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

Proteome analysis is most commonly accomplished by a combination of two-dim ensional gel electrophoresis (2DE) to separate and visualize proteins and m ass spectrometry (MS) for protein identification. Although this technique i s powerful, mature, and sensitive, questions remain concerning its ability to characterize all of the elements of a proteome. In the current study, mo re 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 separa ted. Fifty spots migrating to a region of 4 cm(2) were subjected to MS prot ein identification. Despite the high sample load and extended electrophoret ic separation, proteins from genes with codon bias values of <0.1 (lower ab undance proteins) were not found, even though fully one-half of all yeast g enes 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 we re fractionated and analyzed. We conclude that the large range of protein e xpression levels limits the ability of the 2DE-MS approach to analyze prote ins of medium to low abundance, and thus the potential of this technique fo r proteome analysis is likewise limited.