Related Experiment Video
Updated: Dec 10, 2025
11:54
Detection of Protein Ubiquitination Sites by Peptide Enrichment and Mass Spectrometry
Published on: March 23, 2020
10.1K
Single-Peptide TR-FRET Detection Platform for Cysteine-Specific Post-Translational Modifications
Ville Eskonen1, Natalia Tong-Ochoa1, Leena Mattsson1
1Chemistry of Drug Development, Department of Chemistry, University of Turku, Vatselankatu 2, FI-20014 Turku, Finland.
Analytical Chemistry
|September 3, 2020
Summary
Researchers developed a new assay to detect cysteine modifications, crucial for cell signaling. This sensitive, antibody-free method uses time-resolved Förster resonance energy transfer (TR-FRET) for high-throughput screening of enzymes involved in health and disease.
Area of Science:
- Biochemistry
- Molecular Biology
- Cell Signaling
Background:
- Post-translational modifications (PTMs) regulate cellular processes and are implicated in diseases.
- PTM-modifying enzymes are key drug targets, necessitating efficient detection methods.
- Current assays often lack throughput, rely on antibodies, or are not versatile.
Purpose of the Study:
- To develop a sensitive, high-throughput, antibody-free assay for detecting cysteine-modifying enzymes.
- To establish a single-peptide detection platform using time-resolved Förster resonance energy transfer (TR-FRET).
Main Methods:
- A novel TR-FRET assay was designed utilizing a biotinylated peptide substrate modified at cysteine residues.
- Enzymatic modification prevents thiol coupling to an acceptor dye, altering the TR-FRET signal.
- The assay was validated for S-nitrosylation and ADP-ribosylation using chemical and enzymatic reactions.
Main Results:
- The assay demonstrated high sensitivity with signal-to-background ratios up to 33.0 for S-nitrosylation.
- Effective detection of ADP-ribosylation and enzyme activity was achieved.
- The method showed low nanomolar substrate consumption and proof-of-concept with K-Ras and Gαi peptide substrates.
Conclusions:
- The developed TR-FRET assay is a versatile, antibody-free platform for detecting cysteine-modifying enzymes.
- This method enables high-throughput screening for drug discovery and biological research.
- The assay offers a sensitive and cost-effective approach to study PTMs.
