Unveil plasma proteomics with cutting-edge hybrid-DIA methods utilizing two strategies on the Orbitrap Astral Zoom MS

Significance of the topic

Hybrid-DIA integrates targeted peptide quantitation with data-independent acquisition (DIA) discovery proteomics to address a key trade-off in translational proteomics: achieving sensitive, accurate measurement of predefined biomarkers while retaining broad proteome coverage. This capability is important for biomarker verification, clinical assay development, and workflows that require both discovery context and high-quality targeted quantitation from limited sample material.

Objectives and study overview

The study developed and evaluated two hybrid-DIA acquisition strategies on the Orbitrap Astral Zoom mass spectrometer to combine targeted MS2 (PRM-style) measurements with conventional DIA. The goals were to (1) implement methods that dynamically coordinate targeted scans with DIA windows, (2) assess the impact of adding targeted scans on global proteome depth and quantitative reproducibility, and (3) compare sensitivity and quantitative performance of targeted measurements within hybrid-DIA to standard PRM approaches.

Methods and instrumentation

Two hybrid approaches were implemented:

• tMS2 hybrid-DIA: a sequence of MS1, targeted MS2 (tMS2) using a PRM mass list, then a full DIA experiment within the same cycle.
• SureQuant hybrid-DIA: integration of a standard DIA experiment with on-the-fly SureQuant-triggered PRM scans for predefined targets.

Study materials and sample prep:

• Target peptide panel: PQ500 peptides (Biognosys) spiked and diluted per manufacturer recommendations.
• Biological matrix: pooled, AccelerOme-digested human disease plasma (BioIVT).
• Chromatography: Thermo Vanquish Neo UHPLC with EASY-Spray ES906 column, trap-and-elute workflow, column at 50 °C, mobile phases A = 0.1% formic acid in water, B = 0.1% formic acid in 80% acetonitrile. Gradients optimized for the instrument and sample load.

Acquisition and processing:

• Mass analyzer: Orbitrap Astral Zoom MS operated with combined tMS2/SureQuant and DIA experiments; DIA isolation windows and scan ranges optimized (e.g., precursor mass range ~350–980 m/z in DIA with ~5 m/z windows) and high-resolution MS1/MS2 settings to maximize sensitivity and selectivity.
• Data conversion: HTRMS converter for raw hybrid files.
• DIA data analysis: Spectronaut 20.1.
• Targeted (PRM/SureQuant) processing: Skyline for extraction and quantitative metrics.

Used instrumentation

• Mass spectrometer: Thermo Scientific Orbitrap Astral Zoom MS.
• UHPLC: Thermo Scientific Vanquish Neo UHPLC.
• Column: Thermo Scientific EASY-Spray HPLC ES906 (trap-and-elute configuration).
• Peptide standards: PQ500 (Biognosys).
• Sample provider and digestion kit: BioIVT (plasma) and AccelerOme digestion workflow.
• Software: HTRMS converter, Spectronaut 20.1 (DIA), Skyline (PRM/SureQuant).

Main results and discussion

Impact on proteome depth and peptides detected:

• tMS2 hybrid-DIA incurred an average reduction of ~9.5% in reported protein groups and ~4.2% in peptide identifications compared with a standard DIA-only run.
• SureQuant hybrid-DIA showed a slightly larger average decrease: ~12.8% fewer protein groups and ~6.7% fewer peptides versus standard DIA.
• The number of targeted peptides included in the PRM portion did not further degrade DIA protein-group detection, indicating the method scales with moderate target panel sizes (30–300 targets assessed).

Quantitative precision and correlation:

• Reproducibility for protein-group quantification was strong: median CVs <8% for tMS2 hybrid-DIA and 5–7% for SureQuant hybrid-DIA across triplicate injections, demonstrating robust precision in neat plasma.
• Protein intensity profiles from hybrid-DIA correlated tightly with standard DIA (Pearson R > 0.97), indicating preservation of global quantitative trends despite insertion of targeted scans.

Targeted sensitivity and linearity:

• Targeted peptide intensities measured in hybrid-DIA were modestly reduced relative to standalone PRM approaches: approximately 10% lower versus standard PRM and ~13% lower versus SureQuant PRM in the reported comparisons, yet showed strong linearity and correlation with corresponding PRM datasets.
• Sensitivity was excellent on the Astral platform: over 92% of peptides had a reported limit of detection (LOD) below 25 amol, and more than 82% had a limit of quantification (LOQ) below 50 amol.

Overall interpretation:
Hybrid-DIA successfully balances broad proteome coverage with high-quality targeted quantitation. The modest decreases in protein/peptide identifications are offset by the ability to obtain precise, highly sensitive targeted measurements in the same acquisition, enabling discovery and verification data to be acquired concurrently.

Benefits and practical applications

• Single-run workflow that provides both discovery-level DIA context and targeted, high-sensitivity quantitation — reduces sample consumption and analytical throughput compared with separate discovery and targeted experiments.
• Suitable for biomarker verification pipelines where a limited panel of candidates requires accurate quantification within a broader proteomic context.
• High reproducibility and low LOD/LOQ support clinical and translational studies demanding detection of low-abundance peptides.
• Scalable to moderate target panel sizes without substantial loss of DIA coverage, enabling flexible assay design.

Future trends and applications

• Expansion to larger target panels and optimization of cycle scheduling to further minimize trade-offs between targeted and DIA coverage.
• Improved real-time decision algorithms and instrument firmware to refine on-the-fly triggers (e.g., adaptive SureQuant logic) and prioritize targets dynamically based on precursor intensity or prior runs.
• Integration with machine-learning models to predict and allocate instrument time for the most informative features, maximizing discovery and targeted assay performance simultaneously.
• Application to longitudinal clinical cohorts, multiplexed assays, and low-volume or limited-input specimens (e.g., micro-sampling), leveraging Astral sensitivity.
• Standardization and validation of hybrid-DIA workflows for regulated environments (clinical labs, CROs) to facilitate diagnostic or companion diagnostic use cases.

Conclusion

The hybrid-DIA strategies demonstrated on the Orbitrap Astral Zoom MS provide a practical and reproducible solution to combine unbiased DIA profiling with precise targeted quantitation in a single analysis. They preserve most of the proteome coverage of DIA while delivering near-PRM levels of sensitivity for targeted peptides, enabling streamlined biomarker verification and translational research workflows.

Reference

Qingling Li, Jared Deyarmin, Sophia Steigerwald, Stephanie Samra. Targeted Proteomics: Unveil plasma proteomics with cutting-edge hybrid-DIA methods utilizing two strategies on the Orbitrap Astral Zoom MS. Thermo Fisher Scientific application note, 2026. PO004719-2026-EN.