Large Scale Targeted Protein Quantification using HR/AM Selected Ion Monitoring with MS/MS Confirmation on the Orbitrap Fusion Tribrid MS

Importance of High Resolution Targeted Protein Quantification

This work addresses the growing need for precise and sensitive measurement of selected proteins in complex mixtures. Achieving low attomole detection limits and maintaining reliable quantitation across multiple orders of magnitude are vital for applications such as biomarker validation, industrial quality control and detailed pathway studies.

Study Objectives and Overview

The primary goal was to establish a novel data independent acquisition workflow on the Orbitrap Fusion Tribrid mass spectrometer. This approach integrates high–resolution, accurate–mass (HR/AM) selected ion monitoring (SIM) scans with rapid ion trap MS/MS events in a single run. The performance was benchmarked using spiked peptide standards and complex proteome digests.

Methodology and Instrumentation

Sample Preparation and Experimental Design:

• Seven isotopically labeled yeast peptides were spiked into BSA or Escherichia coli digests at concentrations from 0.01 to 100 fmol/µL.
• Six bovine protein digests covering 10 amol to 1 pmol on–column were spiked into an E. coli background to test five orders of dynamic range.
• A mixture of six protein digests spanning five orders of magnitude was also analyzed to assess large‐scale quantification capabilities.

Liquid Chromatography and Mass Spectrometry:

• Thermo Scientific EASY–nLC 1000 nano–LC system with a PepMap C18 column (2 µm, 75 µm × 50 cm) at 300 nL/min flow.
• Orbitrap Fusion Tribrid MS configured for three sequential SIM windows (400–600, 600–800, 800–1000 m/z) at 240 000 resolution and 200 amu isolation.
• Parallel acquisition of 17 CID MS/MS scans per SIM window using 12 amu isolation in the ion trap for sequence confirmation.

Data Processing

Spectral libraries were built from discovery runs and imported into Thermo Scientific Pinpoint software. Extracted ion chromatograms with 5 ppm mass tolerance were generated for C12 and C13 precursor isotopes. The top eight fragments per peptide were used to confirm identity. Peptides with matching library profiles and P–values below 0.1 were scored as high confidence.

Main Results and Discussion

Detection Limits and Dynamic Range:

• Simultaneous SIM and MS/MS yielded a limit of detection of 10 attomoles on column.
• A linear dynamic range spanning four orders of magnitude demonstrated robust quantification performance.

Complex Sample Quantification:

• All six bovine proteins spanned 10 amol to 1 pmol and were quantified in a single run with 90% of peptides showing CVs below 10%.
• In the same dataset, 771 peptides representing 172 E. coli metabolic proteins were quantified with 69% of peptides exhibiting CVs under 10% and all under 20%.

Benefits and Practical Applications

The presented DIA workflow offers:

• High sensitivity for low–abundance targets without extensive sample enrichment.
• Wide dynamic range capable of quantifying both trace and abundant proteins in one analysis.
• Simultaneous quantitation and confirmation, streamlining targeted assays for research and quality control.

Future Trends and Potential Applications

Advances may include integration with enhanced spectral libraries, real–time instrument control, multiplexed labeling strategies and machine learning for improved data extraction. The approach can be extended to clinical proteomics, biopharmaceutical monitoring and multi‐omics platforms for personalized medicine.

Conclusion

This study demonstrates that coupling HR/AM SIM at 240 000 resolution with parallel CID MS/MS on the Orbitrap Fusion delivers exceptional sensitivity, selectivity and reproducibility for targeted protein quantification. The method supports large‐scale assays with broad dynamic range in a single acquisition.

Used Instrumentation

• Thermo Scientific EASY–nLC 1000 nano–LC system
• Thermo Scientific EASY–Spray PepMap C18 column
• Orbitrap Fusion Tribrid mass spectrometer
• Thermo Scientific Pinpoint v1.3 software

References

• Gillet LC et al Mol Cell Proteomics 2012 11 O111016717
• Egertson JD et al ASMS poster 2012 Multiplexed Data Independent Acquisition for Comparative Proteomics
• Gallien S et al J Proteomics 2012 81 148–158