Updated project metadata. Most of the proteomic studies done so far uses bacterial cells harvested from liquid culture media. However it is widely accepted that many important determinants associated with virulence and host cell adhesion are exclusively expressed during growth on solid media. As an attempt to complement previous studies, in this study we compare the proteome coverage of Escherichia coli K12 from a single colony on solid media with those at different growth phases in liquid culture; i.e. early log, mid log, early - and late stationary growth phases. A total of 2044 protein groups covering approximately 47% of the total proteome were identified across all studied conditions; 1650 number of proteins identified from single colonies and 1679 proteins from liquid cultured cells. Label-free quantitative analysis revealed that single colony proteome in a solid agar differs largely from that in liquid culture. The presence of the Suf-operon, involved in iron mobilisation and swarming motility were associated exclusively with single colony profiles. Whereas proteins involved in motility such as MotA, MotB, fliH, flip, fliD and fliJ were associated exclusively to cells grown in liquid culture. The data presented here provide a valuable resource for understanding the role of key proteins within microenvironments surrounding E.coli single colonies.