A1 Refereed original research article in a scientific journal
Thermal Dissociation Assay for Time-Resolved Fluorescence Detection of Protein Post-Translational Modifications
Authors: Ville Eskonen, Natalia Tong-Ochoa, Salla Valtonen, Kari Kopra, Harri Härmä
Publisher: American Chemical Society
Publication year: 2019
Journal: ACS Omega
Journal name in source: ACS Omega
Volume: 4
Issue: 15
First page : 16501
Last page: 16507
Number of pages: 7
ISSN: 2470-1343
eISSN: 2470-1343
DOI: https://doi.org/10.1021/acsomega.9b02134
Web address : https://pubs.acs.org/doi/10.1021/acsomega.9b02134
Post-translational modifications (PTMs) of proteins provide an important mechanism for cell signal transduction control. Impaired PTM control is a key feature in multiple different disease states, and thus the enzyme-controlling PTMs have drawn attention as highly promising drug targets. Due to the importance of PTMs, various methods to monitor PTM enzyme activity have been developed, but universal high-throughput screening (HTS), a compatible method for different PTMs, remains elusive. Here, we present a homogeneous single-label thermal dissociation assay for the detection of enzymatic PTM removal. The developed method allows the use of micromolar concentration of substrate peptide, which is expected to be beneficial when monitoring enzymes with low activity and peptide binding affinity. We prove the thermal dissociation concept functionality using peptides for dephosphorylation, deacetylation, and demethylation and demonstrate the HTS-compatible flash isothermal method for PTM enzyme activity monitoring. Using specific inhibitors, we detected literature-comparable IC50 values and Z′ factors from 0.61 to 0.72, proving the HTS compatibility of the thermal peptide-break technology.