Orbitrap Exploris 240 mass spectrometer delivers significantly increased lab productivity and reproducibility of human plasma proteome profiling from large human cohort studies

Importance of the Topic

Human plasma is a primary sample for clinical proteomics, offering insights into disease biomarkers and physiological states. High reproducibility and throughput are essential for detecting subtle protein changes in large patient cohorts, which supports reliable biomarker discovery and translational research.

Objectives and Study Overview

This case study evaluates an ultra-high-throughput plasma protein profiling (uHTPPP) workflow designed to process hundreds of human plasma samples rapidly and consistently. Two laboratories, the Precision Medicine Science Center (PMSC) and the University of North Carolina at Greensboro (UNCG), implemented the workflow to compare intra- and inter-laboratory reproducibility and overall productivity.

Methodology and Instrumentation

The uHTPPP workflow combines a 96-well plate digestion kit with automated liquid handling and a high-throughput LC-MS platform. Key steps include:

• Automated dilution, digestion and peptide cleanup using the EasyPep 96-well MS Sample Prep Kit and Hamilton Microlab STARLet script
• Peptide separation on an Evosep One liquid chromatography system with EASY-Spray ionization and column
• Mass analysis on an Orbitrap Exploris 240 mass spectrometer
• Data processing with Thermo Scientific Proteome Discoverer software

Key Results and Discussion

Sample preparation reproducibility was assessed by measuring peptide concentrations in QC serum and plasma. The PMSC observed an average CV of 8.6% over three runs, while the UNCG lab reported 8.4% CV. LC-MS performance, evaluated with a commercial HeLa digest standard and a 60 samples-per-day method, showed less than 5% CV for protein and peptide identifications between labs. Workflow throughput improved dramatically: profiling 300 plasma samples now takes two weeks instead of over a month.

Benefits and Practical Applications

Key advantages of the uHTPPP workflow include:

• Minimal hands-on time with an automated script
• Commercially standardized reagents and data acquisition templates
• High throughput, enabling processing of hundreds to thousands of samples
• Low analytical variability for confident quantitative comparisons
• Robust inter- and intra-laboratory reproducibility

Future Trends and Possibilities

Further developments may include deeper integration of automation, expansion to multi-omics workflows, real-time quality monitoring, and application of machine learning for data interpretation. Scaling to thousands of samples and adapting to clinical diagnostic settings will enhance personalized medicine and large-scale epidemiological studies.

Conclusion

The uHTPPP workflow with the Orbitrap Exploris 240 mass spectrometer delivers a standardized, high-throughput, and highly reproducible platform for human plasma proteome analysis. It meets the demands of large clinical cohorts by combining automated sample preparation, robust LC-MS performance, and streamlined data processing.

Reference

1. Trusiak S, Snovida S, Patel B, Bomgarden R, Chen E. Sampling the Human Population – Ultra-High Throughput Plasma Protein Profiling (uHTPPP) Sample Preparation for Translational Proteomics. Thermo Fisher Scientific Application Note #65727, 2020.