Evaluation of two-dimensional gel electrophoresis-based proteome analysis technology

作者：Steven P. Gygi, Garry L. Corthals, Yanni Zhang, Yvan P. Rochon, Ruedi Aebersold · 发表于：Proceedings of the National Academy of Sciences · 年份：2000 · DOI：10.1073/pnas.160270797 · 被引用次数：1325 · 研究领域：Advanced Proteomics Techniques and Applications、Mass Spectrometry Techniques and Applications、Metabolomics and Mass Spectrometry Studies

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.