Unlocking the plasma proteome with Evotip Pure based, standardized and scalable workflows

Importance of the Topic

Plasma proteomics enables comprehensive characterization of circulating proteins, offering critical insights into physiological and pathological processes. The vast dynamic range and high complexity of the plasma proteome present analytical challenges, yet effective solutions can drive multiplex biomarker discovery and support clinical research in disease monitoring.

Study Objectives and Overview

This application note describes the development of two fully automated, scalable workflows for deep profiling of the plasma proteome. A neat plasma preparation using Protein Aggregation Capture (PAC) and a membrane vesicle enrichment strategy (MagNet) were implemented on an Opentrons OT-2 robot and coupled to Evotip-based downstream analysis to maximize throughput, sensitivity, and reproducibility.

Applied Methodology

The workflows share an end-to-end automation philosophy: sample lysis, reduction, alkylation, on-bead protein capture, and enzymatic digestion are executed without manual intervention.

• Neat plasma workflow: 1 µl plasma diluted in SDS/TCEP/CAA buffer, captured on hydroxyl magnetic beads via acetonitrile-induced aggregation, washed, and digested with LysC and Trypsin.
• MagNet workflow: 4 µl plasma bound to SAX magnetic beads in Bis-Tris Propane buffer, sequential binding and washes, on-deck solubilization in SDS/TCEP/CAA buffer, followed by digestion and direct peptide loading.
• Both workflows deliver peptide mixtures directly onto Evotips for LC-MS analysis, minimizing sample loss.

Instrumentation

• Opentrons OT-2 liquid handling robot for fully automated sample preparation.
• Evotip sample trap integrated with Evosep One system (100 Samples Per Day method, EV1109 column, 40 °C).
• Thermo Scientific Orbitrap Astral mass spectrometer operated at MS1 resolution of 240,000 (380–980 m/z) and HCD fragmentation (25% NCE) in DIA mode.

Main Results and Discussion

Both workflows demonstrated high sensitivity and reproducibility. The neat plasma protocol quantified approximately 1,500 protein groups and over 15,000 precursors per run, while the MagNet strategy expanded coverage to more than 5,000 protein groups and 45,000 precursors. Median coefficients of variation at the protein level were below 10% for both methods, and technical LC-MS variability was under 4%. The MagNet workflow enriched extracellular vesicle markers, detecting 96 out of the top 100 Vesiclepedia proteins, with 82% quantified at CV<20%. Dynamic range analysis showed reliable quantification of low-abundance biomarkers, including 59 of 113 FDA-approved plasma markers and numerous putative disease indicators from the Human Disease Blood Atlas.

Benefits and Practical Applications

• High-throughput capacity: processing up to 192 samples in under 10 hours using minimal plasma volume (≤5 µl).
• Cost-efficient design: reduced reagent consumption, single-tip transfers, and elimination of offline desalting.
• Robust performance: consistent quantitative precision supports large-scale clinical cohort studies and biomarker validation.

Future Trends and Perspectives

Adopting these automated workflows can accelerate multi-cohort plasma proteomics and facilitate integration with other omics layers. Future developments may include real-time quality control, AI-driven data analysis, deeper sub-proteome targeting, and adoption in regulated clinical environments for personalized disease monitoring.

Conclusion

The described PAC and MagNet workflows on the OT-2 platform, combined with Evotip and Orbitrap Astral analysis, establish a robust, scalable, and sensitive pipeline for plasma proteome profiling. This end-to-end solution supports high-throughput biomarker discovery and paves the way for routine clinical implementation.

References

1. Batth TS et al. Protein Aggregation Capture on Microparticles Enables Multipurpose Proteomics Sample Preparation. Mol Cell Proteomics. 2019; mcp.TIR118.001270.
2. Wu CC et al. Mag-Net: Rapid enrichment of membrane-bound particles enables high coverage quantitative analysis of the plasma proteome. bioRxiv. 2023; DOI:10.1101/2023.06.10.544439.
3. Tuck MK et al. Standard Operating Procedures for Serum and Plasma Collection: Early Detection Research Network Consensus. J Proteome Res. 2010; 10.1021/pr800545q.
4. Alvez MB et al. Next generation pan-cancer blood proteome profiling using proximity extension assay. Nat Commun. 2023; 10.1038/s41467-023-39765-y.