Revolutionizing translational research: large-scale targeted PRM proteomics assays enabled by Stellar mass spectrometer

Significance of the topic

Large-scale targeted proteomics is crucial for biomarker verification in translational and clinical research. High-throughput, multiplexed assays enable sensitive and specific quantification of hundreds to thousands of peptides in complex matrices such as plasma, accelerating discovery pipelines and supporting regulatory applications.

Objectives and Study Overview

This work presents a streamlined workflow for developing and validating large-scale parallel reaction monitoring (PRM) assays on the Thermo Scientific Stellar mass spectrometer. The study focuses on creating assays for a 500-peptide reference panel (PQ500) using optimized chromatographic gradients (60SPD and 100SPD) and evaluating analytical performance including reproducibility, linearity, limits of detection (LOD), and limits of quantification (LOQ).

Methodology

• Sample Preparation: Heavy PQ500 peptides spiked into digested plasma (300 ng/µl) with an 11-point serial dilution (100% to 0.005%) to assess analytical figures of merit.
• Data Processing: Skyline for spectral library construction and retention time alignment; PRM Conductor for automated selection and refinement of precursors and transitions.

Used Instrumentation

• Thermo Scientific Vanquish Neo UHPLC with EASY-Spray HPLC ES906A column (trap-and-elute, 45 °C).
• Thermo Scientific Stellar mass spectrometer operated in targeted MSn PRM mode (m/z 200–1500, HCD 30%, AGC standard).
• Skyline software with PRM Conductor external tool for method generation and optimization.

Main Results and Discussion

• Final assays comprised 1 622 precursors and ~13 800 transitions for both 60SPD and 100SPD methods.
• Reproducibility: Over 94% of peptides achieved CV < 20% across 10 plasma replicates.
• Linearity: Calibration curves displayed consistent responses across the 11-level dilution series; example peptide ELLDTVTAPQK demonstrated excellent fit.
• Analytical Sensitivity: LOQs below 50 attomoles for the majority of peptides (≈85% < 500 attomoles); LODs under 50 attomoles for nearly all targets.
• Gradient Comparison: 60SPD gradient outperformed 100SPD with on average 1.6× lower LOQs.

Benefits and Practical Applications

• Rapid assay creation within 3–4 days supports timely biomarker studies.
• Automated workflows increase consistency and reduce manual intervention.
• High sensitivity and specificity facilitate clinical proteomics and QA/QC in pharmaceutical development.
• Scalable platform accommodates large peptide panels for translational research.

Future Trends and Potential Applications

Emerging directions include integration with gas-phase fractionation DIA for seamless discovery-to-validation pipelines, incorporation of novel chromatographic modalities to boost throughput, and application of machine learning for real-time transition selection and method refinement in large-scale targeted assays.

Conclusion

The combination of the Stellar mass spectrometer, Skyline, and PRM Conductor delivers a robust, high-throughput solution for large-scale targeted proteomics. It achieves excellent sensitivity, reproducibility, and streamlined assay development, making it well suited for biomarker verification and translational studies.

References

• [1] PanoramaWeb. PRM Conductor external tool documentation. Thermo Fisher Scientific.
• [2] Remes PM, Jacob CC, Li Q, et al. Large-scale targeted PRM proteomics assays enabled by Stellar mass spectrometer. BioRxiv. 2024 Jun 1; doi:10.1101/2024.05.31.596848.