Large Scale Targeted Protein Quantification Using HR/AM Selected Ion Monitoring with MS/MS Confirmation on a Novel Hybrid, Q-OT-qIT Mass Spectrometer

Significance of the Topic

Large-scale targeted protein quantification provides critical insights into cellular processes, disease biomarkers and biologics quality control. Accurate and reproducible measurement across a wide concentration range is particularly challenging in complex biological matrices due to co-eluting species and wide dynamic range of protein abundances. Advances in mass spectrometry instrumentation and acquisition strategies address these limitations, enabling high-throughput assays in proteomics, clinical and industrial settings.

Study Objectives and Overview

This work aims to create a highly sensitive and selective data independent acquisition (DIA) method for broad-scale targeted proteomics using the Thermo Scientific Orbitrap Fusion Tribrid mass spectrometer. By combining high-resolution, accurate-mass selected ion monitoring (HR/AM SIM) scans with rapid parallel ion trap MS/MS, the approach captures both precise quantification data and sequence-level confirmation in a single analytical run. Analytical performance and throughput were assessed using isotopically labeled peptides and complex proteome digests.

Methodology

• DIA Acquisition: Three SIM scans covering m/z ranges 400–600, 600–800 and 800–1000 with 200 Da isolation windows and 240,000 resolving power were cycled. Each SIM scan was followed by 17 sequential CID MS/MS events (12 Da isolation) in the linear ion trap to interrogate precursors within the same 200 Da window.
• Sample Preparation: Isotope-labeled peptide standards were spiked into E. coli and bovine serum albumin digests across five concentration levels from 0.01 to 1000 fmol/µL. Six bovine proteins were also spiked into E. coli matrix at 10 attomole to 1 pmol on column.
• Data Processing: Thermo Scientific Pinpoint software extracted full SIM chromatograms with a ±5 ppm window for quantification and matched up to eight diagnostic b/y fragment ions against a spectral library for peptide confirmation (p-value <0.1 threshold).

Instrumentation Used

• Mass Spectrometer: Orbitrap Fusion Tribrid (quadrupole mass filter, Orbitrap analyzer, linear ion trap)
• SIM Parameters: Resolution 240,000; AGC target 2×10^4; isolation window 200 Da
• CID MS/MS: Linear ion trap; AGC target 1×10^4; isolation window 12 Da; up to 17 MS/MS per SIM cycle
• Nano-LC Setup: EASY-nLC 1000 with 75 µm × 50 cm C18 column; 300 nL/min flow; 5–35% acetonitrile gradient over 120 min

Results and Discussion

• Sensitivity: SIM scans improved detection sensitivity by 10-fold compared to full-range MS, achieving limits of detection down to 10 attomole for labeled peptides in 500 ng E. coli digest.
• Dynamic Range: A linear response spanning four orders of magnitude was demonstrated for peptide quantitation with precision (%CV) below 10% across most concentration points.
• Complex Samples: Six spiked bovine proteins were reproducibly quantified across five orders of magnitude (10 attomole–1 pmol) in a single run, with >90% of peptides showing %CV <10%.
• Proteome Coverage: Beyond target standards, the same DIA dataset enabled quantification of 771 peptides representing 172 E. coli proteins involved in metabolic pathways, with 69% of peptides exhibiting %CV <10% and 88% <15%.

Benefits and Practical Applications

• High Sensitivity and Selectivity: HR/AM SIM reduces background interference, allowing reliable quantitation of low-abundance peptides in complex matrices.
• Integrated Confirmation: Parallel MS/MS acquisition delivers sequence confirmation without sacrificing throughput or quantification quality.
• Broad Dynamic Range: Single-run assays cover attomole to picomole levels, suitable for biomarker validation, pharmaceutical QC and systems biology studies.
• Reproducibility: Automated data extraction provides consistent output for quality assurance and comparative proteomics.

Future Trends and Applications

Ongoing developments in hybrid MS architectures and data processing algorithms promise even greater multiplexing of quantitative and confirmatory scans. The integration of machine learning for library matching, real-time acquisition decision-making and improved ion optics will further enhance sensitivity and speed. Expanding to post-translational modification mapping and intact protein quantitation will extend the workflow’s utility in clinical proteomics and biologics characterization.

Conclusion

The presented DIA workflow on the Orbitrap Fusion Tribrid platform combines high-resolution SIM and parallel ion trap MS/MS to deliver sensitive, selective and reproducible large-scale targeted protein quantification. Demonstrated across attomole-level detection and broad dynamic range, it offers a robust solution for quantitative proteomics in research and regulated environments.

References

• Gillet LC, et al. Mol Cell Proteomics. 2012; Targeted data extraction of MS/MS spectra from DIA for accurate proteome analysis.
• Egertson JD, et al. ASMS poster. 2012; Multiplexed data independent acquisition for comparative proteomics.
• Gallien S, et al. J Proteomics. 2013; Selectivity of LC-MS/MS analysis: implications for proteomics experiments.