Document Type

Article

Department

​Mathematics

Publication Date

11-2016

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 (t1/2 = 82–103 min) and S. cerevisiae (t1/2 = 279–314 min), but less stable in E. coli (t1/2 = 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.

Comments

Tierney, Pham, and Yang contributed to this article as undergraduate students at Trinity College.

Published as: Allison J. Tierney, Nhat Pham, Kunwei Yang, Brooks K. Emerick, and Michelle L. Kovarik. “Interspecies Comparison of Peptide Substrate Reporter Metabolism Using Compartment-based Modeling.” Analytical and Bioanalytical Chemistry 409, no. 5 (2016): 1173-183. doi:10.1007/s00216-016-0085-9.

Author's accepted manuscript provided by the Trinity College Digital Repository in accordance with publisher's distribution policies.

Publication Title

Analytical and Bioanalytical Chemistry

Volume

409

Issue

5

First Page

1173

Last Page

1183

DOI

10.1007/s00216-016-0085-9

Share

COinS