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

Significance of the Topic

Early and reliable detection of disease biomarkers in blood plasma is critical for improving patient outcomes, especially in oncology where cancer remains a leading cause of death worldwide. Plasma proteomics leverages minimally invasive blood draws to explore protein signatures linked to disease states, but the vast dynamic range of plasma proteins, dominated by abundant species such as albumin and immunoglobulins, makes detection of low-abundance biomarkers challenging. Advances in mass spectrometry instrumentation and streamlined workflows are needed to enhance sensitivity, reproducibility, and throughput in large cohort studies.

Objectives and Study Overview

This study aimed to establish a versatile, high-throughput plasma proteomics platform capable of deep proteome coverage and precise label-free quantitation. By comparing three sample preparation strategies—neat plasma analysis, immuno-depletion of abundant proteins, and nanoparticle-based enrichment using the Seer Proteograph XT Assay—coupled with varying liquid chromatography gradients and data-independent acquisition on the Orbitrap Astral mass spectrometer, the workflow was optimized for throughput rates up to 100 samples per day. The goal was to balance depth of coverage with handling of large sample sets for biomarker discovery.

Methodology and Instrumentation

Sample preparation approaches:

• Neat plasma digestion without depletion for maximum throughput but limited depth.
• High-select immuno-depletion spin columns to remove the top 14 abundant proteins, enhancing detection of lower-abundance species.
• Seer Proteograph XT workflow using engineered nanoparticles to capture a broader range of plasma proteins in an automated one-pot protocol.
• Optional gas-phase fractionation to build a project-specific spectral library.

Chromatography and mass spectrometry:

• Vanquish Neo UHPLC system with EASY-Spray PepMap columns for gradients tailored to 24, 60, or 100 samples per day (SPD).
• Orbitrap Astral mass spectrometer operated in data-independent acquisition mode for high sensitivity and reproducibility.
• Data analysis using DIA-NN for library-free and library-based searches or Proteome Discoverer with the CHIMERYS algorithm.

Main Results and Discussion

Neat plasma assays achieved identification of ~718 proteins with 60 SPD and
6% quantitative CV, highlighting baseline performance. Immuno-depletion increased coverage to ~1,068 proteins at 100 SPD and ~1,380 proteins at 60 SPD, with CVs below 10%. Extending to a 24 SPD gradient and 1,500 ng load yielded ~1,857 proteins. The Seer Proteograph XT approach delivered the deepest profiling, quantifying up to ~4,240 proteins at 100 SPD and ~6,457 proteins at 24 SPD, all with CVs near 6%. Incorporation of a gas-phase fractionation library further boosted identifications by 300–600 proteins. Complementary coverage from depletion and nanoparticle enrichment demonstrated the value of combining methods.

Benefits and Practical Applications

These optimized workflows enable comprehensive plasma proteome analyses at industrial throughput, providing:

• Scalable protocols for large cohort studies with robust quantitation.
• Tradeoffs between speed and depth to match study objectives.
• Deep coverage of low-abundance biomarkers without sacrificing reproducibility.

Future Trends and Possibilities

Emerging developments may include integration of artificial intelligence for automated data interpretation, expansion of nanoparticle chemistries to target specific protein classes, and miniaturized UHPLC formats to further increase sample throughput. Combining orthogonal enrichment strategies with next-generation mass analyzers promises even deeper insights into plasma proteomes, accelerating biomarker discovery and translational research.

Conclusion

The combination of tailored sample preparation strategies and the high sensitivity of the Orbitrap Astral mass spectrometer establishes a powerful platform for high-throughput plasma proteomics. By offering flexible options for throughput and depth, the workflow supports diverse translational applications, from large-scale cohort screening to focused biomarker validation.

References

1. Deutsch E et al Advances and Utility of the Human Plasma Proteome J Proteome Res 2021 20 5241–5263
2. Huang T et al Protein Coronas on Functionalized Nanoparticles Enable Quantitative and Precise Large-Scale Deep Plasma Proteomics bioRxiv 2023
3. Woo J and Zhang Q A streamlined high-throughput plasma proteomics platform with improved coverage J Am Soc Mass Spectrom 2023 34 754–762
4. Geyer P et al Plasma proteome profiling to assess human health and disease Cell Syst 2016 2 185–195