Evaluation of two-dimensional gel electrophoresis-based proteome analysis technology.
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.
Department of Molecular Biotechnology, University of Washington, Seattle, WA 98195-7730, USA.
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Gygi
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Corthals
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Zhang
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Rochon
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Aebersold
D000595:Amino Acid Sequence; D003062:Codon / Q000235:genetics; D015180:Electrophoresis, Gel, Two-Dimensional; D005656:Fungal Proteins / Q000032:analysis* / Q000737:chemistry* / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010449:Peptide Mapping / Q000379:methods*; D020543:Proteome; D012441:Saccharomyces cerevisiae / Q000737:chemistry* / Q000235:genetics; D018401:Sample Size; D012680:Sensitivity and Specificity; D016622:Silver Staining
D003062:Codon; D005656:Fungal Proteins; D020543:Proteome