CYCLIC ION MOBILITY ENABLED MASS SPECTROMETER AND APPLICATION TO HIGH THROUGHPUT PLASMA PROTEOMICS

Significance of the topic

High-throughput plasma proteomics is critical for identifying biomarkers in large patient cohorts, such as those for prostate cancer. Advances in separation technology, including ion mobility, enhance the depth and reproducibility of proteomic analyses.

Objectives and study overview

This work aimed to evaluate a cyclic ion mobility spectrometer coupled to quadrupole time-of-flight mass spectrometry for:

• Qualitative discovery proteomics using nanoscale liquid chromatography.
• High-throughput quantitative proteomic profiling of human plasma.

Methodology and instrumentation

Nanoscale discovery proteomics employed:

• Waters Acquity M-Class system with a 75 μm × 25 cm HSS T3 column.
• Gradient times from 90 to 240 minutes with 0.1% formic acid in water and acetonitrile.
• Cyclic IMS-enabled Q-ToF operated in HDMSE mode (alternating low and elevated energy scans aligned by retention and drift time).

High-throughput plasma proteomics used:

• Waters Acquity Premier I-Class with a 2.1 mm × 10 cm CSH column at 150 μL/min.
• 15-minute gradient from 5% to 35% acetonitrile.

Used instrumentation

• Waters Cyclic IMS Q-ToF mass spectrometer with ion mobility resolution tuning via multiple passes.
• Electrospray ionization source in positive mode.
• Software: ProteinLynx Global Server, Progenesis QI for Proteomics, MetaboAnalyst 5.0.

Main results and discussion

Nanoscale analyses demonstrated an optimum peptide load of 50–75 ng on a 90-minute gradient, yielding approximately 3,700 protein identifications at 1% FDR. Extending to a 240-minute gradient increased IDs to about 4,500, and searching against an expanded homolog-inclusive database reached over 7,200 protein groups. Reproducibility tests showed retention time CVs below 0.2% and signal intensity CVs up to 13%. PCA of peptide data clearly separated sample groups.

High-throughput plasma profiling of eight samples in triplicate (24 runs) identified 369 proteins with statistically significant abundance changes (ANOVA p < 0.05) and 551 proteins detected in at least one injection, covering nearly five orders of dynamic range. Chromatographic overlays across random injections demonstrated robust performance over extended sequences.

Benefits and practical applications of the method

• Enhanced separation: cyclic IMS increases peak capacity and reduces spectral congestion.
• Depth of coverage: thousands of proteins identified in discovery mode.
• High throughput: rapid gradients allow large cohort profiling.
• Robustness and reproducibility: low variation supports longitudinal and multi-site studies.

Future trends and potential applications

Continued improvements in cyclic IMS—including additional mobility passes—will further boost resolving power. Integration with automated sample preparation and multi-omic workflows will expand clinical applicability. Shorter gradients and higher sample throughput will facilitate routine diagnostics and precision medicine studies.

Conclusion

The cyclic ion mobility enabled mass spectrometer demonstrates excellent performance for both deep discovery proteomics and rapid quantitative plasma profiling. Its high resolving power, reproducibility, and throughput make it suitable for large-scale clinical and biomarker research.

References

1. Lennon et al. High-Throughput Microbore UHPLC-Ion Mobility-Enabled-MS-Based Proteomics for Large Cohort Serum Studies. J Proteome Res. 2021;20(3):1705-1715.
2. Pang et al. MetaboAnalyst 5.0: narrowing the gap between raw spectra and functional insights. Nucleic Acids Res. 2021. doi:10.1093/nar/gkab382.
3. Meier et al. Molecular & Cellular Proteomics. 2018;17:2534-2545.