Gygi S P, Corthals G L, Zhang Y, Rochon Y, Aebersold R
Department of Molecular Biotechnology, University of Washington, Seattle, WA 98195-7730, USA.
Proc Natl Acad Sci U S A. 2000 Aug 15;97(17):9390-5. doi: 10.1073/pnas.160270797.
Proteome analysis is most commonly accomplished by a combination of two-dimensional gel electrophoresis (2DE) to separate and visualize proteins and mass spectrometry (MS) for protein identification. Although this technique is powerful, mature, and sensitive, questions remain concerning its ability to characterize all of the elements of a proteome. In the current study, more than 1,500 features were visualized by silver staining a narrow pH range (4.9-5. 7) 2D gel in which 0.5 mg of total soluble yeast protein was separated. Fifty spots migrating to a region of 4 cm(2) were subjected to MS protein identification. Despite the high sample load and extended electrophoretic separation, proteins from genes with codon bias values of <0.1 (lower abundance proteins) were not found, even though fully one-half of all yeast genes fall into that range. Proteins from genes with codon bias values of <0.1 were found, however, if protein amounts exceeding the capacity of 2DE were fractionated and analyzed. We conclude that the large range of protein expression levels limits the ability of the 2DE-MS approach to analyze proteins of medium to low abundance, and thus the potential of this technique for proteome analysis is likewise limited.
蛋白质组分析最常用的方法是结合二维凝胶电泳（2DE）来分离和可视化蛋白质，以及用质谱（MS）进行蛋白质鉴定。尽管这项技术强大、成熟且灵敏，但对于其表征蛋白质组所有成分的能力仍存在疑问。在当前研究中，通过对一块分离了0.5mg酵母总可溶性蛋白的窄pH范围（4.9 - 5.7）二维凝胶进行银染，可视化了1500多个特征点。对迁移至4平方厘米区域的50个点进行了质谱蛋白质鉴定。尽管上样量高且电泳分离时间长，但密码子偏好值<0.1的基因（低丰度蛋白质）所对应的蛋白质却未被发现，即便所有酵母基因中有整整一半都属于这个范围。然而，如果对超过二维凝胶承载能力的蛋白量进行分级分离和分析，就能发现密码子偏好值<0.1的基因所对应的蛋白质。我们得出结论，蛋白质表达水平的巨大差异限制了二维凝胶电泳 - 质谱方法分析中低丰度蛋白质的能力，因此这项技术用于蛋白质组分析的潜力同样有限。
