An in-depth and high throughput plasma proteomics workflow powered by Orbitrap Exploris 480 mass spectrometer using multi nanoparticle-based workflow

Significance of the Topic

Plasma proteomics by mass spectrometry offers unparalleled insights into disease biomarkers and physiological changes. High-throughput and deep coverage workflows are essential to drive large-scale studies with robust quantitative precision and broad dynamic range.

Objectives and Overview

This work presents two complementary LC–MS workflows on the Thermo Scientific Orbitrap Exploris 480 instrument combined with Seer’s Proteograph XT nanoparticle-based sample preparation. The high-throughput method prioritizes speed and reproducibility, while the Max-ID method maximizes proteome depth.

Methodology

Samples (100 µL plasma) are incubated with paramagnetic nanoparticles to generate specific protein coronas, followed by on-particle reduction, alkylation, and one-pot Trypsin/Lys-C digestion. Peptides are desalted via a mixed-media filter plate.
High-throughput LC: two 24 min runs per sample using a PepMap C18 column (150 µm × 15 cm) at 1.5 µL/min (48 min total).
Max-ID LC: pooled peptide wells, single 102 min run on a PepMap Neo C18 column (75 µm × 75 cm) at 0.25 µL/min.
DIA acquisition with 60 000 MS1 and 15 000 MS2 resolution, optimized AGC targets, auto max injection times, and 4 Da isolation windows.

Instrumentation

Thermo Scientific Vanquish Neo UHPLC system
Thermo Scientific EASY-Spray PepMap and PepMap Neo columns
Thermo Scientific Orbitrap Exploris 480 mass spectrometer

Main Results and Discussion

• High-throughput workflow identified >3 000 protein groups and >20 000 peptides per sample with 95% data completeness.
• Max-ID workflow achieved >3 800 protein groups and >27 000 peptides with 99% data completeness.
• Quantitative precision: 87.2% (high-throughput) and 89.2% (Max-ID) of proteins exhibit CV <20%.
• Wide dynamic range enabled reproducible quantitation of high- and low-abundance plasma proteins.

Benefits and Practical Applications

• Scalable nanoparticle enrichment for unbiased plasma proteome coverage.
• Flexible workflows balancing speed and depth for cohort studies and biomarker discovery.
• Robust quantitative performance supports clinical and translational research.

Future Trends and Applications

Emerging machine learning–driven library-free DIA approaches will enhance spectral prediction and reduce reliance on curated libraries. Further automation of nanoparticle sample prep and integration of AI-based data analysis promise to increase throughput and enable real-time biomarker monitoring in clinical settings.

Conclusion

The integration of Seer’s Proteograph XT assay with the Vanquish Neo UHPLC and Orbitrap Exploris 480 MS delivers a highly reproducible, high-throughput and deep plasma proteomics platform, advancing the potential for large-scale biomarker discovery and precision medicine.

References

• PO147-2024-EN