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

Significance of the topic

Plasma proteomics is central to translational research and biomarker discovery because plasma is minimally invasive to collect and contains proteins spanning a large dynamic range that reflect physiological and pathological states. Hybrid acquisition strategies that combine targeted quantitation and data-independent acquisition (DIA) address the common trade-off between broad proteome coverage and the highest-sensitivity measurement of predefined low-abundance peptides. By integrating targeted MS2 (tMS2/PRM-style) or internal-standard-triggered PRM (SureQuant) with DIA discovery scans on a high-sensitivity instrument, researchers can simultaneously quantify prioritized biomarkers and retain unbiased discovery capability across large clinical cohorts.

Objectives and study overview

This technical note developed and evaluated two hybrid-DIA strategies on the Thermo Scientific Orbitrap Astral Zoom MS: a tMS2 hybrid-DIA approach (scheduled targeted MS2 list combined with DIA) and a SureQuant hybrid-DIA approach (heavy-peptide–triggered PRM combined with DIA). Primary goals were to assess (1) proteome depth and peptide identification trade-offs as targeted panel size increases (30–300 peptides), and (2) analytical performance metrics (linearity, LOD, LOQ, precision, accuracy) for targeted peptides across wide dynamic ranges using a PQ500 reference peptide set spiked into pooled neat human plasma digest.

Materials and methods

Key experimental design elements and workflows:

• Two complementary hybrid-DIA workflows were implemented: (A) tMS2 hybrid-DIA: MS1 → scheduled targeted MS2 (PRM-style) → DIA; (B) SureQuant hybrid-DIA: Survey/triggering step for heavy peptides → SureQuant PRM triggered on heavy/light → DIA.
• Performance tests included (i) panel-size experiment with PQ500 heavy peptides spiked 1:20 into pooled plasma digest and panels of 30, 60, 120, 200, and 300 targeted peptides; (ii) serial dilution experiment (1:3 dilutions across 10 levels) to define linearity, LOD, LOQ, accuracy and precision for 300 targeted peptides.
• LC gradient: trap-and-elute scheme on a 150 µm × 15 cm, 2 µm C18 EASY-Spray column at 50 °C; 32-minute gradient optimized for throughput with a 0.8 µL/min flow.
• MS acquisition: Orbitrap Astral Zoom used for all experiments; DIA windowing, MS1 and targeted MS2 parameters were tuned for balance between sensitivity and scan speed (examples: MS1 resolution up to 240,000, DIA precursor range 350–980 m/z, tMS2 isolation widths and injection times adjusted between methods to accommodate targeted panels).
• Quantitation panel and samples: PQ500 reference peptides (Biognosys) spiked into pooled human plasma digest; disease plasma samples sourced from BioIVT for matrix context.

Used instrumentation

• Thermo Scientific AccelerOme Automated Sample Preparation Platform
• Thermo Scientific Vanquish Neo UHPLC System with trap-and-elute
• Thermo Scientific Orbitrap Astral Zoom Mass Spectrometer
• Thermo Scientific Easy-Spray Source and EASY-Spray HPLC column (2 µm C18, 150 µm × 15 cm)
• PQ500 Reference Peptides Kit (Biognosys) and PepMap Neo trap cartridge

Data analysis

• DIA raw files were converted with HTRMS converter and analyzed by Spectronaut (library-free directDIA) for protein/peptide identification and quantitation.
• Targeted quantitation, calibration curves and LOD/LOQ determination were performed using Skyline; LOD/LOQ defined via a three-times signal-to-noise criterion and serial dilution accuracy/precision assessments (triplicate injections).
• Summary statistics, plots and correlation analyses were generated in Python.

Main results and discussion

• Proteome depth: Hybrid-DIA retained substantial discovery depth despite adding targeted scans. For tMS2 hybrid-DIA the number of protein groups decreased by ~9.5% (from 692 to 593–645) and peptides by ~4.2% compared to standard DIA. For SureQuant hybrid-DIA the protein group decrease was ~12.8% and peptide IDs decreased by ~6.7% versus standard DIA.
• Dynamic range and sensitivity: Both hybrid methods detected protein intensities spanning more than six orders of magnitude. Analytical sensitivity on the Orbitrap Astral Zoom was notable—using tMS2 hybrid-DIA, >92% of 300 peptides had LOD <25 amol and >82% had LOQ <50 amol.
• Linearity and quantitative performance: Calibration across serial dilutions showed excellent linearity: 89% of peptides had R2 > 0.99 and 99% had R2 > 0.90. Example peptide (LEYLLLSR) showed linearity and accuracy maintained across ~6 orders of magnitude down to ~6 amol on-column at low levels.
• Precision and reproducibility: Protein quantity CVs were generally low. Reported CVs included ~3.2% for protein group ID counts (tMS2), while quantitative CV for protein quantities across standard DIA and hybrid-DIA experiments ranged ~5–7%; peptide-intensity CVs ranged ~8–13% depending on method and panel size.
• Effect of targeted panels on scan density: Data points per chromatographic peak decreased modestly with large targeted panels. For tMS2 hybrid-DIA mean DIA data points per peak fell from ~5.0–5.3 (30–120 peptides) to ~4.5–4.7 (200–300 peptides). SureQuant hybrid-DIA showed a smaller decrease (mean points ≈5.80 to 5.56 across panel sizes) because PRM acquisition is triggered only when heavy-peptide evidence is present.
• Impact on targeted intensities: Targeted peptide peak intensities in hybrid-DIA were slightly reduced relative to dedicated PRM/SureQuant runs: approximately 8–11% decrease for tMS2 hybrid-DIA (median ratio ~1.08–1.11) and ~13% decrease for SureQuant hybrid-DIA vs standard SureQuant PRM. Despite this, LOD/LOQ and quantitative linearity were not materially affected.
• Correlation with standard methods: Strong agreement was observed between hybrid-DIA and conventional workflows. Pearson correlations for protein quantities between hybrid-DIA and standard DIA and for targeted peptides between hybrid-DIA and PRM/SureQuant were typically r > 0.97–0.99, and cross-validation between the two hybrid approaches also yielded r > 0.97.

Benefits and practical applications

• Simultaneous discovery and robust targeted quantitation: Hybrid-DIA enables concurrent unbiased DIA profiling and sensitive quantification of prioritized low-abundance peptides in the same run, improving throughput for cohort studies and biomarker verification pipelines.
• Flexible trade-offs: The two strategies offer complementary strengths—tMS2 hybrid-DIA is simpler to set up and does not require heavy standards; SureQuant hybrid-DIA increases confidence in peptide identity and quantitation via heavy-peptide triggers at the cost of heavier method complexity and requirements for standards.
• High sensitivity suitable for clinical proteomics: Achieved sub-femtomole to low-attomole detection limits make both hybrid approaches attractive for plasma biomarker studies where target peptides are rare.
• Adaptability: The hybrid methods and parameters are applicable to other sample matrices and can be tuned for panel size, throughput and sensitivity needs.

Future trends and potential uses

• Automation and scaling: Combining automated sample prep with hybrid-DIA is suited to high-throughput clinical studies; further automation of assay setup (e.g., automated transition-list generation and retention-time alignment) will accelerate adoption.
• Intelligent acquisition refinement: On-instrument decision-making (adaptive RT windows, real-time trigger optimization) can maximize data points per peak while supporting larger targeted panels.
• Standardization and multiplexing: Wider use of heavy-peptide panels and community reference kits will improve comparability across labs; hybrid acquisition is promising for multiplexed verification studies that bridge discovery and targeted validation phases.
• Extension to PTMs and modified-peptide panels: Hybrid-DIA could be adapted to targeted phosphopeptide or PTM panels while preserving discovery-level DIA for site-localization and global signaling context.

Conclusion

This study demonstrates that two hybrid-DIA strategies implemented on the Orbitrap Astral Zoom MS—tMS2 hybrid-DIA and SureQuant hybrid-DIA—successfully combine DIA discovery with sensitive targeted quantitation. Both approaches maintain deep proteome coverage, deliver excellent linearity and low attomole-level sensitivity for many peptides, and produce highly correlated protein and peptide quantitation compared to standard DIA and PRM workflows. Choice between methods depends on experimental priorities: method simplicity and adaptive RT (tMS2) versus enhanced identification confidence using internal standards (SureQuant).

References

1. Martínez-Val A, et al. Hybrid-DIA: intelligent data acquisition integrates targeted and discovery proteomics to analyze phospho-signaling in single spheroid. Nature Communications. 2023;14:3599.
2. Gajadhar A. SureQuant intelligence-driven MS: a new paradigm for targeted quantitation. Thermo Fisher Scientific Technical Note 65873.