Development of a method for the high-throughput quantification of cellular proteins

Area of Science:

• Biochemistry and Molecular Biology
• Neuroscience
• Assay Development

Background:

• Quantifying cellular proteins is vital for understanding biological processes.
• Huntington's disease is caused by intracellular mutant huntingtin protein.
• Existing methods for protein quantification may require improvement for specific targets like mutant huntingtin.

Purpose of the Study:

• To develop a sensitive assay for detecting intracellular mutant huntingtin.
• To create a method generally applicable for quantifying other cellular proteins.
• To establish a screening platform for identifying compounds that modulate mutant huntingtin levels.

Main Methods:

• Engineered a small recombinant protein tag recognized by monoclonal antibodies.
• Fused the tag to an inducible fragment of mutant huntingtin.
• Utilized time-resolved Förster Resonance Energy Transfer (TR-FRET) for detection.
• Adapted the assay into a miniaturized, homogeneous format for high-throughput screening (HTS).
• Multiplexed the assay with a cell toxicity readout.

Main Results:

• Successfully detected low intracellular levels of mutant huntingtin using TR-FRET.
• Demonstrated the assay's suitability for miniaturized screening in 1536-well plates.
• Showcased the ability to distinguish protein level changes from cytotoxicity.
• Identified compounds that positively and negatively modulate huntingtin levels.

Conclusions:

• The developed TR-FRET assay is effective for quantifying intracellular mutant huntingtin.
• This assay platform is adaptable for screening other cellular proteins.
• The assay serves as a valuable tool for Huntington's disease drug discovery programs.
• Multiplexing with toxicity assays enhances screening reliability.