High-throughput high-resolution data-independent acquisition workflow on an Orbitrap Exploris 480 mass spectrometer for accurate label-free quantitation

Significance of the Topic

Quantitative proteomics is a cornerstone for deciphering cellular mechanisms and biomarker discovery. However, traditional data-dependent workflows face limitations in reproducibility and throughput. The presented Velocity DIA workflow leverages high-resolution data-independent acquisition on an Orbitrap Exploris 480 to overcome these challenges, balancing depth of coverage and speed for diverse sample types.

Objectives and Study Overview

The study aimed to develop and optimize a label-free quantitation method using DIA on an Orbitrap Exploris 480 MS with varied chromatographic gradients (60, 30, and 9 min). Key goals included maximizing protein identification, ensuring quantitative precision, and assessing the impact of FAIMS Pro interface and sample load.

Methodology and Instrumentation

• Sample Preparation: Thermo Scientific HeLa, E. coli, and yeast peptide digests; three-proteome mixtures mimicking dynamic expression changes.
• Chromatography: Vanquish Neo UHPLC with 50 cm μPAC Neo column; gradients of 60, 30, and 9 min at 350 nL/min; optional AccelerOme automation.
• Mass Spectrometry: Orbitrap Exploris 480 operating in DIA mode; FAIMS Pro interface evaluated at –45 V compensation voltage.
• Data Analysis: Spectronaut (directDIA), DIA-NN, and Proteome Discoverer with CHIMERYS; library construction via Pulsar for targeted search.

Main Results and Discussion

• 30 min gradient with FAIMS yielded over 6,300 proteins and 40,000 peptides with ~5% CV.
• Extending to 60 min achieved ~7,200 proteins and >60,000 peptides, demonstrating deeper coverage.
• Ultra-high throughput 9 min gradient: Max ID mode identified >5,500 proteins; Max Quan mode delivered precise quantitation (CV ~6%).
• Increasing sample load to 500 ng on the 60 min method further boosted identifications to >7,400 proteins with improved CV (~4%).
• Library-based DIA-NN search improved protein identifications by 3–10%, particularly in shorter gradients.
• Quantitative accuracy remained high across all conditions, with median ratios aligning closely with theoretical values.

Benefits and Practical Applications

• Flexible throughput options enable tailored workflows from deep profiling to rapid screening.
• High reproducibility and quantitative precision support biomarker validation and QA/QC operations.
• Optional AccelerOme automation enhances sample handling consistency and reduces manual error.

Future Trends and Potential Applications

• Integration of automated sample preparation and real-time data processing.
• Expansion to single-cell proteomics and large clinical cohorts for translational research.
• Advanced library generation and machine learning-driven algorithms to further boost sensitivity and identification rates.

Conclusion

The optimized Velocity DIA workflow on the Orbitrap Exploris 480 delivers a robust, scalable platform for label-free proteomic quantitation. Varied gradient lengths, FAIMS application, and flexible data processing collectively enable deep proteome coverage, high accuracy, and throughput tailored to diverse analytical needs.

Reference

• Yang K, Kraegenbring J, Saba J, Bromirski M, Hakimi A. High-throughput high-resolution data-independent acquisition workflow on an Orbitrap Exploris 480 mass spectrometer for accurate label-free quantitation. Thermo Fisher Scientific application note P-I-0109, 2024.