Escherichia coli has been used widely in laboratory and the biotech industry. However, the genetic and metabolic characteristics remain inadequately studied, particularly for those strains with extensive genetic manipulations that might have resulted in unknown mutations. Here, we demonstrate a comparative proteomics and genetics approach to identify unknown mutations in E. coli K-12 derivatives. The comparative proteomic and genetic analyses revealed an IS5 disruption of the kdgR gene in two commonly used derivative strains of E. coli K-12, XL1-Blue and DH5α, compared with K-12 wild-type strain W3110. In addition, a controversial deoR mutation was clarified as a wild type in E. coli DH5α using the same approach. This approach should be useful in characterizing the unknown mutations in various mutant strains developed. At the same time, comparative proteomic analysis also revealed the distinct metabolic characteristic of the two derivatives: higher biosynthetic flux to purine nucleotides. This is potentially beneficial for the synthesis of plasmid DNA.