Toward universal protein post-translational modification detection in high throughput format

Area of Science:

• Biochemistry
• Molecular Biology
• Assay Development

Background:

• Post-translational modifications (PTMs) are crucial regulatory mechanisms in various pathological conditions.
• Current assays for PTMs are often specific to certain modifications or groups, limiting their broad applicability.
• There is a need for versatile and efficient assays to facilitate the discovery of modulators for PTM-related diseases.

Purpose of the Study:

• To establish a universal time-resolved luminescence assay for the direct detection of a wide variety of protein PTMs.
• To provide a practical and high-throughput platform for PTM analysis.
• To enable accelerated drug discovery for diseases associated with PTM dysregulation.

Main Methods:

• Development of a universal time-resolved luminescence assay utilizing a peptide-break platform and the leucine zipper concept.
• Utilizing a europium-chelate labeled detection peptide and a non-labeled peptide substrate to form a luminescent dimer.
• Monitoring PTM enzyme activity by the quenching of luminescent signal from detached detection peptide in the presence of soluble quenchers.

Main Results:

• Demonstrated the functionality of the universal assay for monitoring phosphorylation, dephosphorylation, deacetylation, and citrullination.
• The assay operates effectively at sub to low nanomolar enzyme concentrations.
• The developed technique shows high applicability to a broad spectrum of PTMs in a high-throughput format.

Conclusions:

• The established universal luminescence assay provides a versatile tool for detecting diverse protein PTMs.
• This assay platform significantly enhances the efficiency of studying PTMs and discovering related therapeutic agents.
• The method holds promise for broad application in biochemical research and drug discovery for PTM-associated pathologies.