Uncovering biological differences at scale

Significance of the Topic

Plasma proteomics offers minimally invasive insights into systemic biological processes, enabling biomarker discovery and precision medicine applications by capturing dynamic changes in circulating proteins.

Objectives and Study Overview

• Demonstrate integration of Seer Proteograph ONE workflow with Thermo Scientific Orbitrap Astral Zoom mass spectrometer.
• Compare high-throughput (60 samples/day) versus deep-coverage (16 samples/day) methods for plasma proteome profiling.
• Assess quantitative performance, dynamic range, and reproducibility across healthy and disease cohorts.

Methodology and Instrumentation

• Sample cohorts: single-spun and double-spun K₂EDTA plasma from healthy controls, lung cancer, colorectal cancer, and Alzheimer’s disease subjects.
• Automated nanoparticle-based enrichment on the Seer SP200 instrument using the Proteograph ONE Assay Kit.
• Chromatography: Vanquish Neo UHPLC system with trap-and-elute on EASY-Spray C18 or direct injection on IonOpticks Aurora Frontier C18 columns.
• Mass spectrometry: DIA acquisition on Orbitrap Astral Zoom MS with 60 SPD and 16 SPD gradient methods.
• Data analysis: Library-free DIA-NN (1% precursor and protein FDR) and downstream processing in RStudio.

Main Results and Discussion

• Proteome depth: 16 SPD method identified a median of 10,769 protein groups and 152,009 peptides; 60 SPD method yielded 8,213 protein groups and 92,873 peptides.
• Reproducibility: median CVs of 4.2–4.3% for proteins and 8.1–8.9% for peptides across technical replicates.
• Dynamic range: Quantification across 8–9 orders of magnitude demonstrated for both workflows.
• Overlap: 7,625 protein groups shared between methods; unique identifications of 3,144 (16 SPD) and 588 (60 SPD).
• Variance analysis: PCA showed tight clustering of healthy controls versus greater dispersion among patient samples.
• Differential expression: >1,200 proteins dysregulated in lung cancer plasma, including 603 downregulated and 661 upregulated proteins with several FDA-approved biomarkers.
• Pathway enrichment: Immune signaling, chemotaxis, wound healing, tissue remodeling, and hemostasis were prominently affected.

Benefits and Practical Applications

• Unbiased nanoparticle enrichment enhances detection of low-abundance proteins for robust biomarker discovery.
• High throughput supports large-scale clinical and translational studies.
• Deep-coverage method enables comprehensive proteome profiling for mechanistic research.
• Exceptional reproducibility and broad dynamic range ensure reliable quantitative performance.

Future Trends and Opportunities

• Integration with multi-omics and advanced bioinformatics to deepen biological insights.
• Optimization of nanoparticle chemistries for targeted capture of specific protein subsets.
• Scaling workflows for population-level studies in precision medicine.
• Application of AI-driven data interpretation to accelerate biomarker validation.

Conclusion

Combining the Proteograph ONE workflow with Orbitrap Astral Zoom mass spectrometry delivers scalable, reproducible, and sensitive plasma proteomics. Both high-throughput and deep-coverage methods provide robust protein identification, exceptional dynamic range, and reliable quantification, supporting biomarker discovery and disease research.

References

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