We describe a novel LCMS approach to 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. Post-acquisition 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 have 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 which 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 1 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.