One- and two-dimensional SDS-PAGE zymography with quenched fluorogenic substrates provides identification of biological fluid proteases by direct mass spectrometry.
We describe a simple and direct zymographic method for the detection of proteases using quenched fluorescent substrates. The proteases were separated using one- and two-dimensional electrophoresis, and the gel subsequently was incubated with the quenched fluorescent substrate. After a short incubation, the released fluorescence allowed the localization of the proteases directly using UV light. The protease spots could then be cut directly from the gel and processed for identification by mass spectrometry. This method could easily be used to develop or test whether a substrate is specific or not and also to detect the proteases that are able to cleave this substrate in a complex biological fluid. This also allowed direct identification of proteases without complex purification.
Thimon
Véronique
V
Gamètes Males et Fertilité, INRA, CNRS, Haras Nationaux, Université de Tours, Institut National de la Recherche Agronomique, Station de Physiologie de la Reproduction et des Comportements, 37380 Nouzilly, France.
Belghazi
Maya
M
Labas
Valérie
V
Dacheux
Jean-Louis
JL
Gatti
Jean-Luc
JL
D000818:Animals; D001826:Body Fluids / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000378:metabolism*; D013058:Mass Spectrometry / Q000379:methods*; D010447:Peptide Hydrolases / Q000378:metabolism*; D053719:Tandem Mass Spectrometry
D005456:Fluorescent Dyes; D010447:Peptide Hydrolases