High throughput 4D-Proteomics – Application of dia-PASEF® and the Evosep One for short gradients

Significance of the topic

Data-independent acquisition (DIA) methods combined with ion mobility separation have revolutionized proteomics by enabling high sensitivity, reproducibility and throughput. In clinical and large-scale studies, the ability to process hundreds of samples per day with consistent depth of coverage and minimal missing values is critical. The integration of Trapped Ion Mobility Spectrometry (TIMS) with Parallel Accumulation Serial Fragmentation (PASEF), termed dia-PASEF, on the timsTOF Pro platform offers a fourth dimension of separation, enhancing ion utilization, speed and sensitivity. Coupling this with the high‐throughput Evosep One HPLC system addresses the demand for robust, rapid and reproducible proteome profiling in real‐world applications.

Objectives and study overview

The primary aim of this work was to evaluate the performance of dia-PASEF on the timsTOF Pro mass spectrometer in conjunction with the Evosep One HPLC system for ultra‐high‐throughput proteomic analysis. Key targets included:

• Assessing proteome depth and quantitation precision across gradients enabling up to 300 samples per day (SPD).
• Quantifying protein groups and peptide identifications in short (21 min) and ultra‐short (3 min) gradients.
• Evaluating reproducibility and data completeness for triplicate injections.

Methodology and Instrumentation used

Sample preparation

Whole HeLa cell pellets were lysed in trifluoroethanol, reduced with DTT, alkylated with IAA and digested overnight with trypsin. Peptide cleanup was performed using C18 solid‐phase extraction and samples were reconstituted in 0.1% formic acid.

Library generation and DIA workflow

A resource‐specific spectral library was built from 24 high‐pH reversed‐phase fractions processed on the Evosep One (60 SPD). Project‐specific libraries for 100, 200 and 300 SPD workflows were generated via directDIA and combined into hybrid libraries for targeted extraction.

Liquid chromatography

• System: Evosep One HPLC
• Columns: C18, 8 cm×150 µm, 1.5 µm for 60/100 SPD; C18, 4 cm×150 µm, 1.9 µm for 200/300 SPD
• Mobile phases: 0.1% formic acid in water (A), 0.1% formic acid in acetonitrile (B)
• Column temperature: 50 °C
• Gradient methods: 21 min (60 SPD), shorter gradients enabling 100, 200 and 300 SPD

Mass spectrometry

timsTOF Pro operated in dia-PASEF mode with three 25 Da windows per 100 ms scan, covering 400–1,000 m/z over eight frames per cycle (900 ms total). Precursor selection targeted +2/+3 charge states with full ion mobility separation.

Data processing

Spectronaut (v14) with Pulsar search engine was used for library generation and targeted four‐dimensional extraction at 1% FDR on PSM, peptide and protein levels. Hybrid libraries ensured retention‐time precision and depth of coverage across gradients.

Main Results and Discussion

• 60 SPD (21 min): Average of 5,204 protein groups and 39,936 peptides identified per run; identification rate ~4,000 peptides/minute; dynamic range ~5 orders of magnitude; median CV 8.2% (peptides), 5.9% (proteins).
• 300 SPD (4.8 min total): >2,100 protein groups and 10,462 peptides quantified per run.
• Reproducibility: 94% of protein groups identified across three replicates (4915/5213); 81% of proteins quantified with CV <20%, 65% with CV <10%.

The high completeness and precision result from multiplexed precursor fragmentation in the ion mobility dimension and robust chromatography on Evosep One.

Benefits and Practical Applications

• Enables large‐cohort and clinical proteomics with hundreds of samples/day.
• Combines depth of coverage with sub‐5 min gradient times for rapid screening.
• Delivers highly reproducible quantitation and minimizes missing data.
• Offers robust operation suited for regulated environments (QA/QC, biomarker discovery).

Future Trends and Opportunities

• Integration with automated sample-prep and multiplexed isobaric labeling for enhanced throughput.
• Development of AI-driven spectral libraries and real‐time data analysis workflows.
• Extension of 4D‐Proteomics to post‐translational modification profiling and single‐cell analysis.
• Implementation in clinical diagnostics pipelines for personalized medicine.

Conclusion

The combination of dia-PASEF on timsTOF Pro with the Evosep One platform delivers an ideal solution for high‐throughput, in‐depth proteomics. It achieves thousands of protein identifications per run, exceptional reproducibility and quantitation accuracy, even at ultra‐short gradients, meeting the demands of large‐scale and clinical studies.

References

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