Simultaneous Detection of Metalloprotease Activities in Complex Biological Samples Using the PrAMA (Proteolytic Activity Matrix Assay) Method.
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.
Department of Neurosurgery, Marburg University, 35033, Marburg, Germany.|Department of Anesthesiology and Intensive Care Medicine, University Hospital, Albert-Schweitzer Campus 1, 48149, Münster, Germany.
Catharina
C
Conrad
Center for Systems Biology, Massachusetts General Hospital, Boston, MA, USA.
Miles A
MA
Miller
Department of Neurosurgery, Marburg University, 35033, Marburg, Germany. jbartsch@med.uni-marburg.de.
Jörg W
JW
Bartsch
Department of Neurosurgery, Marburg University, 35033, Marburg, Germany.
Uwe
U
Schlomann
Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, 02139, USA.
Douglas A
DA
Lauffenburger
D002460:Cell Line; D057075:Enzyme Assays / Q000379:methods*; D006801:Humans; D020782:Matrix Metalloproteinases / Q000378:metabolism*; D059748:Proteolysis; D013379:Substrate Specificity; D059008:Time-Lapse Imaging; D057188:Workflow
D020782:Matrix Metalloproteinases
ADAM10; Collagenase; Michaelis-Menten; Multiplexed measurement; Network analysis; Sheddase; Singular value decomposition; TACE