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Published on: July 16, 2018
Simultaneous Detection of Metalloprotease Activities in Complex Biological Samples Using the PrAMA (Proteolytic
Catharina Conrad1,2, Miles A Miller3, Jörg W Bartsch4
1Department of Neurosurgery, Marburg University, 35033, Marburg, Germany.
Abstract:
Proteolytic Activity Matrix Analysis (PrAMA) is a method for simultaneously determining the activities of specific Matrix Metalloproteinases (MMPs) and A Disintegrin and Metalloproteinases (ADAMs) in complex biological samples. In mixtures of unknown proteases, PrAMA infers selective metalloproteinase activities by using a panel of moderately specific FRET-based polypeptide protease substrates in parallel, typically monitored by a plate-reader in a 96-well format. Fluorescence measurements are then quantitatively compared to a standard table of catalytic efficiencies measured from purified mixtures of individual metalloproteinases and FRET substrates. Computational inference of specific activities is performed with an easily used Matlab program, which is provided herein. Thus, we describe PrAMA as a combined experimental and mathematical approach to determine real-time metalloproteinase activities, which has previously been applied to live-cell cultures, cellular lysates, cell culture supernatants, and body fluids from patients.
Insights
Proteolytic Activity Matrix Analysis (PrAMA) quantifies specific Matrix Metalloproteinase (MMP) and A Disintegrin and Metalloproteinase (ADAM) activities in biological samples. This method combines experimental assays with computational analysis for real-time protease activity determination.
Area of Science:
- Biochemistry
- Proteomics
- Enzymology
Background:
- Matrix Metalloproteinases (MMPs) and A Disintegrin and Metalloproteinases (ADAMs) are crucial enzymes involved in various physiological and pathological processes.
- Accurate quantification of specific MMP and ADAM activities in complex biological samples remains a challenge.
- Existing methods often lack the specificity or throughput required for comprehensive analysis.
Purpose of the Study:
- To introduce and validate Proteolytic Activity Matrix Analysis (PrAMA) as a novel method for simultaneous determination of MMP and ADAM activities.
- To provide a robust experimental and computational framework for real-time protease activity profiling.
- To demonstrate the applicability of PrAMA across diverse biological matrices.
Main Methods:
- PrAMA utilizes a panel of Förster Resonance Energy Transfer (FRET)-based polypeptide substrates with moderate specificity.
- Enzyme activities are measured in parallel using a plate-reader in a 96-well format.
- Fluorescence data are quantitatively analyzed using a standard table of catalytic efficiencies and a provided Matlab program for computational inference.
Main Results:
- PrAMA successfully determines the activities of specific MMPs and ADAMs in complex biological mixtures.
- The method allows for real-time monitoring of metalloproteinase activities.
- PrAMA has been successfully applied to various biological samples, including live-cell cultures, lysates, supernatants, and patient body fluids.
Conclusions:
- PrAMA offers a powerful and versatile approach for the simultaneous quantification of MMP and ADAM activities.
- This method provides valuable insights into protease function in biological systems.
- PrAMA is a significant advancement for research in areas involving metalloproteinase dysregulation.
