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.T he genomics revolution has changed the paradigm for the comprehensive analysis of biological processes and systems. It is now hypothesized that biological processes and systems can be described based on the comparison of global, quantitative gene expression patterns from cells or tissues representing different states. To test this hypothesis, it is essential that methods for the precise measurement of gene expression be developed and applied.Several methods, including serial analysis of gene expression, oligonucleotide and cDNA microarrays, and large-scale sequencing of expressed sequence tags have been developed to globally and quantitatively measure gene expression at the mRNA level (1, 2). The discovery of posttranscriptional mechanisms that control rate of synthesis and half-life of proteins (3) and the ensuing nonpredictive correlation between mRNA and protein levels expressed by a particular gene (4, 5) indicate that direct measurement of protein expression also is essential for the analysis of biological processes and systems.Global analysis of gene expression at the protein level is now also termed proteomics. The standard method for quantitative proteome analysis combines protein separation by highresolution (isoelectric focusing͞SDS-PAGE) two-dimensional gel electrophoresis (2DE) with mass spectrometric (MS) or tandem MS (MS͞MS) identification of selected protein spots. Important technical advances related to 2DE and protein MS have increased sensitivity, reproducibility, and throughput of proteome analysis while creating an integrated technology.By using 2DE with extended pH range and high-sensitivity protein identification by electrospray ionization and MS͞MS, we have evaluated the potential of the 2DE-MS strategy to serve as the technology base for comprehensive and quantitative pr...

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