记 + CBL/J小鼠　；参考：《生物工程学报》2014年10期

【摘要】：文中探讨了稳定同位素代谢标记(SILAC)定量技术在哺乳动物模型上的应用,建立了可用于疾病模型比较蛋白质组学研究的定量内标。用含1%13C6-Lysine的SILAC鼠粮饲养C57BL/6J雌性小鼠,记为F0代,通过与雄性小鼠交配繁殖出F1代,相同方法繁殖出F2代标记小鼠。分离F2代小鼠的大脑、肺脏、心脏、胃、小肠、肝脏、脾脏、肾脏和肌肉组织,提取各组织的全蛋白,进行蛋白酶Lys-C酶切,利用高效液相色谱串联质谱联用技术(LC-MS/MS)进行鉴定和定量分析。对蛋白质定量结果进行高斯拟合确定均值及标准差,得出不同脏器和小鼠整体的标记效率,肝脏的标记效率最高,为96.34%±0.90%;大脑标记效率最低,为92.62%±1.98%,小鼠整体标记效率为95.80%±0.64%,符合SILAC标记效率不低于95%的国际标准。成功地将SILAC方法从微生物和细胞系水平拓展到哺乳动物模型,这为基于动物模型研究疾病发生、发展机制提供了有力工具。
[Abstract]:In this paper, the application of stable isotope metabolic marker silac in mammalian models was discussed. A quantitative internal standard for comparative proteomics of disease models was established. Female C57BL / 6J mice were fed with SILAC mouse grain containing 1 carton of C6-Lysine for F 0 generation. F1 generation was bred by mating with male mice and F2 generation labeled mice were bred by the same method. The brain, lung, heart, stomach, small intestine, liver, spleen, kidney and muscle tissues of F2 generation mice were isolated, and the whole protein of each tissue was extracted and digested with protease Lys-C enzyme. High performance liquid chromatography tandem mass spectrometry (HPLC / MS / MS) was used to identify and quantify. The mean value and standard deviation of protein quantitative results were determined by Gao Si fitting. The results showed that the labeling efficiency of different organs and mice was the highest, the labeling efficiency of liver was 96.34% 卤0.90, and the labeling efficiency of brain was the lowest. The overall labeling efficiency of mice was 95.80% 卤0.64%, which was in accordance with the international standard of SILAC labeling efficiency of 95%. The SILAC method has been successfully extended from microorganism and cell line to mammalian model, which provides a powerful tool for the study of disease occurrence and development mechanism based on animal model.
【作者单位】：安徽医科大学;军事医学科学院放射医学研究所;北京蛋白质组研究中心;
【基金】：国家重点基础研究发展计划(973计划)(No.2011CB910600) 国家自然科学基金(Nos.31070673,31170780)资助~~
【分类号】：R817.1

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