A robust mass spectrometer for precision medicine – the Orbitrap Exploris 240 mass spectrometer for large-scale plasma protein profiling

Significance of the Topic

Blood plasma proteomics is essential for biomarker discovery in precision medicine to enable routine clinical monitoring and large-scale studies. Robust, reproducible, and high-throughput workflows are key to detecting low-abundance protein biomarkers in diverse patient cohorts and translating findings into clinical practice.

Objectives and Study Overview

This study aimed to demonstrate the reliability and robustness of the Orbitrap Exploris 240 mass spectrometer coupled to Evosep One LC for untargeted plasma protein profiling in large-scale human cohort studies. Key objectives included:

• Evaluating inter-laboratory reproducibility across multiple instruments
• Assessing workflow stability over hundreds of injections
• Benchmarking method performance with preloaded plasma profiling templates
• Investigating scalable throughput and depth of coverage by varying sample loads and gradient lengths

Methodology and Instrumentation

Pooled human serum was depleted of abundant proteins using Top14 spin columns, followed by reduction, alkylation, and trypsin digestion with an automated liquid handling platform. Samples were analyzed using:

• Evosep One LC system with 21 or 44 minute gradients (30 SPD or 60 SPD) and EvoTips
• Orbitrap Exploris 240 mass spectrometer equipped with EASY-Spray ion source and EASY-Spray HPLC columns
• Preloaded plasma profiling LC-MS/MS method template in the Thermo Scientific Method Editor

Data were processed in Proteome Discoverer using a three-stage search workflow combining spectral library and database searches (MSPep, Prosit library, SEQUEST HT), and quantified by label-free quantification with feature mapping. Skyline monitored spiked peptide standards and Prism performed statistical analyses.

Main Results and Discussion

• Inter-laboratory reproducibility on four instruments yielded 2.58% CV for protein identifications and 5.89% CV for peptides.
• Retention time shifts were below 5% CV and mass accuracy remained within ±5 ppm over 200 runs across five weeks.
• From 100 ng HeLa digest, an average of ~2,500 proteins and 14,000 peptides were identified per run.
• Analysis of 200 ng depleted human serum over 200 injections showed <1% change in system suitability metrics without recalibration.
• Quantitative stability was confirmed with >70% of proteins quantified at <20% CV across 100 runs and strong correlation between runs separated by weeks.
• By increasing sample load to 500 ng and using a 44 minute gradient, over 500 plasma proteins including FDA-approved biomarkers were reproducibly detected with CV below 20%.

Benefits and Practical Applications

The described workflow offers:

• High throughput plasma profiling suitable for clinical cohort studies
• Robust inter-instrument and inter-day reproducibility
• Small instrument footprint and simplified operation for routine diagnostics
• Preloaded method templates for harmonized data acquisition and benchmarking
• Scalability to match sample availability, throughput needs, and desired depth of coverage

Future Trends and Potential Applications

Anticipated developments include:

• Integration of automated sample preparation and data acquisition into clinical laboratories
• Deeper proteome coverage through optimized gradients or multiplexed approaches
• Application of advanced data analytics and machine learning for biomarker discovery
• Expansion to multi-omic workflows combining proteomics with metabolomics and genomics
• Implementation in routine diagnostic testing and personalized medicine pipelines

Conclusion

The Orbitrap Exploris 240 mass spectrometer coupled with Evosep One LC demonstrates a robust, reproducible, and scalable workflow for large-scale plasma protein profiling. Preloaded method templates and consistent quantitative performance support its deployment in precision medicine research and clinical applications.