Comprehensive and high-throughput plasma proteome profiling for biomarker discovery

Significance of the topic

Plasma proteomics provides a window into complex physiological and pathological processes by measuring thousands of proteins over a concentration range of over 10 orders of magnitude. High‐resolution LC-MS workflows are essential for discovering novel biomarkers, monitoring disease progression, and advancing personalized medicine. Overcoming limitations in sensitivity, reproducibility, and throughput is critical to translate proteomic insights into clinical applications.

Objectives and study overview

This study evaluates a complete plasma proteome profiling solution combining the Thermo Scientific™ Orbitrap™ Astral™ Zoom mass spectrometer with three sample preparation strategies—neat plasma, immunodepletion of top abundant proteins, and the Seer® Proteograph® ONE Assay—in concert with high‐throughput narrow‐window data‐independent acquisition (nDIA). The goal is to compare proteome coverage, quantitative precision, and scalability across workflows and sample‐per‐day (SPD) throughputs (100, 60, and 24 SPD).

Methodology and instrumentation

Key elements of the workflow include:

• Sample preparation strategies: neat plasma with minimal handling, immunodepletion of 14 high‐abundance proteins using High Select™ spin columns, and dynamic range compression via Proteograph ONE nanoparticles.
• Automation platforms: Thermo Scientific™ AccelerOme™ Automated Sample Preparation and Seer SP200 Automation for Proteograph ONE.
• Chromatography: Vanquish™ Neo UHPLC in trap-and-elute mode; columns—EASY-Spray™ HPLC 2 μm C18 (150 μm×15 cm) for high throughput, PepMap™ Neo 2 μm C18 (75 μm×50 cm) for deeper profiling.
• Mass spectrometry: Orbitrap Astral Zoom MS with MS1 resolution 240 000, narrow DIA windows (2.5–3 m/z), HCD collision energy, and fast scan cycles optimized for throughput.
• Data analysis: Library‐free processing in Seer Proteograph Analysis Suite with 1% FDR at peptide and protein levels, followed by statistical and enrichment analysis in R.

Main results and discussion

Neat plasma yielded a median of 5 500–9 000 peptides and 680–1 165 protein groups (100–24 SPD), with CVs of ~7% for peptides and <5.6% for proteins. Immunodepletion doubled identifications (10 900–18 300 peptides; 1 410–2 403 proteins) at similar precision. Proteograph ONE provided the greatest depth (47 000–75 800 peptides; 5 389–7 185 proteins), with protein CVs improving to <3.7% at 24 SPD. Each method contributed unique protein sets: 185 exclusive to neat, 745 to depleted, and 5 764 to Proteograph ONE. Enrichment analysis revealed immune, coagulation, adhesion, metabolic, and disease‐related pathways across workflows. Compared to the Human Plasma Peptide Atlas (~4 600 proteins), Proteograph ONE identified >50% overlap and ~5 000 additional low‐abundance proteins. High throughput (≥60 SPD) enables profiling >18 000 samples per year on a single instrument.

Benefits and practical applications

• Scalable trade-off between depth and throughput suited to cohort size.
• High quantitative precision and reproducibility via automation and narrow‐window DIA.
• Enhanced detection of low‐abundance biomarkers, including FDA-approved targets.
• Flexible workflows support discovery, clinical validation, and large-scale epidemiological studies.

Future trends and applications

Advances in multiplexed DIA, single-cell proteomics, and multi-omic integration will further deepen insights into disease biology. Continued automation, AI-driven data analysis, and cloud-based workflows will accelerate the translation of plasma proteomics to precision diagnostics and monitoring.

Conclusion

The combination of Orbitrap Astral Zoom MS with neat, immunodepleted, and Proteograph ONE workflows delivers unprecedented depth, precision, and throughput in plasma proteomics. This turnkey solution empowers large‐scale biomarker discovery, robust clinical validation, and personalized medicine applications.

Reference

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