Krijgsveld Jeroen, Ketting René F, Mahmoudi Tokameh, Johansen Janik, Artal-Sanz Marta, Verrijzer C Peter, Plasterk Ronald H A, Heck Albert J R
Center for Biomedical Genetics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Sorbonnelaan 16, 3584 CA Utrecht, The Netherlands.
Nat Biotechnol. 2003 Aug;21(8):927-31. doi: 10.1038/nbt848. Epub 2003 Jul 13.
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 and cells in culture. Here we describe quantitative 15N metabolic labeling of the multicellular organisms Caenorhabditis elegans, a nematode, and Drosophila melanogaster, the common fruit fly, achieved by feeding them on 15N-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.
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。为实现这一点，已经开发了几种方法，其中蛋白质通过代谢标记在体内或通过蛋白质衍生化在体外进行稳定同位素标记。尽管代谢标记是获得所有蛋白质标记的唯一方法，但迄今为止仅应用于单细胞生物和培养细胞。在此，我们描述了多细胞生物秀丽隐杆线虫（一种线虫）和黑腹果蝇（常见的果蝇）的定量15N代谢标记，分别通过用15N标记的大肠杆菌和酵母喂养它们来实现。然后可以通过质谱（MS）确定从不同样品中获得的单个蛋白质的相对丰度。通过比较两种秀丽隐杆线虫菌株（一种有生殖系，一种没有生殖系）的蛋白质表达水平，举例说明了该方法的适用性。所描述的方法为这些模式生物中的准确蛋白质组定量研究提供了工具。
