Peptic Fluorescent "Signal-On" and "Signal-Off" Sensors Utilized for the Detection Protein Post-Translational Modifications

: Kopra K, Eskonen V, Seppala T, Jakovleva J, Huttunen R, Harma H

Publisher: AMER CHEMICAL SOC

: 2019

: ACS Omega

: ACS OMEGA

: 4

: 2

: 4269

: 4275

: 7

: 2470-1343

DOI: https://doi.org/10.1021/acsomega.8b03672

: https://research.utu.fi/converis/portal/detail/Publication/39888075

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.

acsomega.8b03672.pdf