Utilizing the SELECT SERIES Cyclic IMS for High Throughput Plasma Proteomics

Significance of the Topic

Clinical proteomics aims to identify low-abundance biomarkers within high dynamic range samples such as plasma. High throughput and robust analytical workflows are essential to process large patient cohorts and generate statistically meaningful data in studies such as prostate cancer biomarker discovery.

Objectives and Study Overview

This application note investigates the feasibility of using the SELECT SERIES Cyclic IMS Q-Tof platform coupled to analytical scale chromatography for rapid profiling of plasma samples from prostate cancer patients. Eight pooled serum digests representing different disease states or treatments and a quality control pool were analyzed to assess throughput, reproducibility, and proteome coverage.

Methodology and Instrumentation

The workflow employed analytical scale reversed-phase chromatography (2.1 × 100 mm CSH column, 1.7 µm) at 150 µL/min with a 15 minute gradient (5–35% acetonitrile with 0.1% formic acid). Samples (500 ng on-column) were injected in randomized triplicates across cohorts. Mass spectrometry was performed in positive ESI mode using HDMSE acquisition, 50 000 FWHM resolution, ion mobility single pass at 65 FWHM, and Glu-Fibrinopeptide B for lockmass calibration. Data were processed in MassLynx, Progenesis QI for Proteomics, and statistical analysis in MetaboAnalyst.

Main Results and Discussion

• Chromatographic reproducibility: Overlays of 10 representative injections showed consistent retention profiles throughout the 24 injection set, demonstrating system robustness.
• Signal stability: Extracted intensity and retention time for five peptides across three proteins yielded maximum CV of 13% and 0.18% respectively, confirming high analytical precision.
• Protein coverage: A total of 369 proteins were quantified in all replicates, with 551 proteins identified in at least one injection.
• Dynamic range: Peptide intensities spanned nearly five orders of magnitude, illustrating the extended detection range of the Cyclic IMS dual gain detector.
• Biological differentiation: Unsupervised PCA of peptide intensities clearly separated sample groups by disease state or treatment, supporting the method’s ability to discriminate phenotypes.

Advantages and Practical Applications

• High throughput capacity with 15 minute gradients for large cohort studies.
• Analytical flow chromatography enhances robustness over nanoscale setups.
• Ion mobility separation further resolves complex peptide mixtures.
• High mass resolution and extended dynamic range enable low-abundance biomarker detection.

Future Trends and Potential Applications

Emerging trends include integration of shorter gradients and multiplexed sample introduction to further increase throughput, application to other clinical matrices, and leveraging machine learning for pattern recognition in large proteomic datasets. Advances in ion mobility and detector technologies will continue to expand dynamic range and identification rates.

Conclusion

The SELECT SERIES Cyclic IMS combined with analytical scale UPLC delivers a fast, reproducible workflow for high throughput plasma proteomics. The platform’s robustness, sensitivity, and resolution support large-scale clinical studies for biomarker discovery and phenotypic stratification.

References

1. Lennon et al. High-Throughput Microbore Ultra-high Performance Liquid Chromatography-Ion Mobility-Enabled-Mass Spectrometry-Based Proteomics Methodology for the Exploratory Analysis of Serum Samples from Large Cohort Studies, J Proteome Res, 2021.
2. Hughes C., Gethings L.A., Plumb R.S. Qualitative and Quantitative Performance of Cyclic IMS in Nanoscale Proteomic Experiments, Waters Application Note 720007381EN, 2021.