Optimizing LC-MS/MS settings for plasma proteomics analysis with cap-flow LC separation and dia-PASEF

Importance of the Topic

Blood plasma provides a minimally invasive biopsy matrix rich in proteomic information but challenges arise from its high dynamic range and complexity.
High-throughput workflows capable of deep coverage are essential for large-cohort clinical studies.

Study Objectives and Overview

This study aimed to optimize capillary-flow LC-MS/MS settings using dia-PASEF acquisition for rapid and reproducible plasma proteomics.
Researchers compared neat and bead-based enriched plasma digests, targeting throughput up to 220 samples per day while maintaining depth and quantitative fidelity.

Used Methodology

Key procedural steps involved plasma collection, centrifugation (single and double-spun EDTA plasma), enzymatic digestion, and peptide cleanup.
Two workflows were assessed: neat plasma digests via iST-BCT and enriched digests via Enrich-iST kits.

Used Instrumentation

• Waters M-Class HPLC system with novel driver for HyStar control
• Capillary-flow chromatography on Bruker Max RP-C18 column (10 cm×150 µm, 1.5 µm) at 60 °C with a Bruker column toaster
• TimsTOF HT mass spectrometer employing optimized dia-PASEF acquisition

Main Results and Discussion

• Cap-LC at 120 SPD (10 min gradient) and 220 SPD (6 min gradient) achieved narrow peaks and high retention time reproducibility
• Neat plasma runs yielded 450–550 protein groups; enriched plasma achieved 670–1150 groups per run
• Cell lysate analysis reported over 6000 protein groups at 120 SPD
• Low carry-over observed even after multiple injections, ensuring data reliability
• Inter-run overlap was ~80% for neat and ~85% for enriched plasma across single and double-spun samples
• High-abundance proteins demonstrated consistent quantitation across 20 donors

Benefits and Practical Applications

• Turnaround times exceeding 100–200 samples/day enable large-scale clinical proteomics
• Reduced overhead for column washing, equilibration, and sample loading improves lab efficiency
• Short acquisition cycles (<0.5 s) yield robust quantitative data for narrow chromatographic peaks
• Bead-based enrichment enhances proteome depth for biomarker discovery

Future Trends and Possibilities

• Integration of faster LC systems and advanced ion mobility for higher throughput
• Multiplexing and automation to support large cohort real-time analysis
• Extension to clinical diagnostics and personalized medicine applications
• Advancements in AI-driven data processing for automated quality control

Conclusion

Optimized capillary-flow LC-MS/MS with dia-PASEF balances speed, depth, and reproducibility, supporting high-throughput plasma proteomics for clinical research.

References

1 Nolte H MacVicar TD Tellkamp F et al Instant Clue: A Software Suite for Interactive Data Visualization and Analysis Sci Rep 8 12648 2018 DOI 10.1038/s41598-018-31154-6