High-throughput PROTAC compound screening workflow for targeted protein degradation on an Orbitrap Astral mass spectrometer with accurate label-free quantitation

Significance of the Topic

Targeted protein degradation (TPD) represents a transformative strategy for drug discovery by harnessing the cell’s own degradation machinery to selectively eliminate disease-causing proteins. High-throughput and accurate quantitation of proteolysis-targeting chimera (PROTAC) compounds are essential to identify leads that induce desired protein knockdown while minimizing off-target effects.

Aims and Study Overview

The study aimed to establish a robust, label-free quantitative proteomics workflow on the Orbitrap Astral mass spectrometer to enable:

• Ultra-high-throughput screening of PROTAC degraders at 24, 60, 180, and 300 samples per day (SPD).
• Accurate and reproducible quantitation of target and off-target proteins in prostate cancer cells treated with the ARCC-4 PROTAC.
• Deep proteome coverage for in-depth validation using extended chromatographic gradients.

Methodology

VCaP prostate cancer cells were treated in triplicate with 5, 50, or 500 nM ARCC-4 for four hours, followed by lysis, protein quantification (BCA assay), reduction, alkylation, and tryptic digestion using an automated AccelerOme platform.
DIA (data-independent acquisition) methods were implemented with 24- to 300-SPD peptide separations on UHPLC columns, followed by Orbitrap Astral analysis. Data were processed by Spectronaut, DIA-NN, and Proteome Discoverer with CHIMERYS for directDIA analysis.

Used Instrumentation

• Orbitrap Astral mass spectrometer
• Thermo Scientific Vanquish Neo UHPLC system
• 50 cm EASY-Spray PepMap Neo, 15 cm EASY-Spray PepMap, and 5 cm Ionopticks Aurora columns
• Thermo Scientific AccelerOme automated sample preparation
• Spectronaut (Biognosys), DIA-NN, Proteome Discoverer with CHIMERYS algorithms

Main Results and Discussion

The LFQ-DIA workflow yielded:

• Consistent, dose-dependent degradation of androgen receptor (AR) across all throughputs.
• Identification of ~10 000 protein groups at 24 SPD and ~10 400 at 60 SPD using extended gradients.
• Over 8 500 proteins at 180 SPD and ~8 000 at 300 SPD with ultra-fast methods.
• Quantitation precision with median CV ≈ 10% and median abundance ratios matching theoretical values.
• Reproducible AR half-maximal degradation concentration (DC50) values across all throughput levels.

Practical Benefits and Applications

The described workflow allows researchers and QA/QC laboratories to:

• Rapidly screen large PROTAC libraries with reliable on-target/off-target profiling.
• Adjust throughput and gradient length to balance speed versus depth of coverage.
• Integrate high-precision quantitation into lead optimization for TPD drug discovery.

Future Trends and Opportunities

Advancements may include:

• Integration with machine-learning algorithms for automated hit selection.
• Further miniaturization and multiplexing to increase screening capacity.
• Combination with orthogonal biochemical assays to validate degradation mechanisms.

Conclusion

• The Orbitrap Astral platform supports ultra-high-throughput (180/300 SPD) identification of > 8 500 proteins with excellent quantitation accuracy.
• Extended 24/60 SPD gradients enable > 10 000 protein group coverage, providing deep validation of PROTAC activity.
• This LFQ-DIA workflow offers a unified solution for rapid screening and comprehensive analysis in targeted protein degradation drug discovery.