Sakai Jun, Kojima Shinichi, Yanagi Kazunori, Kanaoka Masaharu
Genomic Science Laboratories, Sumitomo Pharmaceuticals Co., Ltd., Osaka 554-0022, Japan.
Proteomics. 2005 Jan;5(1):16-23. doi: 10.1002/pmic.200300885.
We describe a method for simultaneous identification and quantitation of proteins within complex mixtures. The method consists of 18O-labeling, a simple stable isotope-coding that requires merely enzymatic digestion in 18O-water, in combination with a capillary-liquid chromatography electrospray ion-trap mass spectrometer. In a separate experiment using the same sample and a spike test, we demonstrate that the difference ration was calculated accurately using the 18O-labeling method even if the protein was part of a complex mixture. Our data also suggest that the accuracy of the quantitation can be improved by averaging the difference ratios of several peptides. In comparing our method with the isotope-coded affinity tag (ICAT) method, we show that the 18O-labeling method has the advantages of better recovery and fewer isotope effects. Therefore, the 18O-labeling method is a powerful tool for large-scale proteomics applications.
我们描述了一种用于同时鉴定和定量复杂混合物中蛋白质的方法。该方法包括18O标记，这是一种简单的稳定同位素编码，仅需在18O水中进行酶解，再结合毛细管液相色谱电喷雾离子阱质谱仪。在使用相同样品的单独实验和加标测试中，我们证明即使蛋白质是复杂混合物的一部分，使用18O标记法也能准确计算差异比率。我们的数据还表明，通过对几种肽的差异比率求平均值可以提高定量的准确性。在将我们的方法与同位素编码亲和标签（ICAT）方法进行比较时，我们发现18O标记法具有回收率更高和同位素效应更少的优点。因此，18O标记法是大规模蛋白质组学应用的有力工具。
