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

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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.

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

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Acknowledgments

The authors thank the Allbritton Laboratory at the University of North Carolina for generously providing advice and peptide standards, particularly Angela Proctor and Emilie Mainz for helpful discussions and their collaborator Qunzhao Wang for synthesis of the peptides. We also thank Jeremiah Marden and the Graf Laboratory at the University of Connecticut for assistance with the S. cerevisiae and E. coli cultures and lysis. This work was supported by Trinity College.

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Authors and Affiliations

  1. Department of Chemistry, Trinity College, 300 Summit Street, Hartford, CT, 06106, USA

    Allison J. Tierney, Kunwei Yang & Michelle L. Kovarik

  2. Department of Mathematics, Trinity College, 300 Summit Street, Hartford, CT, 06106, USA

    Nhat Pham & Brooks K. Emerick

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  1. Allison J. Tierney
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Correspondence to Michelle L. Kovarik.

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Tierney, A.J., Pham, N., Yang, K. et al. Interspecies comparison of peptide substrate reporter metabolism using compartment-based modeling. Anal Bioanal Chem 409, 1173–1183 (2017). https://doi.org/10.1007/s00216-016-0085-9

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