High-throughput high-resolution data-independent acquisition workflow on an Orbitrap Ascend MultiOmics Tribrid mass spectrometer for accurate label-free quantitation

Importance of the Topic

Data-independent acquisition (DIA) has emerged as a critical approach for large-scale proteomics, delivering reproducible, high-throughput, and deep protein quantitation without the stochastic gaps inherent to data-dependent methods. Optimizing DIA workflows is essential to balance throughput, sensitivity, and quantitative precision, enabling robust biological insights across diverse sample types and experimental designs.

Aims and Overview of the Study

This study introduces and evaluates a Velocity DIA workflow on the Orbitrap Ascend Tribrid mass spectrometer. It assesses label-free quantitation performance across long (60 min), short (30 min), and ultra-high-throughput (9 min) LC gradients, with and without FAIMS Pro interface, and explores methods to maximize protein identification (Max ID) or quantitation precision (Max Quan).

Methodology and Instrumentation

The workflow integrates:

• Automated sample preparation using the Thermo Scientific AccelerOme platform
• Peptide separation on a 50 cm µPAC Neo HPLC column at 350 nL/min via Vanquish Neo UHPLC
• Mass spectrometry on Thermo Scientific Orbitrap Ascend Tribrid with optional FAIMS Pro (CV –45 V)
• Active gradients of 60, 30, and 9 minutes to tailor throughput
• Data processing with Spectronaut directDIA, Proteome Discoverer with CHIMERYS, or DIA-NN, including library-based searches

Main Results and Discussion

Key findings include:

• 30 min gradient yielded >7 000 protein groups and >47 000 peptides with ~5% protein group CV.
• 60 min gradient increased coverage to ~7 800 proteins and >76 000 peptides; loading 500 ng further raised identifications to >8 100 with ~4% CV.
• FAIMS Pro improved protein group IDs by ~5% at 30 min.
• Ultra-high-throughput 9 min Max ID detected >5 400 proteins; Max Quan achieved ~6% CV.
• Library-based DIA-NN searches provided 3–10% additional protein identifications, particularly benefiting shorter gradients.

Benefits and Practical Applications

This Velocity DIA workflow offers:

• Deep proteome coverage from complex mixtures with minimal sample amounts
• Excellent quantitative accuracy and precision across a broad dynamic range
• Scalable throughput for large-cohort studies or rapid screening
• Reduced sample handling variability via automation

Future Trends and Applications

Advancements may include:

• Further acceleration of gradient times with maintained depth and precision
• Integration of AI-driven library generation and data analysis
• Expansion to single-cell and spatial proteomics workflows
• Application to clinical and biopharmaceutical quality control settings

Conclusion

The optimized Velocity DIA workflow on the Orbitrap Ascend Tribrid platform achieves robust label-free quantitation, combining deep proteome coverage, high precision, and flexible throughput to meet diverse analytical requirements.

Reference

• Thermo Fisher Scientific. PO147-2024-EN. 2024.