小鼠饲养过程中蛋白质组的整体重稳定性同位素标记
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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。


Feeding mouse with stable isotope labelled with amino acid
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National Basic Research Program of China (973 Program) (No. 2011CB910600), National Natural Science Foundation of China (Nos. 31070673, 31170780).

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    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/6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:

    The stable isotope labeling by amino acids in culture (SILAC) based quantitative proteomics serves as a gold standard because of the high accuracy and throughput for protein identifications and quantification. In this study, we discussed the application of SILAC technology in mammal model, and developed quantitative internal standard for comparative proteomics of disease model. The C57BL/6 mice fed by special diet containing the 13C6-Lysine and bred F2 generation. We identified and analyzed total proteins of 9 mice tissues of F2 generation, including brain, lung, heart, stomach, intestine, liver, spleen, kidney, and muscle. Quantitative analysis information could evaluate the mice and different tissues’ labeling efficiency. Liver was the most efficient, brain the least, and the labeling efficiency were 96.34%±0.90% and 92.62%±1.98% respectively. The average of the labeling efficiency of F2 generation was 95.80%±0.64%, which met the international standard (≥95%) for SILAC quantitative proteomics effective study. SILAC technology was successfully extended to mammalian model system, which will provide powerful tools for the mechanism study of the pathophysiology process with mouse model.

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樊锋旭,高慧英,徐忠伟,翟琳辉,衣泰龙,张涛,吴飞林,崔春萍,徐平. 小鼠饲养过程中蛋白质组的整体重稳定性同位素标记[J]. 生物工程学报, 2014, 30(10): 1602-1611

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  • 收稿日期:2013-12-23
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  • 在线发布日期: 2014-09-30
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