Relative protein abundances of Escherichia coli growing exponentially on minimal medium with acetate or glucose as the sole carbon source were investigated in a quantitative shotgun proteome analysis with TMT6-plex isobaric tags. Peptides were separated by high resolution high/low pH 2D-LC, using an optimized fraction pooling scheme followed by mass spectrometric analysis. Quantitative data were acquired for 1,994 proteins covering 46 % of the predicted E. coli proteins and 361 differentially abundant proteins were discovered. Differences in protein abundance were observed for proteins in central carbon metabolism, in particular for proteins involved in TCA cycle and glyoxylate shunt, fatty acid metabolism, glycolysis/gluconeogenesis and anaplerotic pathways. Significant changes were observed in proteins relevant for amino acid and protein synthesis, proteins necessary to process environmental information and scavenge for a variety of alternate carbon sources.