Global Plasma Proteome Quantification Using Internal Standard Triggered Targeted Analyses

Significance of the Topic

The ability to quantify the plasma proteome comprehensively is essential for the discovery and validation of protein biomarkers in clinical research and precision medicine. Traditional profiling methods often trade off between depth of coverage and quantitative accuracy. A targeted workflow combining internal standards and high-density acquisition can address these limitations by delivering both broad protein coverage and reliable quantitation.

Objectives and Study Overview

This work presents a global plasma proteome quantification workflow based on the SureQuant method, which uses internal standard-triggered targeted acquisition on next-generation Orbitrap mass spectrometers. The approach leverages the Biognosys PQ500 kit of 804 stable isotopically labeled peptides to systematically screen over 500 plasma proteins in undepleted human plasma.

Methodology and Instrumentation

The SureQuant workflow employs a two-step acquisition:

• MS1 survey scan at 120 k resolution to detect predefined internal standard (IS) precursor ions.
• Low-resolution fast MS/MS of the IS (“watch” mode) to confirm elution, triggering high-resolution PRM (“quant” mode) for endogenous peptides with 60 k resolution and extended fill times.

The method is implemented natively on Orbitrap Exploris 480 and Orbitrap Eclipse Tribrid instruments coupled to UltiMate 3000 RSLC or EASY-nLC 1200 systems. A high-throughput variant uses a Q Exactive HF-X mass spectrometer interfaced with Evosep One via API, achieving a 21-minute gradient.

Main Results and Discussion

• Proteome Coverage: Quantification of ~560 endogenous peptides as surrogates for ~400 plasma proteins in a 70-minute run.
• Sensitivity and Range: Detection from 4 amol to 15 pmol spanning over six orders of magnitude, sufficient for most FDA-approved biomarkers.
• Quantitative Precision: 75 % of peptides achieved CV < 10 % (median CV ~6 %).
• Reproducibility: Excellent correlation (R² ~ 0.97) across instruments, LC setups, and operators.
• Sampling Efficiency: ≥ 6 data points per chromatographic peak ensured robust quantitation.
• High-Throughput Variant: Enabled up to 5-fold increase in sample throughput with marginal performance loss.

Benefits and Practical Applications

• Merges the quantitative rigor of targeted methods with the broad coverage of discovery workflows.
• Preconfigured PQ500 SureQuant methods support plug-and-play deployment in diverse laboratories.
• Robust, time-independent acquisition enhances portability and ease of use.
• Suitable for large-scale clinical studies, quality control, and biomarker validation.

Future Trends and Potential Applications

Further integration of native high-throughput API-driven acquisition into instrument software will boost throughput without compromising data quality. Advances in real-time data processing, AI-assisted peak detection, and emerging MS platforms will extend sensitivity, reduce sample requirements, and enable even larger cohort studies. Combining such workflows with automated sample preparation and cloud-based analytics will accelerate translational proteomics efforts.

Conclusion

The SureQuant internal standard-triggered workflow provides a robust, sensitive, and high-throughput solution for global plasma proteome quantification. Its native implementation on Orbitrap Exploris and Eclipse systems, along with a high-throughput variant on Q Exactive HF-X, offers a versatile platform for clinical biomarker research and large-scale studies.

References

• Gallien S., Wang J., Gajadhar A.S., Patel B., Kellmann M., Arrey T.N., Harder A., Huguet R., McAlister G., Bailey D., Eliuk S., Xuan Y., Huhmer A., Chen E.L. Global Plasma Proteome Quantification Using Internal Standard Triggered Targeted Analyses. Thermo Fisher Scientific Application Note.