An Ultra High-throughput Plasma Protein Profiling (uHTPPP) Workflow Using a Modified Quadrupole-Orbitrap Mass Spectrometer

Significance of the Topic

Plasma proteomics is essential for discovering biomarkers in translational and clinical research, yet it faces challenges such as the high dynamic range of plasma proteins and the need to analyze large cohorts with statistical rigor. A robust, high-throughput workflow is critical to achieve consistent sensitivity, reproducibility, and scalability required for biomarker discovery and therapeutic monitoring in diverse patient populations.

Objectives and Study Overview

This study introduces an Ultra High-throughput Plasma Protein Profiling (uHTPPP) workflow that integrates automated sample preparation, high-throughput liquid chromatography, and a modified Quadrupole-Orbitrap mass spectrometer (Orbitrap Exploris 240). The goal was to evaluate the performance, reproducibility, and sensitivity of the uHTPPP workflow for large-scale plasma and serum proteomic analysis, supporting translational research and biomarker discovery.

Methodology

The uHTPPP workflow consists of:

• Automated depletion of abundant proteins (Top14) from human plasma or serum using High Select Midi columns.
• Sample digestion and clean-up with the EasyPep 96-well MS Sample Prep Kit on a Hamilton STARLet liquid handling system, reducing per-sample processing time from 20 minutes (manual) to 2.5 minutes (automated).
• Peptide separation on the EvoSep One LC system employing short gradients (21 or 44 minutes) and EvoTips for high throughput.
• Mass spectrometric analysis on the Orbitrap Exploris 240 using data-dependent acquisition, with consistent mass accuracy and retention time stability over multiple days.
• Data processing and feature mapping with Thermo Scientific Proteome Discoverer 2.5 for library search, retention time alignment, and quantitative analysis.

Used Instrumentation

• Thermo Scientific Orbitrap Exploris 240 mass spectrometer.
• EvoSep One high-throughput liquid chromatography system.
• EASY-Spray ES806A column with integrated emitter and heater.
• Hamilton STARLet robotic liquid handling workstation.
• Thermo Scientific Proteome Discoverer 2.5 software.

Main Results and Discussion

The uHTPPP workflow demonstrated:

• High peptide recovery (>80%) from automated preparation with <10% CV plate-to-plate variability.
• Excellent retention time consistency (<6% CV) and stable mass accuracy (<5 ppm drift) over five days.
• Reproducible identification of over 5,000 protein groups and 49,000 peptides from Top14-depleted serum, with >60% of 500 quantified plasma proteins showing <20% CV.
• Robust performance across hundreds of sequential injections, supporting up to 60 samples per day with minimal instrument downtime.

Benefits and Practical Applications

The uHTPPP workflow offers:

• Scalable sample throughput to support large cohort studies and population variability analysis.
• Automated sample preparation that reduces hands-on time and technical variability.
• High reproducibility and quantitative accuracy for reliable biomarker discovery and validation.
• Compatibility with clinical and industrial QA/QC environments requiring consistent, high-quality proteomic data.

Future Trends and Opportunities

Advancements to consider include:

• Integration of deeper depletion or enrichment strategies to expand proteome coverage.
• Adoption of real-time quality control and AI-driven data analysis to accelerate biomarker identification.
• Development of multiplexed workflows and microflow LC methods to further increase throughput.
• Translation of uHTPPP protocols into regulated clinical settings and diagnostic pipelines.

Conclusion

The uHTPPP workflow combining automated sample preparation, EvoSep One high-throughput LC, and the Orbitrap Exploris 240 mass spectrometer delivers exceptional reproducibility, sensitivity, and scalability for plasma proteomics. This streamlined approach supports robust biomarker discovery and translational research across large sample cohorts.

References

No formal reference list was provided in the original text.