High Definition Multiple Reaction Monitoring: The Application of Quadrupole Ion Mobility Time-of-Flight Mass Spectrometry for Targeted Proteomics Studies

Significance of the Topic

Targeted quantification of peptides and proteins in complex samples is essential for applications ranging from biomarker validation to quality control. High sensitivity, specificity, and a broad dynamic range are critical for reliable results.

Objectives and Study Overview

This study evaluated the High Definition Multiple Reaction Monitoring (HD-MRM) workflow on the SYNAPT G2-Si quadrupole ion mobility time-of-flight mass spectrometer for targeted proteomics. The aim was to demonstrate improvements in sensitivity, selectivity, and acquisition flexibility compared with conventional MRM approaches.

Methodology and Instrumentation

• Sample Preparation: Tryptic digestion of E. coli cytosolic proteins, bovine serum albumin (BSA), and alcohol dehydrogenase (ADH).
• Chromatography: nanoACQUITY UPLC with a BEH 1.7 μm, 15 cm × 75 μm column.
• Mass Spectrometry: SYNAPT G2-Si operated in HD-MRM mode combining T-Wave ion mobility separation and Q-TOF MS/MS.
• Data Processing: TargetLynx and Skyline software for quantification and method development.

Results and Discussion

• Wideband Enhancement synchronized ion push events with ion mobility separation, yielding 5–10× product ion signal increases versus conventional CID MS/MS.
• Constant infusion of [Glu1]-Fibrinopeptide B showed >10-fold improvement in signal-to-noise ratio.
• Summation of multiple product ions from BSA and ADH peptides further lowered detection limits.
• A dynamic range of at least four orders of magnitude was achieved for the CCTESLVNR peptide, with linearity (R²=0.995) down to amol levels.
• Scheduled acquisition allowed monitoring of hundreds of transitions in a single LC/MS run.

Benefits and Practical Applications

• Ultralow limits of detection and quantification for all product ions in a single acquisition.
• Improved selectivity through ion mobility separation minimizes chemical noise and interferences.
• Post-acquisition selection of product ions enhances workflow flexibility.
• Simultaneous full-scan and targeted data acquisition with HD RADAR aids matrix effect evaluation and assay optimization.

Future Trends and Opportunities

Integration of ion mobility with targeted workflows is expected to expand multiplexing capabilities and analytical robustness. Automated collision energy prediction and advanced data analysis pipelines will streamline assay development and enhance reproducibility. Potential applications include clinical diagnostics, environmental analysis, and biopharmaceutical quality control.

Conclusion

HD-MRM on the SYNAPT G2-Si platform offers a powerful targeted proteomics solution, delivering high sensitivity, broad dynamic range, and enhanced selectivity. Ion mobility-enhanced acquisition ensures comprehensive product ion coverage without duty cycle losses, supporting reliable quantification in complex matrices.

References

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2. Duncan MW, Yergey AL, Patterson SD. Quantifying proteins by mass spectrometry: the selectivity of SRM is only part of the problem. Proteomics. 2009;9(5):1124–1127.
3. MacLean B, Tomazela DM, Shulman N, et al. Skyline: an open source document editor for creating and analyzing targeted proteomics experiments. Bioinformatics. 2010;26(7):966–968.