Rapid Proteomic Analysis Using 1 mm Scale Chromatography, Providing the Required Throughput for Large Cohort Studies

Significance of the Topic

Proteomic analysis is critical for understanding biological processes, disease mechanisms, and biomarker discovery. High-throughput methods are essential to process large population cohorts efficiently while retaining data quality. Traditional nanoscale chromatography offers sensitivity but suffers from long runtimes and operational complexities, limiting its utility in clinical and large-scale studies.

Objectives and Study Overview

This study evaluates the performance of one millimeter scale chromatography coupled to the SYNAPT XS Mass Spectrometer using HDMSE acquisition. The primary aim is to achieve a six-fold increase in sample throughput (15-minute gradients) compared to nanoscale setups, without compromising peptide and protein identification numbers in moderately complex samples such as E. coli digests and human plasma.

Methodology and Instrumentation

• Sample Preparation: Waters ProteinWorks protocol for reduction, alkylation, tryptic digestion, and SPE cleanup. iRT peptides spiked for retention time reference.
• Chromatographic Configurations: 1 mm × 100 mm CSH C18 column at 70 µL/min for 15 min; 300 µm × 100 mm at 7 µL/min for 45 min; 75 µm × 250 mm at 0.35 µL/min for 90 min.
• Mobile Phases: 0.1% formic acid in water (A) and acetonitrile (B); gradient 3–35% B.
• Mass Spectrometry: SYNAPT XS with HDMSE in positive ion mode; data processing with Progenesis QI for Proteomics v4.2 and Skyline v20.1.

Main Results and Discussion

• Chromatographic Performance: Peak widths of 0.03–0.04 min at half-maximum and 0.05–0.07 min at 10% height for 1 mm columns demonstrate high resolution within 15 min gradients.
• Identification Metrics: Comparable E. coli protein and peptide counts across 75 µm, 300 µm, and 1 mm columns (FDR <1%), indicating minimal loss of coverage despite faster runs.
• Reproducibility: Human plasma QC replicates showed median retention time CV of 0.05% and peak area CV <20% for iRT peptides. A total of 533 plasma proteins were identified (FDR <1%).

Benefits and Practical Applications

One millimeter scale chromatography significantly reduces analysis time and increases throughput, making it suitable for large cohort proteomic studies. The method balances sensitivity, reproducibility, and robustness, facilitating clinical research, biomarker validation, and quality control workflows.

Future Trends and Applications

Continued development of microflow and midflow chromatography, in combination with improved mass spectrometer sensitivity and AI-driven data analysis, will enable deeper proteome coverage at high throughput. Integration with automated sample prep and cloud-based analytics will further streamline large-scale proteomic investigations.

Conclusion

The 1 mm scale chromatography approach coupled with SYNAPT XS HDMSE achieves a six-fold increase in throughput, maintains identification performance, and offers high reproducibility for mid-complexity proteomic samples. This strategy addresses key challenges in large-cohort studies and opens avenues for efficient clinical proteomics workflows.

References

• Bruderer R et al. Analysis of 1508 plasma samples by capillary flow data-independent acquisition profiles proteomics of weight loss and maintenance. Mol Cell Proteom. 2019;18(6):1242–1254.
• Bennike TB et al. A Cost-Effective High-Throughput Plasma and Serum Proteomics Workflow Enables Mapping of the Molecular Impact of Total Pancreatectomy with Islet Autotransplantation. J Proteome Res. 2019;17(5):1983–1992.
• Hughes C et al. Rapid Qualitative Analysis and Absolute Quantification of Plasma Proteins Using SONAR with Biognosys PQ500 for Proteomic Clinical Research Studies. Waters Application Note. 2018;720006388EN.