High sensitivity and high-throughput label free interaction proteomics using PASEF® on the timsTOF Pro mass spectrometer

Importance of the Topic

Protein interactions underpin cellular functions and regulatory networks in living organisms. High-sensitivity and high-throughput interaction proteomics enable comprehensive mapping of these networks without relying on labeling strategies. Label-free methods reduce experimental complexity, lower costs and preserve native conditions of protein complexes. Advances in mass spectrometry, particularly the timsTOF Pro platform with PASEF acquisition, promise significant improvements in sensitivity, speed and sample throughput. These capabilities support large-scale interactome studies in demanding systems such as mammalian cell cultures and facilitate multi-condition experimental designs.

Study Objectives and Overview

This study demonstrates the application of PASEF-enabled timsTOF Pro mass spectrometer for label-free interaction proteomics in mammalian cell models. The main goals are to assess sensitivity gains, throughput improvements and depth of protein coverage when scaling down sample input to 6- and 12-well plate formats. A proof-of-concept experiment compares performance metrics between standard workflows and PASEF-based workflows using 90-minute and 45-minute gradients. The study also explores the detection of direct and indirect interaction partners of a bait protein under native pull-down conditions.

Methodology

Cell lysates corresponding to 250 µg total protein from 6-well plates were incubated with beads coupled to either bait peptide or scrambled control peptide. After affinity capture and washing, on-bead digestion yielded peptide samples. Peptides were desalted using StageTips and one-quarter of the digests, equivalent to one well from a 12-well format, were analyzed per run. Liquid chromatography was performed on a Thermo Easy nLC 1200 system equipped with a 15 cm pulled-emitter column. Data acquisition employed standard PASEF methods in oTOFControl with 90-minute and 45-minute gradients. Raw data were processed with MaxQuant (version 1.6.7.0) enabling MaxLFQ quantification and four-dimensional matching including collision cross section values. Statistical analysis and visualization were conducted in Perseus.

Instrumentation Used

• timsTOF Pro mass spectrometer (Bruker Daltonics)
• Parallel accumulation–serial fragmentation (PASEF) acquisition mode
• Trapped ion mobility spectrometry (TIMS) for ion time focusing
• Thermo Easy nLC 1200 liquid chromatography system with 15 cm pulled-emitter column

Key Results and Discussion

PASEF on the timsTOF Pro achieved rapid sequencing speed by synchronizing ion mobility separation with quadrupole precursor selection. TIMS-based time focusing enhanced sensitivity, enabling low sample input measurements. When comparing 90-minute PASEF runs to previous platform data, protein and peptide identifications increased significantly. Even with the 45-minute gradient, protein coverage exceeded earlier benchmarks. Median sequence coverage improved by approximately 24 % for 90-minute runs and 15 % for 45-minute runs. Label-free quantification revealed high reproducibility across replicates and clear separation between bait and control samples in principal component analysis. The workflow identified both known direct binders and novel candidate interactors, as well as indirect interactors recruited via intermediate proteins. These findings highlight the potential of fast, low-input PASEF workflows to dissect complex interaction networks under varying buffer conditions.

Benefits and Practical Applications of the Method

• Significant sensitivity gain allows sample scaling to 6- and 12-well plate formats
• High throughput enabled by short gradients and PASEF acquisition supports multi-condition studies
• Improved protein coverage facilitates detection of post-translational modifications
• Label-free quantification simplifies experimental design and reduces cost
• Capability to distinguish direct versus indirect interactors using buffer variations and rapid analysis

Future Trends and Potential Applications

Emerging workflows aim to further shorten gradients, for example to 21 minutes using the Evosep system, enabling up to 60 analyses per day or 15 interactome profiles in quadruplicate. Integration with advanced bioinformatics and targeted assays will deepen interaction network insights and accelerate validation of novel interactors. Combined with automated sample preparation and robotic liquid handling, these developments will facilitate high-content screening of protein interactions in diverse biological contexts, including drug discovery, biomarker research and systems biology.

Conclusion

The timsTOF Pro mass spectrometer with PASEF acquisition offers a powerful platform for label-free interaction proteomics, delivering exceptional sensitivity, high throughput and deep proteome coverage from minimal sample amounts. This approach streamlines large-scale interactome studies in mammalian systems and opens new avenues for high-resolution mapping of protein networks under multiple experimental conditions.

References

1. Meier F et al. Journal of Proteome Research. 2015;14(9):3800–3810.
2. Meier F et al. Molecular & Cellular Proteomics. 2018;17(12):2534–2545.
3. Prianichnikov N et al. Molecular & Cellular Proteomics. 2020;19(5):1056–1068.