Thermo Scientific™ Orbitrap Exploris™ 240 mass spectrometer - Plasma profiling–reliable large cohort analysis with a fully automated workflow

Importance of the Topic

High-throughput and reproducible plasma protein profiling is critical for translational proteomics studies, supporting biomarker discovery and clinical research. Automated workflows with integrated quality controls address challenges in large-scale cohort analysis by ensuring consistent sample processing, stable mass accuracy, and retention time over extended operation.

Study Objectives and Overview

This work introduces an Ultra-High Throughput Plasma Protein Profiling (uHTPPP) workflow combining automated sample preparation, capillary flow LC, and high-resolution accurate-mass MS using the Orbitrap Exploris 240. The goal is to demonstrate stability, reproducibility, and scalability for large cohorts, processing up to 96 samples in four hours and analyzing 30 to 100 samples per day.

Methodology and Used Instrumentation

• Sample Preparation: Depletion of abundant proteins from pooled human serum using High Select Top14 resin and EasyPep 96 MS Sample Prep Kit on Hamilton Microlab StarLet, enabling 96 samples per four-hour run.
• Liquid Chromatography: EvoSep One system with preformed gradient, 21-minute method, capillary flow, and EASY-Spray HPLC column (150 mm × 150 μm, 3 μm).
• Mass Spectrometry: Thermo Scientific Orbitrap Exploris 240 operating in data-dependent acquisition (DDA) mode with high-resolution accurate-mass detection.
• Data Analysis: Thermo Scientific Proteome Discoverer software version 2.4 with 1% PSM false discovery rate.

Main Results and Discussion

The workflow achieved stable mass accuracy (within ±5 ppm) and retention time across five days of uninterrupted operation. Peptide recovery and protein identification demonstrated intra- and inter-day reproducibility with coefficients of variation below 5% and dynamic range exceeding five orders of magnitude. Over 100 depleted serum samples were analyzed with consistent performance. Automated sample preparation reduced hands-on time by 12× and combined data acquisition and processing time by 4× compared to manual methods.

Practical Applications and Benefits

• Enables standardized plasma profiling for large cohort studies in translational and clinical proteomics.
• Delivers high throughput (up to 500 samples per day) with minimal bench footprint and low variability.
• Supports robust biomarker discovery by integrating QA/QC procedures throughout the workflow.

Future Trends and Applications

Anticipated developments include scaling the workflow for thousands of samples, integrating automated robotics for end-to-end proteomic analysis, and combining with machine learning for deeper data interpretation. Compact, high-throughput platforms will advance personalized medicine and large-scale biomarker validation studies.

Conclusion

The described uHTPPP workflow, leveraging the Orbitrap Exploris 240 mass spectrometer, delivers reliable, reproducible, and scalable plasma proteomic profiling. It meets the demands of large cohort analyses by ensuring mass accuracy stability, retention time consistency, and rapid throughput with integrated quality control.