A proteome and acetylome study under different culture conditions was carried out by quantitative label-free mass spectrometry analysis. E. coli K12 BW25113 strain was grown employing TB7 complex medium or M9 minimal medium, supplemented with glucose 20 mM or glycerol 40 mM as carbon source. Therefore, 4 culture conditions (TB7-glucose, TB7-glycerol, MM9-glucose, and MM9-glycerol) were used. Samples were taken in exponential and in stationary growth phase, resulting in 8 experimental samples: TB7-glucose in exponential phase, TB7-glucose in stationary phase, TB7-glycerol in exponential phase, TB7-glycerol in stationary phase, MM9-glucose in exponential phase, MM9-glucose in stationary phase, MM9-glycerol in exponential phase, and MM9-glycerol in stationary phase, with 4 biological replicates from each. This work emphasises the importance of the culture conditions choice, as they determine both, location of acetylation, and acetylation level, which may have an impact on regulation of the central metabolism.