Enhanced sensitivity of the Orbitrap Astral Zoom mass spectrometer for deeper proteome coverage in single-cell proteomics applications

Importance of the Topic

Single-cell proteomics enables detailed analysis of protein composition and function at the individual cell level. High sensitivity and throughput are critical to overcome limited sample amounts and achieve deep proteome coverage in low-input experiments.

Objectives and Study Overview

This study evaluates the performance of the Orbitrap Astral Zoom mass spectrometer for low-input and single-cell proteomics. It compares depth of coverage, sensitivity, acquisition speed, and reproducibility against the previous Orbitrap Astral instrument using HeLa and K562 cell digests and HEK single cells.

Methodology and Instrumentation

• Chromatography: Vanquish Neo UHPLC system with Aurora Ultimate 25 cm and Aurora Rapid 8 cm C18 columns in direct injection mode.
• Mass Spectrometry: Orbitrap Astral Zoom operated in data-independent acquisition with FAIMS Pro Duo at a compensation voltage of -48 V and carrier gas flow of 3.5 L/min.
• Acquisition parameters: Up to 270 Hz scan rate, low-input application mode, DIA isolation widths and injection times adjusted by sample amount.
• Data Analysis: Proteome Discoverer with Chimerys intelligent search and Spectronaut 19.6 directDIA against human UniProt database, with 1% false discovery rate at precursor, peptide, and protein levels.

Key Results and Discussion

• K562 digest dilution series: Over 5,200 protein groups identified from 50 pg and 6,100 from 250 pg at 50 samples per day (SPD).
• HeLa digest performance: At 50 SPD, 3,700 protein groups from 50 pg and 5,600 from 250 pg; at 120 SPD, 3,700 from 50 pg and over 5,100 from 250 pg.
• Comparison with previous Orbitrap Astral: 10% gain in protein IDs for 50 pg HeLa and 5% for 250 pg at 50 SPD.
• HEK single-cell analysis: Average of 5,800 protein groups using a library-free workflow at 50 SPD.
• Reproducibility: Median protein group coefficient of variation below 6% over multiple technical replicates; 85% of identified proteins showed CV below 20% across dilution series.

Benefits and Practical Applications

The enhanced ion optics and faster scan rates deliver deeper proteome coverage for low-input samples and single cells. Library-free DIA workflows simplify data processing and enable high-throughput profiling in research, quality control, and clinical applications.

Future Trends and Opportunities

Advancements may include further improvements in ion transmission, multiplexed single-cell workflows, integration of machine learning for data analysis, and expanded applications in immunology, oncology, and biomarker discovery.

Conclusion

The Orbitrap Astral Zoom mass spectrometer significantly advances single-cell proteomics by improving sensitivity, throughput, and reproducibility. It outperforms its predecessor in low-load experiments and supports robust library-free workflows for deep proteome profiling.

References

1. Tabiwang N. Arrey et al. Deeper proteome coverage and faster throughput for single-cell samples on the Orbitrap Astral mass spectrometer, Technical note 002780, Thermo Fisher Scientific, 2025.