Stable isotope labeling with amino acids in Drosophila for quantifying proteins and modifications.
Stable isotope labeling with amino acids in Drosophila for quantifying proteins and modifications.
复制标题
果蝇中氨基酸的稳定同位素标记用于定量蛋白质和修饰。
DOI：
10.1021/pr300613c
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发表时间：
2012-09-07
影响因子：
4.4
通讯作者：
Peng J
中科院分区：
文献类型：
--
作者：
Xu P;Tan H;Duong DM;Yang Y;Kupsco J;Moberg KH;Li H;Jin P;Peng J
果蝇是遗传学研究中常用的动物模型，其定量蛋白质组学研究正在兴起。在这里，我们详细介绍了一个程序的发展，将稳定的同位素标记的氨基酸到苍蝇蛋白质组。在果蝇（Drosophila melanogaster）（SILAC fly）中用氨基酸稳定同位素标记的方法中，用在改良培养基中生长的SILAC标记的酵母喂养果蝇，使得能够在单代中几乎完全标记。在一系列无效实验中评估了个体苍蝇之间蛋白质组的生物学变异。我们进一步应用SILAC苍蝇方法来分析脆性X综合征模型中的蛋白质，脆性X综合征是人类遗传性智力低下的最常见原因。该分析在疾病模型中确定了许多改变的蛋白质，包括肌动蛋白结合蛋白profilin和微管蛋白相关蛋白futsch。通过免疫印迹分析验证两种蛋白的变化。此外，我们扩展了SILAC果蝇策略，通过测量两个主要的多聚泛素链（K48和K63键）的泛素沿着水平，研究果蝇寿命期间（从第1天到第30天）蛋白质泛素化的动态。结果表明，蛋白质泛素化的丰度和两个主要的连接在测量的年龄范围内没有显着变化。总之，这些数据证明了SILAC原理在D.黑腹果蝇，促进强大的苍蝇基因组学与新兴蛋白质组学的整合。
Drosophila melanogaster is a common animal model for genetics studies, and quantitative proteomics studies of the fly are emerging. Here, we present in detail the development of a procedure to incorporate stable isotope-labeled amino acids into the fly proteome. In the method of stable isotope labeling with amino acids in Drosophila melanogaster (SILAC fly), flies were fed with SILAC-labeled yeast grown with modified media, enabling near complete labeling in a single generation. Biological variation in the proteome among individual flies was evaluated in a series of null experiments. We further applied the SILAC fly method to profile proteins from a model of fragile X syndrome, the most common cause of inherited mental retardation in human. The analysis identified a number of altered proteins in the disease model, including actin-binding protein profilin and microtubulin-associated protein futsch. The change of both proteins was validated by immunoblotting analysis. Moreover, we extended the SILAC fly strategy to study the dynamics of protein ubiquitination during the fly life span (from day 1 to day 30), by measuring the level of ubiquitin along with two major polyubiquitin chains (K48 and K63 linkages). The results show that the abundance of protein ubiquitination and the two major linkages do not change significantly within the measured age range. Together, the data demonstrate the application of the SILAC principle in D. melanogaster, facilitating the integration of powerful fly genomics with emerging proteomics.
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影响因子：
3.4
作者：
Alonso, J;Santarén, JF
通讯作者：
Santarén, JF
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Bateman, Randall J.;Munsell, Ling Y.;Holtzman, David M.
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Holtzman, David M.
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Chien, S;Reiter, LT;Gribskov, M
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Gribskov, M
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Krijgsveld, J;Ketting, RF;Heck, AJR
通讯作者：
Heck, AJR
影响因子：
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作者：
Kirkpatrick, Donald S.;Hathaway, Nathaniel A.;Gygi, Steven P.
通讯作者：
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