A1 Journal article – refereed
Peptic Fluorescent "Signal-On" and "Signal-Off" Sensors Utilized for the Detection Protein Post-Translational Modifications




List of Authors: Kopra K, Eskonen V, Seppala T, Jakovleva J, Huttunen R, Harma H
Publisher: AMER CHEMICAL SOC
Publication year: 2019
Journal: ACS Omega
Journal name in source: ACS OMEGA
Journal acronym: ACS OMEGA
Volume number: 4
Issue number: 2
Number of pages: 7
ISSN: 2470-1343
eISSN: 2470-1343

Abstract
Protein post-translational modifications (PTMs) are typically enzyme-catalyzed events generating functional diversification of proteome; thus, multiple PTM enzymes have been validated as potential drug targets. We have previously introduced energy-transfer-based signal-modulation method called quenching resonance energy transfer (QRET), and utilize it to monitor PTM addition or removal using the developed peptide-break technology. Now we have reinvented the QRET technology, and as a model, we introduced the tunable fluorescent "signal-on" and "signal-off" detection scheme in the peptide-break PTM detection. Taking the advantage of time-resolved fluorescence-based single-label detection technology, we were able to select the signal direction upon PTM addition or removal by simply introducing different soluble Eu3+-signal-modulating molecule. This enables the selection of positive signal change upon measurable event, without any additional labeling steps, changes in assay condition or Eu3+-reporter. The concept functionality was demonstrated with four Eu3+-signal modulators in a high-throughput compatible kinase and phosphatase assays using signal-on and signal-off readout at 615 nm or time-resolved Forster resonance energy transfer at 665 nm. Our data suggest that the introduced signal modulation methodology provides a transitional fluorescence-based single-label detection concept not limited only to PTM detection.

Downloadable publication

This is an electronic reprint of the original article.
This reprint may differ from the original in pagination and typographic detail. Please cite the original version.




Last updated on 2019-21-08 at 21:09