Standardized, fully automated neat plasma and Mag-Net enrichment workflows enabled by the Evotip Pure

Importance of the Topic

High-throughput proteomics is essential for large clinical and industrial studies where reproducibility, sample integrity and automated workflows reduce manual intervention and variability. Plasma proteomics in particular enables minimally invasive biomarker discovery for disease monitoring and drug development. Integrating robust sample preparation with high-speed LC-MS analysis drives both research and QA/QC applications toward greater scale and consistency.

Study Objectives and Overview

This study presents four fully automated, end-to-end sample preparation workflows combining Evotip Pure desalting cartridges with the Biomek i5 liquid handler. The workflows target:

• Bead-based PAC digestion for cell lysates (HeLa samples)
• Neat plasma peptide desalting and loading
• Mag-Net enrichment of membrane-bound vesicles from plasma
• Interchangeable modules for flexible cohort processing

Each workflow can process up to 576 samples in parallel, integrating automated desalting, clean-up, digestion and storage prior to LC-MS analysis.

Methodology and Instrumentation

The automated platform includes:

• Beckman Coulter Biomek i5 liquid handler with custom deck layout
• Evotip Pure cartridges for peptide capture and storage
• On-bead protein aggregation capture (PAC) using magnetic microparticles, trypsin/Lys-C digestion at 37 °C
• Neat plasma workflow using a prototype sealing adapter for desalting 1 µl plasma on Evotips
• Mag-Net workflow for rapid enrichment of vesicle-associated proteins from 4 µl plasma
• LC-MS acquisition on timsTOF HT using a standard 200 samples-per-day (SPD) method

The sample volumes ranged from 1 µg HeLa lysate to 4 µl plasma, with overnight digestion and automated Evotip loading in under five minutes per sample.

Key Results and Discussion

All workflows demonstrated high reproducibility and minimal carry-over:

• 576 HeLa samples (1 µg each) processed in 47 hours, averaging less than two minutes per sample
• Average of 3 500 protein identifications per plate with PAC workflow and 500 SPD LC-MS
• Plate-to-plate Pearson correlation coefficients ≥0.84 and digestion efficiency above 85% zero-missed cleavages
• Neat plasma workflow identified over 500 unique proteins from 1 µl input with consistent quantification
• Mag-Net workflow yielded approximately 2 700 proteins from 4 µl plasma, including 40 vesicle markers with CVs below 20%

Blank wells and LC-MS blanks showed negligible signal, confirming absence of cross-contamination and positional bias. Modular design allowed seamless swapping of enrichment modules without compromising throughput.

Benefits and Practical Applications

The integrated workflows deliver:

• Scalable, hands-free processing of hundreds of samples with minimal manual handling
• High sample utilization and efficient reagent consumption through online Evotip loading
• Modularity to adapt to diverse sample types including cell lysates, neat plasma and vesicle fractions
• Reliable performance suitable for clinical proteomics, biomarker validation and QA/QC laboratories

Future Trends and Applications

Advances may include:

• Adapting modules for other biofluids (urine, cerebrospinal fluid) and low-input samples
• Integration with faster MS acquisition modes and data-independent analysis
• Machine-learning–driven method optimization for dynamic workflow adjustments
• Clinical deployment in regulated environments for diagnostic assays and longitudinal studies

Conclusion

The combination of Evotip Pure cartridges and the Biomek i5 liquid handler offers a robust, fully-automated solution for high-throughput proteomics. Four interchangeable workflows meet the needs of large-scale HeLa and plasma studies with excellent reproducibility, throughput and ease of use, paving the way for broader adoption in research and clinical laboratories.

References

1. Batth TS, Tollenaere M, Rüther P, et al. Protein Aggregation Capture on Microparticles Enables Multipurpose Proteomics Sample Preparation. Mol Cell Proteomics. 2019;18(6):1027–1043.
2. Wu CC, Tsantilas KA, Park J, et al. Mag-Net: Rapid enrichment of membrane-bound particles enables high coverage quantitative analysis of the plasma proteome. bioRxiv. 2023:2023.06.10.544439.