Global protein quantification of mouse heart tissue based on the SILAC mouse.
Global protein quantification of mouse heart tissue based on the SILAC mouse.
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DOI：
10.1007/978-1-62703-386-2_4
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发表时间：
2013-01-01
期刊：
影响因子：
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通讯作者：
Kruger, Marcus
中科院分区：
文献类型：
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作者：
Konzer, Anne;Ruhs, Aaron;Kruger, Marcus
利用稳定的同位素对活体进行代谢标记已成为全球蛋白质定量的有力工具。SILAC(稳定同位素标记细胞培养中的氨基酸)方法是基于将非放射性标记的氨基酸同位素形式掺入细胞蛋白质中。对永生化细胞和单细胞生物(例如酵母和细菌)的有效SILAC标记最近扩展到更复杂的生物，包括蠕虫、苍蝇，甚至啮齿动物。给一代小鼠喂食含(13)C6-赖氨酸(重)的饲料会导致天然(轻)同位素(12)C6-赖氨酸的完全交换。SILAC标记的生物体主要被用作非标记生物体的重“尖峰”标准，与高性能质谱仪的结合允许进行全球蛋白质组筛选。在这里，我们使用全标记的SILAC小鼠从分离的心肌细胞中识别基于SILAC对的蛋白质，并分析β-Parvin缺陷的心脏。我们的方法证实了β-Parvin的缺失，同时揭示了心脏组织中α-Parvin的明显调节。在这个方案中，我们描述了SILAC小鼠群体的产生，并展示了两种对心脏组织进行蛋白质组学分析的方法。因此，SILAC小鼠插入方法是一种容易获得的程序，并允许对疾病模型和基因敲除小鼠进行直接的系统分析。
Metabolic labeling of living organisms with stable isotopes has become a powerful tool for global protein quantitation. The SILAC (stable isotope labeling with amino acids in cell culture) approach is based on the incorporation of nonradioactive-labeled isotopic forms of amino acids into cellular proteins. The effective SILAC labeling of immortalized cells and single-cell organisms (e.g., yeast and bacteria) was recently extended to more complex organisms, including worms, flies, and even rodents. The administration of a (13)C6-lysine (heavy) containing diet for one mouse generation leads to a complete exchange of the natural (light) isotope (12)C6-lysine. SILAC-labeled organisms are mainly used as a heavy "spike-in" standard into nonlabeled counterparts, and the combination with high-performance mass spectrometers allows for global proteomic screening. Here we used the fully labeled SILAC mice to identify proteins based on SILAC pairs from isolated cardiomyocytes, and we analyzed beta-parvin-deficient hearts. Our approach confirmed the absence beta-parvin and revealed simultaneously a clear up regulation of alpha-parvin in heart tissue. In this protocol, we describe the generation of a SILAC mouse colony and show two approaches to perform a proteome-wide analysis of heart tissue. Thus, the SILAC mouse spike-in approach is a readily available procedure and allows for a straightforward systematic analysis of disease models and knockout mice.