A high-throughput PROTAC compound screening workflow for targeted protein degradation with the Orbitrap Astral mass spectrometer for accurate label-free quantitation

Significance of the Topic

The emergence of targeted protein degradation (TPD) by PROTACs has revolutionized drug discovery, enabling irreversible removal of disease-associated proteins and expanding the druggable proteome beyond traditional inhibition. High-throughput and high-sensitivity mass spectrometry is essential to profile proteome-wide degradation, confirm specificity, and detect off-target effects.

Objectives and Study Overview

This study presents a robust, ultra-high-throughput workflow for screening PROTAC compounds using the Thermo Scientific Orbitrap Astral mass spectrometer. A proof-of-concept screen evaluated ARCC-4–induced degradation of the androgen receptor in VCaP prostate cancer cells across four throughput regimes (24, 60, 180, 300 samples per day) to balance proteome depth and speed.

Methodology

VCaP cells were treated with ARCC-4 (5–500 nM) or DMSO control for 4 hours. Protein extracts underwent reduction, alkylation, and tryptic digestion via the AccelerOme platform. Peptide samples were analyzed by label-free data-independent acquisition (LFQ-DIA) on the Orbitrap Astral MS coupled to a Vanquish Neo UHPLC. Four LC gradients (24, 60, 180, 300 SPD) employed different column lengths and flow rates to adjust proteome coverage versus throughput. Data were processed using directDIA with Spectronaut, DIA-NN, and Proteome Discoverer with CHIMERYS, controlling FDR at 1%.

Used Instrumentation

• Orbitrap Astral mass spectrometer
• Vanquish Neo UHPLC system
• IonOpticks Aurora C18 and Thermo Scientific EASY-Spray PepMap columns
• Thermo Scientific AccelerOme sample preparation and EasyPep lysis kits

Results and Discussion

Ultra-fast 180 and 300 SPD methods quantified ~8 000–8 500 protein groups and ~60 000–130 000 peptide groups with median CV ~10%. Extended 24 and 60 SPD gradients yielded deeper coverage of ~10 000–10 400 proteins and ~120 000–142 000 peptides, maintaining high quantitative accuracy. ARCC-4 induced a consistent, dose-dependent androgen receptor degradation (DC50 ~20–30 nM, R2>0.95) across all throughputs, demonstrating method specificity and reproducibility. Three-proteome mixture tests confirmed precise quantitation against theoretical ratios.

Benefits and Practical Applications

• Enables rapid screening of PROTAC libraries with reliable identification of on-target effects.
• Supports in-depth validation by selectable gradient lengths for comprehensive proteome profiling.
• Delivers high sensitivity to detect low-abundance targets and off-target events.

Future Trends and Applications

The integration of ultra-high-speed MS with advanced DIA algorithms and AI-driven data analysis will further accelerate TPD discovery. Scaling to larger compound libraries and coupling with orthogonal assays may enable real-time decision-making in early drug development. Emerging instruments with higher acquisition rates promise deeper coverage at even faster throughputs.

Conclusion

The Orbitrap Astral MS–based LFQ-DIA workflow provides a versatile platform for TPD compound screening and validation, achieving a balance between speed and proteome depth. Its high sensitivity, quantitative precision, and throughput adaptability position it as a key tool in PROTAC-driven drug discovery pipelines.

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