Mass-spectrometry-linked screening of protein fractions for enzymatic activities--a tool for functional genomics.
A simple and rapid strategy is described to screen protein fractions for defined enzymatic activity. A protein fraction from a porcine kidney extract was immobilized by covalent coupling to activated affinity beads. The immobilized proteins were incubated with probes specific for different enzyme activities. The reaction products were analyzed by matrix-assisted laser desorption/ionization (MALDI)-mass spectrometry. The MALDI spectra indicate the presence of 5'-nucleotidase, phosphatase, kinase, glutathione reductase, and renin activities in the kidney protein extract. Furthermore, the method can be used to screen for inhibitors of enzymatic reactions. The method is adaptable to high-throughput sample handling and automated mass spectrometric analysis and therefore suited for functional genomics.
Medizinische Klinik IV, Universitätsklinikum Benjamin Franklin, Freie Universität Berlin, Hindenburgdamm 30, 12200 Berlin, Germany.
J
J
Jankowski
N
N
Stephan
M
M
Knobloch
S
S
Fischer
D
D
Schmaltz
W
W
Zidek
H
H
Schlüter
D000818:Animals; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000032:analysis; D004798:Enzymes / Q000032:analysis*; D007668:Kidney / Q000201:enzymology*; D008863:Microspheres; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D013379:Substrate Specificity; D013552:Swine; D013997:Time Factors
D004791:Enzyme Inhibitors; D004798:Enzymes