NDLI: Interspecies comparison of peptide substrate reporter metabolism using compartment-based modeling

Content Provider	Springer Nature Link
Author	Tierney, Allison J. Pham, Nhat Yang, Kunwei Emerick, Brooks K. Kovarik, Michelle L.
Copyright Year	2016
Abstract	Peptide substrate reporters are fluorescently labeled peptides that can be acted upon by one or more enzymes of interest. Peptide substrates are readily synthesized and more easily separated than full-length protein substrates; however, they are often more rapidly degraded by peptidases. As a result, peptide reporters must be made resistant to proteolysis in order to study enzymes in intact cells and lysates. This is typically achieved by optimizing the reporter sequence in a single cell type or model organism, but studies of reporter stability in a variety of organisms are needed to establish the robustness and broader utility of these molecular tools. We measured peptidase activity toward a peptide substrate reporter for protein kinase B (Akt) in E. coli, D. discoideum, and S. cerevisiae using capillary electrophoresis with laser-induced fluorescence (CE-LIF). Using compartment-based modeling, we determined individual rate constants for all potential peptidase reactions and explored how these rate constants differed between species. We found the reporter to be stable in D. discoideum (t 1/2 = 82–103 min) and S. cerevisiae (t 1/2 = 279–314 min), but less stable in E. coli (t 1/2 = 21–44 min). These data suggest that the reporter is sufficiently stable to be used for kinase assays in eukaryotic cell types while also demonstrating the potential utility of compartment-based models in peptide substrate reporter design. Graphical abstract Cell lysates from several evolutionarily divergent species were incubated with a peptide substrate reporter, and compartment-based modeling was used to determine key steps in the metabolism of the reporter in each cell type
Starting Page	1173
Ending Page	1183
Page Count	11
File Format	PDF
ISSN	16182642
Journal	Analytical and Bioanalytical Chemistry
Volume Number	409
Issue Number	5
e-ISSN	16182650
Language	English
Publisher	Springer Berlin Heidelberg
Publisher Date	2016-11-29
Publisher Place	Berlin, Heidelberg
Access Restriction	One Nation One Subscription (ONOS)
Subject Keyword	Amino acids/peptides Capillary electrophoresis/electrophoresis Modeling Bioanalytical methods Biological samples Enzymes Analytical Chemistry Biochemistry Laboratory Medicine Characterization and Evaluation of Materials Food Science Monitoring/Environmental Analysis
Content Type	Text
Resource Type	Article
Subject	Analytical Chemistry Biochemistry

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