Application of peptides as reaction specific probes for the automated mass spectrometry-assisted enzyme screening (MES)

Abstract

Peptides play a central role as reaction specific probes for screening protein libraries for target enzymes with protein-modifying activities with the automated platform technology named mass spectrometry assisted enzyme screening (MES). The protein libraries are generated by fractionating protein extracts and by immobilizing the proteins covalently to activated affinity beads. The innovative idea of the MES-system is to incubate the fractions of the protein library with special designed peptides that serve as substrates for protein modifying enzymes and to detect the resulting reaction products by mass spectrometry, if the target enzyme is present in a fraction of the protein library. The peptide sequences, selected for the reaction specific probes, are usually parts of the endogenous protein substrates. The main advantage of the MES approach is that even complex protein mixtures can be screened for enzymatic activities. The MES technique is suited for the search for unknown target enzymes with defined catalytic reaction profiles, for investigating the metabolism of defined substrates in cells or tissues and for comparing defined enzyme activities in organisms in different states. Therefore the application of the MES system includes a wide area, from the identification of new drug targets to the identification of enzymes relevant for biotechnological processes. Here the MES system is demonstrated for the screening for urotensin-II (U-II) metabolising enzymes in renal tissue. With MES des-Val-U-II was determined as major metabolite of U-II of renal tissue proteins.