High-throughput proteomics using narrow window DIA on the Orbitrap Astral Zoom MS

Importance of the Topic

Data-independent acquisition (DIA) proteomics has become the method of choice for comprehensive, reproducible proteome profiling. High-throughput workflows with very short chromatographic gradients benefit from narrow isolation windows to reduce precursor interference and improve identification specificity. The new Orbitrap Astral Zoom mass spectrometer expands acquisition speed, sensitivity, and dynamic range, addressing critical challenges in rapid, single-shot, label-free proteomics.

Objectives and Study Overview

This study aimed to demonstrate the performance of Thermo Scientific’s Orbitrap Astral Zoom MS for narrow-window DIA (nDIA) across throughputs of 30 to 300 samples per day (SPD) using a HeLa cell digest. Key goals included:

• Optimizing MS parameters for high-throughput MS2 quantitation.
• Comparing identification depth and quantitative precision against the predecessor Orbitrap Astral MS.
• Evaluating instrument flexibility in balancing MS1 and MS2 data points per peak by adjusting isolation window width and range.

Methodology and Instrumentation

HeLa digest standards (20–200 ng load) were analyzed using narrow-window DIA methods on Thermo Scientific Vanquish Neo UHPLC systems coupled to either the Orbitrap Astral Zoom MS or the Orbitrap Astral MS. Gradient lengths were tuned to achieve 30–300 SPD. Data were processed with Spectronaut 19 against the Homo sapiens SwissProt database at 1% FDR.

Used Instrumentation

• Orbitrap Astral Zoom mass spectrometer
• Orbitrap Astral mass spectrometer
• Thermo Scientific Vanquish Neo UHPLC (NanoCap Trap&Elute or Direct Injection)
• EASY-Spray and PepMap Neo columns
• IonOpticks Aurora Ultimate XT column for 30 SPD
• Spectronaut 19 software for data analysis

Main Results and Discussion

Across throughputs and loads, the Orbitrap Astral Zoom MS consistently outperformed its predecessor:

• Identification gains of 10–15% more peptides and precursors, and 5–10% more protein groups, especially at 300 SPD.
• Deep proteome coverage: ~8,000 protein groups (>100,000 peptides) in 4.8 min gradients at 200 ng load and 300 SPD; over 11,000 groups with longer 30 SPD gradients.
• High quantitative precision: MS1 data points per peak at 240 k resolution every 0.6 s; MS2‐based precision with <10% CV for the majority of peptides and protein groups.
• Flexibility to widen isolation windows (3–5 Th) for greater MS2 data points per peak while maintaining selectivity.

Benefits and Practical Applications

High-throughput nDIA on the Orbitrap Astral Zoom MS delivers:

• Rapid, single‐shot proteome profiling with deep coverage suitable for clinical and translational studies.
• Robust quantitation across a wide dynamic range, supporting quality control and biomarker discovery.
• Adaptable acquisition schemes to prioritize either maximum identifications or enhanced MS2 quantitation precision.

Future Trends and Potential Applications

Further improvements may focus on even faster MS2 cycle times and advanced spectral processing to push proteome depth at ultrahigh throughput. Integration with multiplexed and ion mobility methods could expand applications in large-scale clinical cohorts, systems biology, and real-time proteomics.

Conclusion

The Orbitrap Astral Zoom MS sets a new benchmark for high-throughput narrow-window DIA proteomics. By combining enhanced acquisition speed, sensitivity, and flexible window schemes, it achieves deeper coverage and precise quantitation across a wide range of sample throughputs, making it an outstanding instrument for modern proteomic workflows.