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

Significance of the Topic

Data-independent acquisition (DIA) with label-free quantitation (LFQ) offers a powerful solution for large-scale proteomics by combining consistent peptide sampling with high throughput. Optimizing DIA methods on advanced platforms such as the Orbitrap Ascend Tribrid mass spectrometer enhances sensitivity, reproducibility and proteome coverage, supporting applications from biomarker discovery to quality control in complex samples.

Objectives and Study Overview

This study aimed to develop and evaluate a high-resolution, high-throughput LFQ-DIA workflow—termed “Velocity DIA”—on an Orbitrap Ascend Tribrid MS coupled with a Vanquish Neo UHPLC. Three active gradients (60 min, 30 min, 9 min) were tested to balance proteome depth and throughput. Two modes were compared: Max ID for maximal protein identifications and Max Quan for optimized quantitative precision.

Methodology

• Sample Preparation: HeLa protein digest standard spiked with E. coli and yeast peptides at defined ratios, dissolved in 0.1% formic acid.
• Chromatography: Direct injection onto a 50 cm µPAC Neo column at 350 nL/min on a Vanquish Neo UHPLC, operated with 9 min, 30 min and 60 min active gradients.
• Mass Spectrometry: Orbitrap Ascend Tribrid with FAIMS Pro interface, optimized source parameters and DIA acquisition settings.
• Data Analysis: Spectronaut directDIA, DIA-NN and Proteome Discoverer with CHIMERYS, evaluating identification rates, coefficient of variation (CV) and quantitative accuracy.

Main Results and Discussion

• Proteome Coverage: The 9 min Max ID method identified over 5,400 proteins. Extending gradients to 30 min and 60 min increased identifications to 7,000+ and nearly 7,800 proteins, respectively.
• Quantitative Precision: The 9 min Max Quan mode achieved ~6% protein group CV, while 30 min and 60 min gradients yielded ~5% and ~4% CV, supporting robust quantitation.
• FAIMS Impact: Inclusion of the FAIMS Pro interface improved protein group identifications by ~5% across methods.
• Dynamic Range: Mixed proteome samples demonstrated accurate measurement of theoretical abundance ratios with narrow distribution around medians, confirming quantitation reliability for bacterial, fungal and human proteins.

Benefits and Practical Applications of the Method

• High throughput: Sub-10 min runs enable rapid sample turnover for large-scale studies.
• Deep coverage: 7,000+ protein identifications from 200 ng HeLa digest support comprehensive profiling.
• Accurate quantitation: Low CVs and faithful abundance ratios facilitate biomarker validation and comparative analyses.
• Automation compatibility: Integrates with AccelerOme for streamlined sample prep and reduced handling variability.

Future Trends and Applications

Continued advances may include single-cell proteomics by further reducing sample requirements, integration with machine-learning algorithms for enhanced data interpretation, coupling with ion mobility or novel separation techniques, and adaptation for clinical diagnostics and real-time monitoring.

Conclusion

The Velocity DIA workflow on the Orbitrap Ascend Tribrid MS with Vanquish Neo UHPLC delivers a flexible platform for LFQ proteomics, achieving a balance between throughput, depth and quantitative accuracy. This method supports diverse research and QC applications in analytical chemistry and proteomics.

Instrument Used

• Thermo Scientific Orbitrap Ascend Tribrid Mass Spectrometer with FAIMS Pro interface
• Thermo Scientific Vanquish Neo UHPLC system
• Thermo Scientific µPAC Neo 50 cm HPLC column
• Thermo Scientific AccelerOme automated sample preparation platform (optional)

References

• Yang K., Kraegenbring J., Huang J., Saba J., Hakimi A. High-throughput high-resolution data-independent acquisition workflow on an Orbitrap Ascend Tribrid mass spectrometer for accurate label-free quantitation. Thermo Fisher Scientific, 2024.