Short gradients with Bruker’s nanoElute coupled to the timsTOF Pro with PASEF — Throughput and deep proteome measurements

Importance of the Topic

Rapid, high-throughput proteomic analysis is critical for large cohort studies, biomarker discovery, and clinical research. Combining short nano-LC gradients with advanced trapped ion mobility and PASEF on the timsTOF Pro addresses the demand for faster turnaround without compromising proteome depth.

Objectives and Study Overview

This work aims to configure the Bruker nanoElute and timsTOF Pro for 28.8 min injection-to-injection cycles, achieving:

• Up to 50 proteomic runs per day
• Maximized sequencing speed via TIMS-PASEF
• Deep coverage in both single-shot and fractionated samples

Methodology and Instrumentation Used

The optimized platform includes:

• Chromatography: nanoElute with 100 mm × 75 µm ID columns packed with 1.9 µm C18, 500 nl/min flow
• Gradient: 21.4 min peptide elution, 28.8 min total cycle
• Mass Spectrometry: timsTOF Pro with captive spray, TIMS separation (100 ms accumulation/ramp), PASEF at > 120 Hz
• Samples: 200–250 ng HeLa digest and high-pH reversed-phase fractionated murine cerebellum
• Data Analysis: PEAKS Studio, 1% PSM FDR

Main Results and Discussion

Single-shot runs (21.4 min gradient):

• ~4 180 protein groups and ~26 000 unique peptides identified per run
• Reproducibility: protein ID CV = 1.3%, peptide ID CV = 2.7%; ~90% overlap across runs
• Quantitative precision: Pearson r ≈ 0.97, median protein CV ≈ 10.3%

Deep profiling with 24-fraction offline high-pH RP (< 12 h total):

• 9052 protein groups from HeLa; 10 299 from murine cerebellum
• Generation of > 100 000 peptide CCS values for library creation

Benefits and Practical Applications

This workflow delivers exceptional throughput and robustness, enabling large-scale biomarker validation, clinical proteomics, and rapid creation of spectral libraries for DDA/DIA methods.

Future Trends and Applications

• Integration with DIA for enhanced quantitation and reproducibility
• Automated CCS-based library generation to improve peptide identification
• Adoption in clinical pipelines demanding high sample numbers and fast results
• Further development of multiplexed and miniaturized LC workflows

Conclusion

By minimizing LC overhead and exploiting TIMS-PASEF, sub-30 min proteomic cycles on the timsTOF Pro achieve deep coverage and high throughput, supporting expansive studies without sacrificing analytical depth.

Reference

1. Meier F, Beck S, Grassl N, Lubeck M, Park MA, Raether O, Mann M. Parallel accumulation–serial fragmentation (PASEF): Multiplying sequencing speed and sensitivity by synchronized scans in a trapped ion mobility device. J Proteome Res. 2015; DOI:10.1021/acs.jproteome.5b00932
2. Meier F, Brunner AD, Koch S, Koch H, Lubeck M, Krause M, Goedecke N, Decker J, Kosinski T, Park M, Bache N, Hoerning O, Cox J, Räther O, Mann M. Online parallel accumulation–serial fragmentation (PASEF) with a novel trapped ion mobility mass spectrometer. Mol Cell Proteomics. 2018; DOI:10.1074/mcp.TIR118.000900