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

Significance of the Topic

High-performance label-free quantitative proteomics provides critical insights into protein expression, biological pathways and disease mechanisms. Data-independent acquisition (DIA) workflows on high-resolution instruments address the challenges of reproducibility and depth in large-scale studies, enabling consistent profiling across hundreds to thousands of samples.

Objectives and Study Overview

This study aimed to develop and evaluate a robust LFQ-DIA workflow—termed Velocity DIA—on the Thermo Scientific Orbitrap Exploris 480 mass spectrometer. The research focused on optimizing chromatographic gradients of 9, 30 and 60 minutes to balance throughput and proteome coverage and to assess qualitative and quantitative performance across varied sample loads and complexity.

Used Instrumentation

• Orbitrap Exploris 480 mass spectrometer with FAIMS Pro interface
• Thermo Scientific Vanquish Neo UHPLC system
• 50 cm µPAC Neo HPLC separation column
• AccelerOme automated sample preparation platform

Methodology

• Sample Preparation: HeLa, E. coli and yeast digests were dissolved in 0.1% formic acid and mixed at defined ratios to evaluate quantitative accuracy.
• Chromatography: Direct injection at 350 nL/min on a 50 cm µPAC column using active gradients of 9, 30 and 60 minutes.
• Mass Spectrometry: DIA acquisition with FAIMS Pro to reduce chemical noise; sample loads ranged from 200 ng to 500 ng.
• Data Analysis: Raw data processed using Spectronaut, DIA-NN and Proteome Discoverer with CHIMERYS for directDIA-based identification and quantitation.

Main Results and Discussion

Integration of FAIMS Pro increased protein group identifications by ~5%. Velocity DIA achieved over 5,500 protein groups in a 9 min gradient, more than 6,300 in 30 min and over 7,200 in 60 min, with protein group CVs between 4% and 6%. In a three-proteome mixture, measured abundance ratios were within 5% of theoretical values, demonstrating high accuracy across a broad dynamic range. Increasing sample loading to 500 ng further elevated identifications above 7,400 proteins with ~4% CV.

Benefits and Practical Applications of the Method

• High-throughput proteome profiling suitable for large sample cohorts.
• Label-free quantitation with excellent precision and accuracy.
• Flexible gradient design to tune depth versus throughput.
• Compatibility with automated sample preparation to minimize variability.

Future Trends and Applications

Advances in ion mobility separation, shorter chromatographic methods and AI-driven data analysis are expected to further enhance throughput and sensitivity. The extension of Velocity DIA to single-cell proteomics, clinical biomarker discovery and real-time monitoring of biological processes represents promising future directions.

Conclusion

The optimized Velocity DIA workflow on the Orbitrap Exploris 480 coupled to µPAC chromatography delivers robust, reproducible and high-depth label-free quantitation, providing a versatile platform for diverse proteomics applications.