Unveiling hidden protein depths: a high-throughput plasma proteomics workflow for enhanced biomarker discovery on Orbitrap Astral MS

Importance of the Topic

Blood plasma is a rich source of potential biomarkers for early disease detection due to its non-invasive collection and complex proteome composition. However, the wide dynamic range of plasma proteins poses challenges for comprehensive analysis. Integrating high-resolution mass spectrometry with advanced sample preparation strategies is crucial to revealing low-abundance proteins and supporting translational biomarker research.

Objectives and Study Overview

• Develop robust high-throughput plasma proteomics workflows combining varied sample preparation techniques.
• Compare neat plasma, nanoparticle-based enrichment (Proteograph XT), and immunodepletion of abundant proteins.
• Assess performance across multiple LC–MS throughputs (100, 60, 24, 16 samples/day) using label-free DIA on the Orbitrap Astral MS.

Methodology and Instrumentation

The study employed pooled healthy donor plasma processed in three ways: direct digest (neat), nanoparticle enrichment via Seer Proteograph XT, and depletion of top-14 abundant proteins using high-select spin columns. After trypsin/Lys-C digestion, peptides were separated on various columns (15 cm, 50 cm EASY-Spray PepMap; 60 cm IonOpticks) and resolved on a Vanquish Neo UHPLC system. The Orbitrap Astral MS operated in DIA mode enabled comprehensive protein quantitation. Data were analyzed with Spectronaut, DIA-NN, and Proteome Discoverer with CHIMERYS.

Key Results and Discussion

• Deep proteome coverage: Over 4,000, 5,000, 6,000, and 8,000 protein groups identified at 100, 60, 24, and 16 samples/day, respectively, with CVs of 4–6%.
• Neat plasma workflow yielded ~720 proteins at 60 SPD (CV ~6%), balancing simplicity and throughput.
• Top-14 depletion improved coverage to 1,100–1,850 proteins (100–24 SPD) with CVs around 10%.
• Proteograph XT enrichment identified ~3,900–4,900 proteins at 100–60 SPD (CV ~6%), delivering the deepest automated workflow.
• Incorporating a spectral library expanded identifications by 300–600 proteins in high-throughput runs.
• Complementarity observed: ~20–25% of depletion-unique proteins were not captured by enrichment, supporting combined approaches.

Benefits and Practical Applications

• Scalable workflows tailored to study size and depth requirements.
• High precision (CV <6%) ensures reliable quantitation in large cohorts.
• Automated sample preparation and rapid DIA acquisition accelerate biomarker discovery pipelines.
• Flexible instrument configurations enable laboratories to optimize throughput versus depth.

Future Trends and Opportunities

Advancements in spectral library generation, AI-driven data analysis, and further optimization of sample preparation will enhance sensitivity and proteome coverage. Integration with multi-omics platforms and expansion to clinical cohorts promises to advance personalized diagnostics and therapeutic monitoring.

Conclusion

The presented high-throughput plasma proteomics workflows, leveraging Orbitrap Astral MS and varied preparation strategies, achieve unmatched depth and reproducibility. These methods empower large-scale biomarker studies and support translational research by uncovering low-abundance proteins in complex plasma samples.

References

• Yang K., Flora A., Motamedchaboki K., et al. Unveiling hidden protein depths: a high-throughput plasma proteomics workflow for enhanced biomarker discovery on Orbitrap Astral MS. Thermo Fisher Scientific Application Note PO147-2024-EN.
• Spectronaut, Biognosys AG.
• CHIMERYS intelligent search algorithm, MSAID.