Sampling the human population – ultra-high- throughput plasma protein profiling (uHTPPP) sample preparation for translational proteomics

Significance of the Topic

Large-cohort proteomic profiling of human plasma demands high-throughput, reproducible sample preparation to minimize manual errors and enable translational research. uHTPPP addresses these needs by automating the EasyPep 96 MS kit on a robotic platform, ensuring robust, scalable analyses for population studies.

Objectives and Study Overview

The study aims to develop and validate an automated, ultra-high-throughput plasma protein profiling workflow (uHTPPP) that maximizes efficiency and reproducibility for mass spectrometry-based proteomics of large human cohorts. It compares automated versus manual methods regarding peptide recovery, processing time, and variability.

Methodology and Instrumentation

• Sample Preparation: EasyPep 96 MS kit automated on Hamilton Microlab STARLet with MPE2 positive-pressure and evaporation modules (uHTPPP).
• QA/QC: Sample dilution QC using fluorescein in glycerol to verify pipetting accuracy across 48- and 96-well formats.
• Digestion: In-solution Trypsin/LysC digestion for 1.75 hours following lysis, reduction, and alkylation on-deck at 37°C.
• Peptide Clean-up: Mixed-bed resin in 96-well filter plate, purified via MPE2 positive pressure, dried and reconstituted for LC-MS.
• Analysis: Evosep One LC 60 samples/day method with 21-minute gradient coupled to EASY-Spray source and Q Exactive HF-X mass spectrometer.
• Data Processing: Proteome Discoverer 2.4 with 1% FDR against the reviewed UniProt human database.

Results and Discussion

• Automation achieved over 70% peptide recovery for serum and plasma with 8-fold faster processing (2.5 versus 20 minutes per sample) compared to manual spin columns.
• Well-to-well reproducibility: 48 replicates of serum showed 7.2% CV and consistent LC-MS/MS identifications with protein and peptide ID CVs below 5%.
• Plate-to-plate reproducibility: Three plates of pooled serum and plasma yielded recoveries above 88% with CVs of 8.6% (pooled serum) and 20.4% (individual patient serum).
• Liquid handling optimization produced CVs below 3% for 50 μL transfers and below 1% for 300 μL buffers.
• Sample dilution QC confirmed pipetting accuracy with CVs below 5% for fluorescein dilutions.

Benefits and Practical Applications

• High-throughput capacity enables processing of hundreds to thousands of plasma samples with minimal hands-on time.
• Improved reproducibility and scalability support translational proteomics, clinical biomarker studies, and QA/QC workflows.
• Built-in QA/QC scripts facilitate routine monitoring of robotic performance and sample consistency.

Future Trends and Potential Applications

• Integration with isobaric labeling approaches (e.g., TMT) and peptide fractionation for deeper proteome coverage.
• Adaptation to other LC–MS platforms and incorporation of multi-omic sample types.
• Application to diverse biofluids and clinical sample matrices for large-scale studies.

Conclusion

The uHTPPP workflow provides a fully automated, high-throughput solution for plasma and serum sample preparation. By combining the EasyPep 96 MS kit with robotic automation, positive-pressure peptide clean-up, and comprehensive QA/QC, it achieves rapid processing, high peptide recovery, and reproducibility. uHTPPP is well suited for scaling translational proteomics and future methodological expansions.