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

Importance of the topic

The ability to perform deep, accurate, and precise proteome profiling at high sample throughput is essential for drug discovery, translational biology, clinical cohort studies, and high-throughput screening. Narrow-window data-independent acquisition (nDIA) addresses the specificity and chimericity limitations of wide-window DIA, enabling improved peptide identification, PTM site localization, and quantitative performance even on very short chromatographic gradients.

Objectives and overview of the study

This technical note evaluates the performance of the Thermo Scientific Orbitrap Astral Zoom mass spectrometer for high-throughput proteomics using narrow-window DIA (nDIA). The goals were to: benchmark identification depth and quantitative precision/accuracy across throughputs from 30 to 300 samples per day (SPD); compare the Astral Zoom to the preceding Orbitrap Astral MS; and illustrate method trade-offs (isolation width, MS2 injection time, and data points per peak) for MS1- versus MS2-based quantitation.

Methodology

Key experimental design elements:

• Samples: HeLa digest standard and three-proteome mixes (human:yeast:E. coli) prepared from lyophilized digests; dilution series from 20 ng to 1000 ng per injection.
• LC setups: IonOpticks Aurora Ultimate 25 cm × 75 µm (30 SPD direct injection) and Thermo EASY-Spray 2 µm C18, 150 µm × 150 mm with a PepMap Neo trap (60–300 SPD trap-and-elute).
• DIA strategy: narrow isolation windows (2–5 Th) across typical precursor ranges (commonly 380–980 m/z; alternative reduced ranges such as 400–800 m/z or 385–585 m/z tested to increase MS2 DPPP).
• MS acquisition: Astral detector with HCD, MS1 full scan at 240k resolution, DIA MS2 acquisition at extremely high scan rates (up to ~270 Hz nominal), and optional bent-trap pre-accumulation to add ~1 ms effective ion fill in parallel.
• Data processing: Spectronaut 19.5 directDIA (library-free); peptide and protein identifications reported as unique (stripped) peptides and protein groups; normalization by human FASTA for HYE mixtures.

Used instrumentation

• Mass spectrometer: Thermo Scientific Orbitrap Astral Zoom mass spectrometer (Astral detector) with EASY-Spray ion source.
• UHPLC: Thermo Scientific Vanquish Neo UHPLC system (NanoCap direct injection and trap-and-elute configurations).
• Columns: IonOpticks Aurora Ultimate (25 cm × 75 µm, 1.7 µm) for 30 SPD and EASY-Spray 2 µm C18 (150 µm × 150 mm) for 60–300 SPD (PepMap Neo trap cartridge used for trap-and-elute).
• Consumables and standards: Pierce HeLa protein digest, Promega yeast digest, Waters MassPREP E. coli digest; optima LC-MS solvents and common lab consumables.

Main results and discussion

Identification depth and throughput:

• At 30 SPD and 200 ng HeLa, the Astral Zoom identified ~11,117 protein groups and >200,000 unique peptides; at 300 SPD and 200 ng, nearly 8,000 protein groups (~100,000 peptides) were reported.
• Compared to the previous Orbitrap Astral MS, the Astral Zoom consistently delivered 10–15% more peptides/precursors and ~5% more protein groups overall, with the greatest gains (15–25% peptides/11–17% proteins) on very short gradients.

Instrument improvements driving gains:

• Faster acquisition (nominal up to 270 Hz; ~210 Hz at 3 ms MS2 IT) due to improved ion optics and transfers.
• Bent-trap pre-accumulation increases ions available per MS2 scan (reported up to ~45% more ions), providing flexible trade-offs between scan rate and sensitivity.
• Enhanced spectral processing and detector parallelization reduce the need for spectral averaging and enable robust single-scan spectra at high speed.

Quantitative precision and accuracy:

• For HeLa and three-proteome mixtures the platform produced excellent precision: median protein-level CVs around 4–4.5% across loads; peptide-level median CVs ~12–14% (slightly higher on Astral Zoom due to additional low-abundance peptides identified).
• Proteome ratio accuracy in three-proteome mixes yielded median errors within ~7.5% of expected values and median log2 ratios close to theoretical values (human ~0, yeast ~-1, E. coli ~+2) across loads from 20 ng to 1 µg.
• Methods tuned for MS2 quantitation (wider windows 3–5 Th and restricted precursor ranges) improved MS2 data points per peak (DPPP) and maintained strong quantitation even for 300 SPD short runs.

DPPP trade-off study (100 ng, 30 SPD):

• Three DIA schemes evaluated: 2 Th/3 ms (median MS2 DPPP = 3), 3 Th/5 ms (DPPP = 4), and 5 Th/10 ms (DPPP = 5).
• The 3 Th/5 ms method delivered the best balance of identifications and precision for 100 ng: highest protein IDs and best median CV at protein level; 5 Th/10 ms modestly improved peptide-level CV but reduced identifications (~500 fewer proteins, ~20,000 fewer peptides).
• All three methods produced accurate proteome ratios within ~6% relative error.

Instrument reproducibility:

• Across 25 Orbitrap Astral Zoom instruments measured over 4 months, protein group identifications varied by less than 5%, demonstrating strong instrument-to-instrument repeatability in real-world lab deployments.

Benefits and practical applications

• High-throughput proteome profiling: Enables 30–300 SPD operation with deep proteome coverage suitable for large cohort studies, phenotypic screening, and time-sensitive workflows.
• Flexible quantitation modes: Can prioritize MS1-based quantitation for maximum IDs or adjust DIA windows and MS2 injection times to favor MS2-based precision and PTM site localization.
• Improved sensitivity and coverage: Pre-accumulation and faster scan rates increase identifications, particularly benefitting low-abundance proteins and complex samples.
• Scalable to clinical/industrial settings: Robust instrument repeatability and fast cycle times support routine, high-throughput deployments.

Future trends and potential uses

• Further optimization for PTM-centric workflows: nDIA with Astral Zoom should improve phosphoproteomics and glycoproteomics site localization while preserving throughput.
• Shorter gradients and AI-driven method optimization: Combining rapid acquisition with machine learning for dynamic window placement or adaptive filling could further increase identifications and quantitation robustness.
• Integration with large-cohort clinical proteomics: The demonstrated throughput and reproducibility make the platform suitable for population-scale studies and biomarker validation pipelines.

Conclusion

The Orbitrap Astral Zoom mass spectrometer significantly advances high-throughput nDIA proteomics by combining faster MS2 acquisition, bent-trap pre-accumulation, and enhanced spectral processing. It delivers measurable gains in peptide and protein identifications, retains excellent quantitative precision and accuracy across a range of throughputs and loads, and provides practical flexibility to trade identification depth versus MS2-based quantitation performance. Its demonstrated instrument-to-instrument reproducibility supports deployment in large-scale and routine laboratories.

References

• Lou R.; Shui W. Acquisition and analysis of DIA-based proteomic data: A Comprehensive survey in 2023. Molecular & Cellular Proteomics 2024, 23(2), 100712.
• Lancaster N.M.; Sinitcyn P.; Forny P. et al. Fast and deep phosphoproteome analysis with the Orbitrap Astral mass spectrometer. Nat. Commun. 2024, 15, 7016.
• Jager S.; Zeller M.; Pashkova A. et al. In-depth plasma N-glycoproteome profiling using narrow-window DIA on the Orbitrap Astral mass spectrometer. Nat. Commun. 2025, 16, 2497.
• Guzman U.H.; Martinez-Val A.; Ye Z. et al. Ultra-fast label-free quantification and comprehensive proteome coverage with narrow-window DIA. Nat. Biotechnol. 2024, 42, 1855.
• Serrano L.R.; Peters-Clarke T.M.; Arrey T.N. et al. The one hour human proteome. Molecular & Cellular Proteomics 2024, 23(5), 100760.