Simultaneous Qualitative and Quantitative Analysis of theEscherichia coli Proteome

We describe a novel LCMS approach to the relative quantitation and simultaneous identification of proteins within the complex milieu of unfractionated Escherichia coli. This label-free, LCMS acquisition method observes all detectable, eluting peptides and their corresponding fragment ions. Postacquisition data analysis methods extract both the chromatographic and the mass spectrometric information on the tryptic peptides to provide time-resolved, accurate mass measurements, which are subsequently used for quantitation and identification of constituent proteins. The response of E. coli to carbon source variation is well understood, and it is thus commonly used as a model biological system when validating an analytical method. Using this LCMS approach, we characterized proteins isolated from E. coli grown in glucose, lactose, and acetate. The change in relative abundance of the corresponding proteins was measured from peptides common to both conditions. Protein identities were also determined for those peptides that were unique to each condition, and these identities were found to be consistent with the underlying biochemical restrictions imposed by the growth conditions. The relative change in abundance of the characterized proteins ranged from 0.1- to 90-fold among the three binary comparisons. The overall coverage of the characterized proteins ranged from 10 to 80%, consisting of one to 34 peptides per protein. The quantitative results obtained from our study were comparable to other existing proteomic and transcriptional profiling approaches. This study illustrates the robustness of this novel LCMS approach for the simultaneous quantitative and comprehensive qualitative analysis of proteins in complex mixtures. We describe a novel LCMS approach to the relative quantitation and simultaneous identification of proteins within the complex milieu of unfractionated Escherichia coli. This label-free, LCMS acquisition method observes all detectable, eluting peptides and their corresponding fragment ions. Postacquisition data analysis methods extract both the chromatographic and the mass spectrometric information on the tryptic peptides to provide time-resolved, accurate mass measurements, which are subsequently used for quantitation and identification of constituent proteins. The response of E. coli to carbon source variation is well understood, and it is thus commonly used as a model biological system when validating an analytical method. Using this LCMS approach, we characterized proteins isolated from E. coli grown in glucose, lactose, and acetate. The change in relative abundance of the corresponding proteins was measured from peptides common to both conditions. Protein identities were also determined for those peptides that were unique to each condition, and these identities were found to be consistent with the underlying biochemical restrictions imposed by the growth conditions. The relative change in abundance of the characterized proteins ranged from 0.1- to 90-fold among the three binary comparisons. The overall coverage of the characterized proteins ranged from 10 to 80%, consisting of one to 34 peptides per protein. The quantitative results obtained from our study were comparable to other existing proteomic and transcriptional profiling approaches. This study illustrates the robustness of this novel LCMS approach for the simultaneous quantitative and comprehensive qualitative analysis of proteins in complex mixtures. Escherichia coli is a microbial symbiote found in the colon and large intestine of most warm blooded animals that plays a critical role in vertebrate anabolism and catabolism. The environment in which E. coli lives is subject to rapid changes in the availability of the carbon and nitrogen compounds necessary to provide its energy and primary building blocks. E. coli survival hinges on the ability to successfully control the expression of genes coding for enzymes and proteins required for growth in response to environmental changes. Because of its simple cellular structure and its relative ease of maintenance and manipulation in the laboratory, E. coli has become the “workhorse host” for most research in molecular biology and microbiology. As a result, it is regarded as one of the most completely characterized organisms in all biology. The ease with which recombinant proteins can be expressed in E. coli has made this bacterium useful in the study of many basic biological processes as well as in the production of heterologous proteins for research and therapeutic purposes. For these reasons, E. coli has become a model system for testing new analytical technologies. 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