A nascent proteome study combining click chemistry with 2DE
A nascent proteome study combining click chemistry with 2DE
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DOI：
10.1002/pmic.201200393
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发表时间：
2013-01-01
期刊：
影响因子：
3.4
通讯作者：
Dontsova, Olga A.
中科院分区：
文献类型：
--
作者：
Osterman, Ilya A.;Ustinov, Alexey V.;Dontsova, Olga A.
为了研究细胞对条件变化的动态反应，新生蛋白质组的选择性标记是必要的。在这里，我们报告了一种结合点击化学蛋白质标记和二维DIGE的方法。为了测试该方法的相关性，我们比较了活跃生长的细菌细胞与暴露于蛋白质合成抑制剂红霉素的细胞的新生蛋白质组。将细胞与甲硫氨酸类似物，高炔丙基甘氨酸一起孵育，并用专门为此目的合成的单磺化中性Cy3和Cy5叠氮化物选择性标记其新生蛋白质组。在荧光标记后，将蛋白质样品混合并进行标准2D DIGE分离。该方法使我们能够揭示红霉素处理后新合成的蛋白质急剧减少，而总蛋白质组没有受到显着影响。此外，还鉴定了几种合成对红霉素具有抗性的蛋白质。
To investigate the dynamic cellular response to a condition change, selective labeling of the nascent proteome is necessary. Here, we report a method combining click chemistry protein labeling with 2D DIGE. To test the relevance of the method, we compared nascent proteomes of actively growing bacterial cells with that of cells exposed to protein synthesis inhibitor, erythromycin. Cells were incubated with methionine analog, homopropargyl glycin, and their nascent proteome was selectively labeled with monosulfonated neutral Cy3 and Cy5 azides specially synthesized for this purpose. Following fluorescent labeling, the protein samples were mixed and subjected to standard 2D DIGE separation. The method allowed us to reveal a dramatic reduction of newly synthesized proteins upon erythromycin treatment, while the total proteome was not significantly affected. Additionally, several proteins, whose synthesis was resistant to erythromycin, were identified.