Proteolysis of whole cell extracts with immobilized enzyme columns as part of multidimensional chromatography

This paper describes the efficacy of immobilized trypsin columns in the digestion of cellular extracts that contained thousands of proteins. Effectiveness of proteolysis was evaluated with extracts of Escherichia coli by size-exclusion chromatography. Immobilized trypsin columns were operated in either the continuous-flow or stopped-flow mode at temperatures ranging from ambient to 37 degrees C with incubation times of 0-2 h. The results of these studies indicate that reduced and alkylated extracts of proteins from E. coli can be digested in 20 min when the enzyme column is operated at elevated temperature. The advantage of immobilized enzyme columns is that they can be easily incorporated into multidimensional separation systems for automated proteomics.