Uncovering biological differences at scale: high-throughput and in-depth plasma proteomics with the Seer Proteograph ONE workflow and Orbitrap Astral Zoom Mass Spectrometer

Importance of the Topic

Plasma proteomics offers a minimally invasive route to discover disease‐related molecular signatures, but its application has been constrained by the vast dynamic range of protein concentrations. Integrating high‐throughput workflows with advanced mass spectrometry is critical to unlock deep, reproducible profiling needed for large‐scale clinical biomarker discovery and precision medicine.

Objectives and Study Overview

This study evaluates the performance of the Seer Proteograph ONE sample preparation workflow combined with the Thermo Scientific Orbitrap Astral Zoom mass spectrometer. It aims to demonstrate both rapid, high‐throughput analysis and ultradeep proteome coverage in plasma samples from healthy controls and patients with lung cancer, colorectal cancer, and Alzheimer’s disease.

Methodology

Twenty human K2EDTA plasma samples (five per condition) matched for age, gender, and ethnicity were obtained and processed using the Seer Proteograph ONE workflow. Two LC–MS approaches were compared:

• High‐throughput method: 60 samples per day (SPD) using an EASY-Spray HPLC ES906 column and direct trap-elute configuration.
• Max-ID (deep profiling) method: 16 SPD using an IonOpticks Aurora Frontier column in direct injection mode.

Peptides were quantified, normalized to 50 ng/µL, separated on a Thermo Vanquish Neo UHPLC, and analyzed by data‐independent acquisition (narrow‐window DIA) on the Orbitrap Astral Zoom MS. Data were processed with the Proteograph Analysis Suite using a library‐free DIA-NN search.

Used Instrumentation

• Seer Proteograph ONE sample preparation kit with SP200 automation.
• Thermo Scientific Vanquish Neo UHPLC system.
• Orbitrap Astral Zoom mass spectrometer.
• EASY-Spray HPLC ES906 column (150 µm × 15 cm, 2 µm).
• IonOpticks Aurora Frontier column (75 µm × 60 cm, 1.7 µm).
• Pierce Peptide Fluorometric Quantitative Assay.

Main Results and Discussion

Both workflows achieved robust, reproducible profiling:

• High‐throughput (60 SPD) identified >8,200 protein groups and achieved median peptide and protein %CVs below 12% across triplicate preparations.
• Max‐ID (16 SPD) reached over 10,800 protein groups, capturing a dynamic range spanning 7–8 orders of magnitude.
• Workflow overlap: the 60 SPD method covered 64% of proteins in the 16 SPD run and retained 96% of FDA-approved biomarker proteins.
• PCA highlighted tight clustering of healthy controls versus wider dispersion among patient groups, reflecting biological heterogeneity.
• In lung cancer versus healthy controls, 1,264 proteins were differentially expressed (603 downregulated, 661 upregulated), including key FDA-approved markers and pathways linked to immune signaling and tissue remodeling.

Benefits and Practical Applications

• Scalable sample preparation and MS acquisition support large clinical cohorts.
• High reproducibility enables reliable biomarker discovery and validation.
• Flexible throughput options optimize either speed or depth of coverage depending on study goals.
• Exceptional dynamic range allows detection of low‐abundance, clinically relevant proteins.

Future Trends and Potential Applications

Emerging directions include integration with artificial intelligence and machine learning for enhanced data interpretation, further multiplexing to increase throughput, automated sample processing to minimize manual error, and coupling with multi‐omics approaches or single‐cell proteomics for comprehensive translational studies.

Conclusion

The combination of the Seer Proteograph ONE workflow and Orbitrap Astral Zoom MS delivers a powerful, scalable platform for plasma proteomics. It reconciles the demands of rapid, high‐throughput screening with the need for ultradeep, high‐precision analysis, supporting robust biomarker discovery and precision medicine applications.