Customer Insights - Accelerating phosphoproteomics research with trapped ion mobility mass spectrometry (TIMS)

Significance of the Topic

The analysis of protein phosphorylation is central to understanding cellular signaling pathways, disease mechanisms and therapeutic targets. Low abundance and structural isomers of phosphopeptides pose significant analytical challenges that require high sensitivity, high throughput and enhanced separation power. Trapped ion mobility spectrometry (TIMS) combined with modern mass spectrometry platforms offers a fourth dimension of separation, improving the identification and quantitation of phosphopeptides and related post-translational modifications.

Objectives and Study Overview

This study from Kyoto University’s Laboratory of Molecular and Cellular BioAnalysis (MCBA) under Professor Yasushi Ishihama aims to accelerate phosphoproteomics research by leveraging TIMS on the Bruker timsTOF Pro. Key goals include:

• Developing nanoLC-MS workflows with ultra-high throughput and sensitivity
• Applying 4D-Proteomics to global phosphoproteome profiling and kinase inhibitor studies
• Implementing rapid clinical metaproteomics to explore host-microbiome interactions
• Integrating computational and bioinformatics approaches for large-scale data analysis

Methodology and Instrumentation

The laboratory employs advanced separation chemistry, mass spectrometry and bioinformatics:

• Sample preparation techniques: StageTip enrichment, HAMMOC for phosphopeptides, PTS-aided digestion for membrane proteins
• NanoLC systems: meter-long capillary columns and rapid gradients (1 minute runs)
• TIMS-QTOF platform: Bruker timsTOF Pro with parallel accumulation–serial fragmentation (PASEF)
• Data handling: CCS-aware workflows, jPOST proteome repository and collaboration on computational pipelines

Main Findings and Discussion

Implementation of TIMS-PASEF enabled:

• Deep phosphoproteome coverage from less than 200 ng of material
• Identification of tens of thousands of phosphopeptides in single-shot LC-MS runs
• Discovery that over 70 % of human proteins undergo phosphorylation, refining prior estimates
• High-throughput kinase inhibitor profiling with 864 runs per day using 100-second gradients
• Rapid metaproteomics of clinical fecal samples to probe host-microbiome proteome interactions

Benefits and Practical Applications

The integration of TIMS and PASEF delivers significant practical advantages:

• Enhanced separation of isobaric and positional isomer phosphopeptides
• Reduced sample requirements and preparation time
• Robust operation supporting large clinical cohorts
• Improved throughput for drug discovery, biomarker profiling and precision oncology studies

Future Trends and Potential Applications

Proteomics is evolving toward greater automation, depth and data completeness. Emerging directions include:

• Data-independent acquisition (DIA) workflows for unbiased MS2 coverage
• Proteogenomics combining next-generation sequencing with high-resolution proteomics
• AI-driven computational analysis to extract insights from multi-omics datasets
• Development of dedicated proteome sequencers to achieve near-complete proteome coverage

Conclusion

Trapped ion mobility mass spectrometry on the timsTOF Pro significantly advances phosphoproteomics by delivering ultra-high sensitivity, throughput and structural resolution. These capabilities empower researchers to dissect cellular signaling, profile kinase networks and explore clinical and metaproteomic applications, driving progress in drug discovery and precision medicine.

References

1. Sugiyama N et al. Mol Syst Biol 2008;4:193
2. Olsen JV et al. Sci Signal 2010;3(104):ra3
3. Sharma K et al. Cell Reports 2014;8(5):1583-1594
4. Glover MS et al. J Am Soc Mass Spectrom 2018;25(5):786-794
5. Fernandez-Lima F. Int J Ion Mobil Spec 2016;19:65-67
6. Hu Y et al. J Med Chem 2015;58(1):30-40
7. Wu P et al. Drug Discovery Today 2016;21(1):5-10
8. Sugiyama N & Ishihama Y. J Pharm Biomed Anal 2016;130:264-272
9. Sugiyama N et al. Sci Rep 2019;9:10503
10. Ang MY et al. Clin Chim Acta 2019;498:38-46