Parallel accumulation – serial fragmentation combined with data-independent acquisition (diaPASEF)

Significance of the topic

Mass spectrometry–based proteomics aims to identify and quantify thousands of proteins in complex biological samples. Traditional data-dependent acquisition (DDA) strategies suffer from stochastic precursor selection, leading to missing values and limited reproducibility across runs. Data-independent acquisition (DIA) addresses these issues by systematically fragmenting wide mass windows, but requires fast scanning, high resolution and accuracy. The introduction of trapped ion mobility spectrometry (TIMS) further enhances separation in four dimensions (m/z, retention time, intensity and ion mobility), while correlating collisional cross section (CCS) values improves alignment of precursor and fragment data. diaPASEF combines Parallel Accumulation–Serial Fragmentation (PASEF) with DIA on the timsTOF Pro, enabling near-complete ion utilization and deep proteome coverage in a single run.

Objectives and study overview

The primary goal was to evaluate diaPASEF for comprehensive, reproducible analysis of whole-cell proteomes. Specifically, the study assessed how different DIA window placement schemes in the m/z–ion mobility plane affect peptide and protein identification, quantitative precision and overall proteome coverage in single 120-minute LC–MS runs of 200 ng HeLa digest. A novel software pipeline, Mobi-DIK, was applied to process the four-dimensional data and extract targeted fragment ion chromatograms using an ion mobility–enabled spectral library.

Methodology and instrumentation

• Sample preparation: HeLa S3 cells were lysed and digested with LysC and trypsin (1:100 w/w) following established protocols.
• Chromatography: Peptides were separated on a 50 cm × 75 µm reversed-phase column packed with 1.9 µm C18 particles, employing a 95 min gradient from 5% to 30% organic, followed by 5 min to 60%.
• Mass spectrometry: A Bruker timsTOF Pro with dual TIMS device was operated in diaPASEF mode, defining up to 32 DIA windows per 100 ms TIMS scan. Four isolation schemes (schemes 1–4) covering diagonal trajectories in the m/z–CCS space were tested, each achieving a ~2 s cycle time.
• Data analysis: The Mobi-DIK toolkit (based on OpenMS) generated ion mobility–resolved spectral libraries from 48 high-pH fractions acquired in DDA PASEF mode. Targeted extraction and scoring used PyProphet for 1% protein FDR control and CCS alignment within 2% deviation.

Main results and discussion

• Window schemes 3 and 4, which sample two diagonal trajectories per cycle, yielded superior coverage compared to single-trajectory schemes 1 and 2.
• Scheme 3 achieved the highest identifications: over 61 000 peptide peak groups and a median of 7 371 proteins per run at 1% FDR, corresponding to ~83% of the library.
• Quantitative precision was excellent, with a median coefficient of variation of 10% and pairwise Pearson correlation > 0.96 across triplicates.
• The high duty cycle of diaPASEF captures a vast majority of multiply charged precursors in each TIMS separation, maximizing sensitivity and reproducibility.

Benefits and practical applications

• Deep single-shot proteome coverage without extensive prefractionation.
• High reproducibility and quantitative accuracy for large-cohort studies.
• Efficient use of ion beam, improving sensitivity for low-abundance species.
• Library-based workflows leveraging CCS values reduce false identifications and facilitate inter-run alignment.

Future trends and potential applications

The integration of ion mobility into DIA workflows promises further enhancements in multiplexing, throughput and depth. Future directions include:

• Adaptive window placement driven by real-time ion populations to optimize specificity.
• Expansion of spectral libraries with CCS annotation for diverse organisms and post-translational modifications.
• Combination with direct-DIA and library-free approaches to reduce dependency on prebuilt libraries.
• Applications in clinical proteomics and systems biology requiring robust quantification across hundreds of samples.

Conclusion

diaPASEF on the timsTOF Pro delivers unprecedented proteome depth and quantitative reliability in single runs by coupling high-speed PASEF with systematic DIA isolation in four dimensions. The optimized acquisition schemes and Mobi-DIK analysis achieve reproducible identification of over 7 000 proteins from 200 ng of digest, marking a significant advance toward the ideal mass spectrometer for proteomics.

References

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