timsTOF Pro with PASEF and Evosep One: Maximizing throughput, robustness and analytical depth for shotgun proteomics

Importance of the Topic

The growing demand for large-scale shotgun proteomics in clinical research requires workflows that combine high analytical depth with rapid sample turnover. Traditional nano-LC-MS/MS platforms often lack the speed and robustness needed to analyze hundreds of samples with sufficient statistical power.

Objectives and Study Overview

This work assesses the performance of the Bruker timsTOF Pro mass spectrometer employing Parallel Accumulation Serial Fragmentation (PASEF) coupled to the Evosep One HPLC system. Using 5.6, 11.5 and 21 minute gradients on 50 ng HeLa cell digests, the study examines protein identification rates, quantitative reproducibility and instrument robustness at throughputs up to 200 samples per day.

Methodology

Predefined gradient methods corresponding to 200, 100 and 60 injections per day were applied. The 5.6 minute gradient produced peptide peaks of ~2 seconds FWHM. PASEF acquisition was optimized to 0.5 second cycles (one full MS TIMS scan plus four MS/MS scans), achieving >100 MS/MS spectra per second while ensuring adequate full MS sampling for label-free quantification. Data processing utilized Bruker DataAnalysis and PEAKS Studio software.

Used Instrumentation

• Evosep One HPLC: uses disposable Evotips, low-pressure pumps for gradient formation and offset, storage loop, and a 4 cm column for high-throughput or 8 cm column for deeper coverage.
• Bruker timsTOF Pro: orthogonal electrospray interface, trapped ion mobility spectrometry (100 ms accumulation/ramp), rapid quadrupole switching and time-of-flight analysis enabling PASEF at >120 Hz.

Main Results and Discussion

• 5.6 minute gradient: identification of >1 400 protein groups and >7 000 unique peptides per run; throughput of 200 runs/day with >90% MS utilization.
• 11.5 minute gradient: identification of >2 300 protein groups.
• 21 minute gradient: identification of >2 800 protein groups.
• Quantitative reproducibility across 96 injections demonstrated R² ≈0.97 and TIC CV of 2.3%; dynamic range spanned three orders of magnitude.
• TIMS adds a gas-phase separation dimension (Ω/z) orthogonal to m/z and retention time, increasing peak capacity and yielding cleaner MS/MS spectra.

Benefits and Practical Applications

The integrated workflow delivers high proteome coverage with rapid cycle times, suitable for large cohort studies, biomarker discovery and validation. Low sample requirements reduce contamination and maintenance needs, enhancing long-term robustness.

Future Trends and Applications

Advancements in real-time ion mobility filtering and data processing may further boost identification rates and quantification accuracy. Expanded automation and multiplexing could elevate throughput beyond current limits, enabling high-content studies in precision medicine.

Conclusion

The combination of Evosep One and timsTOF Pro with PASEF establishes a robust, high-speed proteomics platform capable of identifying thousands of proteins in minutes per sample, effectively meeting the dual demands of depth and throughput in modern proteomics.

References

1. Meier F et al. Journal of Proteome Research 2015; PMID:26538118.