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

This paper describes the efficacy of immobilized trypsin columns in the dig estion of cellular extracts that contained thousands of proteins. Effective ness of proteolysis was evaluated with extracts of Escherichia coli by size -exclusion chromatography. Immobilized trypsin columns were operated in eit her the continuous-how or stopped-flow mode at temperatures ranging from am bient to 37 degreesC with incubation times of 0-2 h. The results of these s tudies indicate that reduced and alkylated extracts of proteins from E. col i 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 b e easily incorporated into multidimensional separation systems for automate d proteomics. (C) 2001 Published by Elsevier Science B.V.