Hybrid DIA on the Orbitrap Excedion Pro mass spectrometer: Bridging global depth and targeted sensitivity for proteomic analysis

Significance of the topic

Hybrid data-independent acquisition (hybrid DIA) integrates broad discovery proteomics with high-sensitivity targeted quantitation in a single LC–MS experiment. This approach addresses a common analytical trade-off: DIA offers extensive proteome coverage but can lack sensitivity for low-abundance targets, while targeted methods (PRM) give reliable quantitation but sacrifice global depth. Hybrid DIA reduces sample consumption and run time, improves reproducibility for precious or limited samples, and enables simultaneous discovery and verification workflows critical for biomarker research, clinical proteomics, and complex biological studies.

Goals and overview of the study

The technical note evaluated hybrid DIA implemented on the Thermo Scientific Orbitrap Excedion Pro Hybrid Mass Spectrometer coupled to an OptiSpray μPAC Neo column and Vanquish Neo UHPLC. Using a HeLa digest dilution series (0.5–500 ng on-column) spiked with a 6×5 peptide reference mix (0.05–500 fmol), the work aimed to demonstrate: high proteome depth in a single 35-minute run, reproducible quantitation across a wide dynamic range, and sensitive targeted peptide measurement via integrated PRM (tMS2) scheduling enabled by adaptive retention time (adaptive RT) alignment.

Methodology

Samples: HeLa Protein Digest Standard was prepared at five concentrations (0.5, 5, 50, 100, 500 ng) with a constant spike-in of the 6×5 peptide mix spanning 0.05–500 fmol. Three technical replicates were acquired per concentration.

Separation and acquisition: Peptides were separated on a 50 cm OptiSpray μPAC Neo cartridge (direct injection) using a 35-minute gradient on a Vanquish Neo UHPLC. Hybrid acquisition combined conventional DIA scans with scheduled PRM (tMS2) events driven by an imported precursor list and an .RTbin retention-time alignment file (adaptive RT) to focus PRM sampling in narrow windows while preserving DIA coverage.

Data processing: DIA data were analyzed with Spectronaut 20.1 using directDIA and 1% FDR filtering. PRM results were processed in Skyline-Daily v25.1 using y-ion transitions to calculate fragment areas. Linearity, limit of detection (LOD) and limit of quantitation (LOQ) assessments used bilinear regression with criteria including R² and CV thresholds.

Used instrumentation

• Mass spectrometer: Thermo Scientific Orbitrap Excedion Pro Hybrid MS.
• UHPLC: Thermo Scientific Vanquish Neo UHPLC System.
• Ion source: Thermo Scientific OptiSpray ion source with plug-and-play cartridge and automated emitter positioning.
• Separation column: OptiSpray μPAC Neo 50 cm cartridge (micro pillar array technology).
• Software: Biognosys Spectronaut 20.1 (directDIA) and Skyline-Daily v25.1; data handling used .RTbin adaptive retention-time files for dynamic scheduling.

Main results and discussion

Proteome depth and reproducibility:

• Protein identifications scaled with load: from ~2,694 proteins at 0.5 ng to >6,400 proteins at 50 ng (and similar high counts up to 500 ng), while peptide IDs ranged from ~15,000 to >68,000 across loads.
• High intra-run reproducibility: three replicates at 50 ng HeLa showed ~99% protein overlap and ~96% peptide overlap; median peptide CVs were <15% across all concentrations, with a median CV of ~9.9% for 50 ng replicates.

Quantitative dynamic range and linearity:

• Global protein abundances covered more than five orders of magnitude in a 50 ng HeLa run.
• Linear quantitation across sample loads was observed: 370 proteins showed R² >0.8 across all concentrations; >1,300 proteins (≈20% of the dataset) showed linear behavior (R² >0.8) in at least three concentration levels.

Targeted sensitivity (PRM within hybrid DIA):

• The 6×5 peptide standard (six groups × five concentrations from 0.05 to 500 fmol) was used to evaluate targeted performance. PRM quantitation produced R² >0.9 for targeted peptides, with median CVs <10% (n=3) across concentration points.
• PRM-derived LOQ was 50 attomole for the tested peptides, with detection down to 5 attomole in some experiments. PRM spectra and narrow isolation windows increased sensitivity and specificity relative to the wider isolation DIA MS² acquisitions.

Adaptive RT and workflow efficiency:

• Adaptive RT (.RTbin) enabled dynamic scheduling of PRM events, narrowing retention windows and increasing the number of achievable targets without manual list adjustments.
• Method setup was reported as straightforward: adding a tMS2 experiment and loading the precursor list and RTbin file (implemented in a few clicks) before acquisition.

Practical benefits and applications

• Single-run discovery plus targeted quantitation reduces sample consumption and instrument time versus separate DIA and PRM experiments — advantageous for limited or precious samples.
• Robust electrospray (OptiSpray) and cartridge columns increase run-to-run stability and simplify maintenance, improving throughput and reproducibility in routine and regulated labs.
• Hybrid DIA is suitable for workflows requiring both broad profiling (biomarker discovery, system biology) and sensitive quantitation of predefined targets (verification, clinical candidate monitoring).

Future trends and potential uses

• Wider adoption of hybrid acquisition strategies for clinical proteotyping, targeted biomarker panels, and PTM-focused studies; potential to streamline discovery-to-validation pipelines.
• Integration with advanced retention-time prediction, real-time decision algorithms and machine learning for adaptive acquisition optimization and increased multiplexing.
• Application to low-input and single-cell proteomics as instrument sensitivity and chromatography continue to improve.
• Standardization and automation of hybrid-DIA workflows could enhance reproducibility across laboratories and support regulated use cases.

Conclusion

Hybrid DIA on the Orbitrap Excedion Pro, combined with adaptive RT scheduling and OptiSpray μPAC chromatography, provides a practical, single-run solution that bridges deep proteome coverage with high-sensitivity targeted quantitation. The approach demonstrated >6,000 protein IDs in 35 minutes at 50 ng with strong reproducibility (median CVs <15%) and PRM-targeted LOQs down to 50 attomole, reducing the need for separate discovery and targeted experiments and enabling efficient workflows for both research and translational proteomics.

References

1. Martínez-Val A., et al. Hybrid DIA: intelligent data acquisition integrates targeted and discovery proteomics to analyze phospho-signaling in single spheroids. Nature Communications. 2023;14:3599.
2. Goetze S., et al. Simultaneous targeted and discovery-driven clinical proteotyping using hybrid-PRM/DIA. Clinical Proteomics. 2024;21:26.
3. Remes P., et al. Highly multiplex targeted proteomics enabled by real-time chromatographic alignment. Analytical Chemistry. 2020;92:11809.