Ultrahigh-Throughput Liquid Chromatography with Tandem Mass Spectrometry Method for Targeted Protein Degradation...

Area of Science:

• Proteomics and Mass Spectrometry
• Drug Discovery and Development
• Molecular Biology and Biochemistry

Background:

• Targeted protein degradation (TPD) is a promising therapeutic strategy for eliminating disease-causing proteins.
• High-throughput screening is crucial for validating the efficacy and selectivity of novel TPD compounds across numerous proteins.
• Existing proteomic methods face challenges in achieving the necessary throughput and precision for large-scale TPD screening.

Purpose of the Study:

• To develop and validate an ultrahigh-throughput liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for targeted protein degradation (TPD) compound screening.
• To assess the proteome coverage, reproducibility, and quantitation accuracy of the developed method at high throughput.
• To apply the method to study dose-dependent degradation patterns of TPD compounds and identify potential therapeutic benefits.

Main Methods:

• Development of a 300 samples per day (SPD) LC-MS/MS method utilizing the Orbitrap Astral mass spectrometer.
• Evaluation of proteome coverage and reproducibility using coefficient of variation (CV) analysis.
• Assessment of quantitation accuracy through multi-proteome mixture experiments (two- and three-species digests).

Main Results:

• The method achieved identification of nearly 8000 protein groups from a single cell line with a CV < 10% at 300 SPD.
• Demonstrated high quantitation accuracy for small and large fold changes in three-proteome mixtures (R² of 0.999 for a two-proteome mixture).
• Studied dose-dependent degradation of pomalidomide, iberdomide, and mezigdomide, suggesting mezigdomide's enhanced efficacy in T cells via IKZF2 degradation.

Conclusions:

• An ultrahigh-throughput LC-MS/MS method with excellent proteome coverage and quantitation accuracy was successfully developed.
• The method is highly suitable for rapid chemoproteomics screening of targeted protein degradation (TPD) drug libraries.
• Findings suggest mezigdomide's potential for boosting anticancer immunity through T cell-specific protein degradation.