Deep proteome coverage at scale combined with reproducible quantitation on the timsTOF HT

Importance of the Topic

The accurate quantitation of proteins is essential for understanding biological processes and disease mechanisms.
Data independent acquisition using dia-PASEF on the timsTOF HT platform delivers deep proteome coverage with high reproducibility and reduced missing data.

Study Objectives and Overview

The study aimed to optimize a dia-PASEF acquisition strategy for short LC gradients and assess its performance on single proteome samples and complex mixtures, benchmarking coverage, accuracy and reproducibility.

Methodology and Instrumentation

• Tryptic digests of yeast, HeLa and E. coli prepared at defined ratios.
• Separation on a 15 cm C18 column (75 μm ID, 1.6 μm particles, Aurora, IonOpticks) with a 15-minute ACN gradient using nanoElute 2 nano HPLC.
• Mass spectrometry analysis on timsTOF HT via CaptiveSpray 2 ion source.
• Window placement optimized with py-diAID; data processed in Spectronaut v18 directDIA+ mode with 1% FDR at peptide and protein levels.

Results and Discussion

• Yeast proteome: average identification of 4408 protein groups and 50 450 peptides in 15-minute runs with 95% of proteins quantified at CV < 20%.
• HeLa digest: nearly 8000 protein groups and 105 000 stripped peptides identified, demonstrating scalability to complex human samples.
• Complex mixture (HeLa/yeast/E. coli): 12 893 protein groups from 173 851 precursors, ~10 peptides per protein, median CV ~5% and accurately recovered theoretical abundance ratios across species.

Benefits and Practical Applications

This optimized dia-PASEF workflow enables high-throughput label-free quantitation with minimal instrument time, making it suitable for large-scale studies such as biomarker discovery, clinical proteomics and systems biology investigations.

Future Trends and Possibilities for Use

Anticipated advancements include integration with AI-driven data analysis, expansion to single-cell proteomics, further reduction of gradient times, and multi-omic workflows that leverage library-free DIA strategies.

Conclusion

The combination of dia-PASEF on the timsTOF HT with short gradients affords near-complete proteome coverage and robust quantitation in under 15 minutes, offering a powerful solution for high-throughput proteomic research.