An Inflection Point in High-Throughput Proteomics with Orbitrap Astral: Analysis of Biofluids, Cells, and Tissues

Area of Science:

• Proteomics
• Mass Spectrometry
• Biotechnology

Background:

• Advancements in mass spectrometry technology are crucial for comprehensive proteome analysis.
• Efficient and reproducible sample preparation is essential for high-throughput proteomics.
• Translational research requires robust workflows for analyzing diverse biological samples.

Purpose of the Study:

• To present a comprehensive proteomics workflow integrating Orbitrap and Astral mass analyzers.
• To demonstrate the effectiveness of Adaptive Focused Acoustics (AFA) and automated SP3 for sample preparation.
• To highlight the workflow's utility in biomarker discovery and disease pathology.

Main Methods:

• Utilized Orbitrap and Astral mass analyzers for proteomics analysis.
• Integrated Adaptive Focused Acoustics (AFA) for high-throughput cell and tissue lysis.
• Automated the detergent-compatible single-pot, solid-phase-enhanced sample Preparation (SP3) method for protein digestion.

Main Results:

• Achieved identification of over 10,000 proteins in cells and tissues (heart, liver, lung, intestine) with a 24-minute gradient using data-independent acquisition.
• Demonstrated high reproducibility, with over 80% of proteins showing a coefficient of variation (CV) less than 20% for HeLa digest injections.
• A 45-minute run identified approximately 90% of the expressed proteome, showcasing broad coverage.

Conclusions:

• The presented proteomics workflow is robust, high-throughput, and compatible with diverse sample types.
• This integrated approach significantly enhances protein identification capabilities for translational research.
• The workflow holds substantial potential for advancing biomarker discovery and understanding disease pathology.