One- and two-dimensional SDS-PAGE zymography with quenched fluorogenic substrates provides identification of biological fluid proteases by direct mass spectrometry

Analytical Biochemistry
Véronique ThimonJean-Luc Gatti

Abstract

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.

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Citations

Jun 28, 2008·Analytical and Bioanalytical Chemistry·H SchlüterM Trusch
Feb 28, 2013·Nature Methods·Jennifer VandoorenGhislain Opdenakker
Nov 26, 2009·Molecular Biology and Evolution·Jingjing LiZhaolei Zhang
Sep 30, 2014·International Journal of Biological Macromolecules·Danielle S AragãoDulce Elena Casarini

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