The available Escherichia coli genome sequences offer an opportunity to further expand our understanding of this bacterium. In the current study, we present a rapid method for the isolation of bacterial alkaline proteins using acid incubation, purification and protein array by 2-DE, followed by protein identification using MS. Fifty-seven proteins were randomly chosen, in which 55 were identified by a database searching of MS data. The searching results showed that most of these alkaline proteins were involved in special functions within the cell, suggesting that alkaline proteome is an ideal fraction for an understanding of their special functions. Furthermore, alkaline proteomes were compared between the period of majority live bacteria (18-h culture), the period of similar amount of live and dead bacteria (30-h culture) and the period of majority dead bacteria (48-h culture). Six proteins were identified as differentially expressed targets, in which putative transcriptional regulator and superoxide dismutase genes were cloned and expressed for antiserum preparations. The antisera were applied for the confirmation of results obtained from 2-DE. The presented data clearly reveal that alkaline proteome analysis by 2-DE with MS plays an important role in the understanding of protein functions within the cell, and six alkaline proteins are determined as key ones in an overgrowth-mediated growth cycle of E. coli.