AbstractMining the emerging abundance of microbial genome sequences for hypotheses is an exciting prospect of “functional genomics”. At the forefront of this effort, we compared the predictions of the complete Escherichia coli genomic sequence with the observed gene products by assessing 381 proteins for their mature N‐termini, in vivo abundances, isoelectric points, molecular masses, and cellular locations. Two‐dimensional gel electrophoresis (2‐DE) and Edman sequencing were combined to sequence Coomassie‐stained 2‐DE spots representing the abundant proteins of wild‐type E. coli K‐12 strains. Greater than 90% of the abundant proteins in the E. coli proteome lie in a small isoelectric point and molecular mass window of 4–7 and 10–100 kDa, respectively. We identified several highly abundant proteins, YjbJ, YjbP, YggX, HdeA, and AhpC, which would not have been predicted from the genomic sequence alone. Of the 223 uniquely identified loci, 60% of the encoded proteins are proteolytically processed. As previously reported, the initiator methionine was efficiently cleaved when the penultimate amino acid was serine or alanine. In contrast, when the penultimate amino acid was threonine, glycine, or proline, cleavage was variable, and valine did not signal cleavage. Although signal peptide cleavage sites tended to follow predicted rules, the length of the putative signal sequence was occassionally greater than the consensus. For proteins predicted to be in the cytoplasm or inner membrane, the N‐terminal amino acids were highly constrained compared to proteins localized to the periplasm or outer membrane. Although cytoplasmic proteins follow the N‐end rule for protein stability, proteins in the periplasm or outer membrane do not follow this rule; several have N‐terminal amino acids predicted to destabilize the proteins. Surprisingly, 18% of the identified 2‐DE spots represent isoforms in which protein products of the same gene have different observed pI and Mr, suggesting they are post‐translationally processed. Although most of the predicted and observed values for isoelectric point and molecular mass show reasonable concordance, for several proteins the observed values significantly deviate from the expected values. Such discrepancies may represent either highly processed proteins or misinterpretations of the genomic sequence. Our data suggest that AhpC, CspC, and HdeA exist as covalent homomultimers, and that IcdA exists as at least three isoforms even under conditions in which covalent modification is not predicted. We enriched for proteins based on subcellular location and found several proteins in unexpected subcellular locations.