Cost-efficient and scalable end-to-end workflow on the Opentrons OT-2 for neat plasma utilizing Evotip Pure

Importance of the topic

Plasma proteomics provides a minimally invasive window into disease biology and biomarker discovery. High-throughput, reproducible, and cost‐effective sample preparation is essential to support large clinical studies and routine diagnostics.

Objectives and Study Overview

This study introduces a fully automated workflow on the Opentrons OT-2 liquid handler, directly integrated with Evotip Pure, to process neat plasma from raw sample to ready‐to‐analyze tips. The goal is to enable scalable, hands‐off preparation of up to 192 samples in eight hours while maintaining analytical robustness.

Methodology

The end‐to‐end protocol employs Protein Aggregation Capture (PAC) on magnetic beads within a 96‐well format:

• Add 1 µL plasma to each well and perform one‐pot reduction (5 mM TCEP) and alkylation (10 mM CAA) in 1% SDS for 1 hour at ambient temperature.
• Capture proteins on MagReSyn Hydroxyl beads by adding acetonitrile to 80%, wash with 100% acetonitrile for 10 minutes.
• Digest on‐bead with 10 ng LysC and 40 ng trypsin for 4 hours at room temperature.
• Load 40% of the peptide digest onto Evotip Pure using the OT-2 deck for immediate online desalting and storage.

Instrumentation Used

• Opentrons OT-2 liquid handling robot with custom Python script and HTML‐form interface.
• Evotip Pure for sample loading and on‐tip desalting.
• Evosep One LC system with 100 SPD (EV1109 column at 40 °C) and 200 SPD (EV1107 column at ambient) methods.
• timsTOF HT mass spectrometer (Bruker) operated in dia-PASEF mode.
• DIA-NN software (v1.8.1) in library-free mode against the reviewed human proteome.

Main Results and Discussion

• Digest efficiency: ~65% fully cleaved peptides across 16 replicates, identifying 4 000 precursors mapping to ~400 protein groups with median CV of 13.4%.
• Proteome coverage spans four orders of magnitude, including multiple FDA‐approved biomarkers quantified with CVs below 20%.
• Plate‐wide evaluation with the 200 SPD method yielded an average of ~300 proteins per well, data completeness above 95%, and no positional bias by row or column in PCA analysis.

Benefits and Practical Applications

• Minimal sample volume (1 µL) and reduced enzyme consumption lower costs for large cohorts.
• Fully automated hands‐off workflow increases reproducibility and throughput (up to 192 samples in 8 hours).
• Direct integration with LC-MS via Evotip Pure streamlines sample transfer and desalting.

Future Trends and Possibilities of Use

Modular expansion of this workflow could include temperature‐controlled digestion, additional cleanup steps, or adaptation to other sample types. Transferring the concept to alternative automation platforms may further increase throughput and flexibility for clinical and industrial proteomics.

Conclusion

The standardized OT-2/Evotip Pure workflow delivers a robust, scalable, and cost‐efficient solution for high‐throughput plasma proteomics. Its minimal hands‐on time, low sample consumption, and excellent quantitative performance establish a foundation for broader adoption and future methodological advancements.

References

1. Batth TS., Tollenaere M., Rüther P., Gonzalez-Franquesa A., Prabhakar BS., Bekker-Jensen S., Deshmukh AS., Olsen JV. Protein Aggregation Capture on Microparticles Enables Multipurpose Proteomics Sample Preparation. Mol Cell Proteomics. 2019; mcp.TIR118.001270.