Development and Application of the Metalloprotease Activity Multiplexed Bead-Based Immunoassay (MAMBI)
Author(s)Chopko Ahrens, Caroline; Chiswick, Evan; Ravindra, Kodihalli C.; Miller, Miles Aaron; Ramseier, Julie Y.; Isaacson, Keith B.; Lauffenburger, Douglas A; Griffith, Linda G; ... Show more Show less
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Metalloproteinases (MMPs) are zinc-dependent endopeptidases that cleave various proteins to regulate normal and diseased cellular functions, and as such, they play significant roles in human tissue development, homeostasis, and the pathogenesis of many diseases, including cancers, endometriosis, arthritis, etc. Most MMPs are produced as zymogenic latent enzymes that must be cleaved to activate their catalytic regions, and localized endogenous protein inhibitors further regulate activity. Accordingly, they operate within recursive networks to degrade extracellular matrix proteins and regulate cell signaling by cleaving growth factors and receptors at the cell surface and in the local pericellular environment. Thus, high-resolution information about the concentrations of specific active MMPs, revealing their intricate regulatory networks, may improve disease diagnosis and treatment. Here, we introduce a new and readily mastered method for measuring MMP activities in a multiplex fashion. We integrate aspects of activity-based enzyme labeling with commercial high-throughput, multiplexed protein quantification to yield the metalloproteinase activity multiplexed bead-based immunoassay (MAMBI). Assays of recombinant active MMP-1, -2, -3, -7, -8, -9, -12, and -13 establish the sensitivity and selectivity of MAMBI detection. Levels of active native MMPs are similarly characterized in conditioned cell culture medium, menstrual effluent, and uterine tissue. In a single MAMBI (5 μL), we achieve sensitivities equal to those from leading single-plex MMP activity detection strategies (e.g., 10-15 M for MMP-1). We also demonstrate high-throughput inhibitor screening via the MAMBI approach in complex, patient-derived samples.
DepartmentMassachusetts Institute of Technology. Department of Biological Engineering
American Chemical Society (ACS)
Ahrens, Caroline C. et al. "Development and Application of the Metalloprotease Activity Multiplexed Bead-Based Immunoassay (MAMBI)." Biochemistry 58, 38 (September 2019): 3938–3942 © 2019 American Chemical Society
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