High throughput proteomics Application of dia PASEF and Evosep One for short gradients

Importance of the topic

High-throughput proteomic analyses that deliver deep coverage and reproducible quantification are essential for large-scale biological and clinical studies. Short LC gradients with robust data-independent acquisition enable rapid turnaround while maintaining data quality.

Objectives and study overview

This work evaluates the integration of dia-PASEF (data-independent acquisition Parallel Accumulation–Serial Fragmentation) with the Evosep One chromatography system for very short gradients. The aim is to assess throughput, proteome depth, and quantitation precision in single-shot analyses of complex samples.

Methodology

An in-house tryptic HeLa cell digest served as a complex model. Key steps included:

• Coupling the Evosep One system to a timsTOF Pro mass spectrometer.
• Implementing a dia-PASEF scheme with 24 windows of 25 Da covering m/z 400–1,000 and a total cycle time of 900 ms.
• Generating a resource-specific library by DDA-PASEF fractionation (24 fractions, ~10 hours at 60 samples per day).
• Creating hybrid spectral libraries for 100, 200, and 300 SPD methods by combining resource-specific data with directDIA runs.

Used instrumentation

• Evosep One liquid chromatography system (Evosep Biosystems)
• timsTOF Pro mass spectrometer with trapped ion mobility and PASEF (Bruker Daltonics)

Main results and discussion

• At 60 samples per day (SPD) with a 6-minute gradient, an average of 5 204 protein groups and 39 936 peptides were identified at 1% FDR.
• Shorter gradient formats (100, 200, 300 SPD) retained high identification rates and quantitative precision.
• The fastest method (300 SPD: 3-minute gradient, 5-minute total run) yielded over 2 100 protein groups and 10 462 peptides per run.
• Median coefficients of variation were 8.2% at the peptide level and 5.9% at the protein level across triplicates.
• Quantitative dynamic range spanned approximately five orders of magnitude.

Benefits and practical applications

• Enables single-shot deep proteome profiling for hundreds of samples per day.
• Delivers high reproducibility and quantitative accuracy suitable for cohort studies.
• Reduces instrument time and operational costs through ultra-short gradients.

Future trends and possibilities

• Integration into clinical and industrial workflows for large-scale biomarker discovery.
• Improvements in library generation and real-time data processing to further enhance speed and depth.
• Extension to diverse sample types and potential use in point-of-care proteomic diagnostics.

Conclusion

The combination of dia-PASEF on the timsTOF Pro with the Evosep One system achieves unprecedented throughput and robust proteome coverage using very short chromatographic gradients. This platform is well suited for large-scale quantitative proteomics applications.