Metabolic labeling of C-elegans and D-melanogaster for quantitative proteomics
Metabolic labeling of C-elegans and D-melanogaster for quantitative proteomics
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DOI：
10.1038/nbt848
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发表时间：
2003-08-01
影响因子：
46.9
通讯作者：
Heck, AJR
中科院分区：
文献类型：
--
作者：
Krijgsveld, J;Ketting, RF;Heck, AJR
比较蛋白质组学中的一个关键问题是准确量化蛋白质表达水平的差异。为了实现这一点，已经开发了几种方法，在体内通过代谢标记或通过蛋白质衍生化在体外用稳定的同位素标记蛋白质。虽然代谢标记是获得所有蛋白质标记的唯一方法，但到目前为止，它仅应用于单细胞生物体(1，2)和培养中的细胞(2，3)。在这里，我们描述了多细胞生物线虫线虫和常见果蝇黑腹果蝇的N-15代谢定量标记，分别以N-15标记的大肠杆菌和酵母喂养它们。从不同样品中获得的单个蛋白质的相对丰度可以通过质谱分析(MS)来确定。通过比较两个线虫菌株的蛋白质表达水平，证明了该方法的适用性，一个菌株有胚系，另一个没有胚系。所描述的方法学为在这些模式生物中进行准确的定量蛋白质组学研究提供了工具。
A crucial issue in comparative proteomics is the accurate quantification of differences in protein expression levels. To achieve this, several methods have been developed in which proteins are labeled with stable isotopes either in vivo via metabolic labeling or in vitro by protein derivatization. Although metabolic labeling is the only way to obtain labeling of all proteins, it has thus far only been applied to single-celled organisms(1,2) and cells in culture(2,3). Here we describe quantitative N-15 metabolic labeling of the multicellular organisms Caenorhabditis elegans, a nematode, and Drosophila melanogaster, the common fruit fly, achieved by feeding them on N-15-labeled Escherichia coli and yeast, respectively. The relative abundance of individual proteins obtained from different samples can then be determined by mass spectrometry (MS). The applicability of the method is exemplified by the comparison of protein expression levels in two C. elegans strains, one with and one without a germ line. The methodology described provides tools for accurate quantitative proteomic studies in these model organisms.