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

Significance of the Topic

The analysis of individual cells at the proteome level addresses heterogeneity masked in bulk measurements. Achieving deep coverage from minute sample amounts is crucial for understanding cell‐specific functions, biomarkers and therapeutic targets. Enhanced sensitivity and higher throughput are key to unlocking the full potential of single‐cell proteomics.

Objectives and Study Overview

This study evaluates the performance of the Thermo Scientific Orbitrap Astral Zoom mass spectrometer in low‐input and single‐cell workflows. Key aims include:

• Comparing proteome coverage and repeatability against the previous Orbitrap Astral MS
• Assessing identification rates across dilution series of K562 and HeLa digests
• Demonstrating library‐free single‐cell analysis of HEK293F cells

Methodology and Instrumentation

Samples included Promega HeLa and K562 digests diluted to 50–2 000 pg and FACS‐sorted HEK293F single cells. Chromatographic separations were performed on a Vanquish Neo UHPLC system with IonOpticks Aurora C18 columns (25 cm XT or 8 cm Rapid). The Orbitrap Astral Zoom MS was operated in data‐independent acquisition (DIA) ‘Low Input’ mode with a FAIMS Pro Duo interface. Data processing employed Proteome Discoverer with CHIMERYS search and Spectronaut 19.6 directDIA workflow, both using 1 % FDR at precursor, peptide and protein levels.

Used Instrumentation

• Vanquish Neo UHPLC system
• Aurora Ultimate 25 cm XT C18 and Aurora Rapid 8 cm C18 columns (IonOpticks)
• Orbitrap Astral Zoom mass spectrometer (Thermo Scientific)
• FAIMS Pro Duo ion mobility interface
• Proteome Discoverer and Spectronaut v19.6 software

Main Results and Discussion

Comparison of Orbitrap Astral Zoom MS versus the earlier Astral MS revealed:

• A 10 % increase in protein group identifications at 50 pg HeLa and a 5 % increase at 250 pg using 50 samples per day (SPD)
• Identification of >5 200 protein groups from 50 pg K562 and >6 100 from 250 pg with median CV < 6 %
• For 50 pg HeLa digest: 3 700 protein groups at 120 SPD and >5 600 at 50 SPD
• For 250 pg HeLa: >5 100 protein groups at 120 SPD and >6 800 at 50 SPD with median CV < 5 %
• High repeatability over 20 injections (10 + 10) of 250 pg K562 and HeLa, maintaining >6 100 and >6 500 protein groups respectively
• HEK293F single cells yielded >5 800 protein groups per cell using a library‐free directDIA approach at 50 SPD

Benefits and Practical Applications

The Orbitrap Astral Zoom MS delivers enhanced ion transfer and faster acquisition (up to 270 Hz), enabling deeper proteome coverage from sub‐nanogram inputs. Library‐free DIA workflows simplify data analysis and enable high‐throughput single‐cell studies, supporting applications in cell biology, biomarker discovery and quality control in biomanufacturing.

Future Trends and Applications

Continued advances may include further optimizations in ion optics, integration of real‐time AI‐based acquisition strategies and expansion into clinical proteomics. Combining ultrafast, high‐sensitivity MS with automated sample preparation and cloud‐based data processing will drive broader adoption in translational research and diagnostics.

Conclusion

The Thermo Scientific Orbitrap Astral Zoom mass spectrometer significantly enhances sensitivity and throughput for low‐input and single‐cell proteomics. It outperforms the previous Astral MS by 5–10 % in protein identifications, maintains excellent repeatability and supports robust, library‐free analyses.

Reference

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.