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Updated: Jun 25, 2025
A Plasma Sample Preparation for Mass Spectrometry using an Automated Workstation
Published on: April 24, 2020
Fully Automated Workflow for Integrated Sample Digestion and Evotip Loading Enabling High-Throughput Clinical
Anders H Kverneland1, Florian Harking2, Joel Mario Vej-Nielsen3
1Faculty of Health Sciences, Novo Nordisk Foundation Center for Protein Research, University of Copenhagen, Copenhagen, Denmark; Department of Oncology, National Center of Cancer Immune Therapy, Copenhagen University Hospital - Herlev and Gentofte, Herlev, Denmark.
Automated sample preparation using the Opentrons OT-2 robot significantly speeds up protein analysis for large cohorts. This efficient workflow enhances biomarker discovery by processing up to 192 samples in 6 hours.
Area of Science:
- Proteomics
- Biomarker Discovery
- Automation in Life Sciences
Background:
- Protein identification and quantification are crucial for biomarker discovery.
- High-throughput mass spectrometry requires efficient sample preparation, which is currently a bottleneck for large cohort studies.
Purpose of the Study:
- To develop and evaluate a fully automated, efficient workflow for protein sample preparation on the Opentrons OT-2 robot.
- To assess the workflow's performance in terms of speed, reproducibility, sensitivity, and cost-effectiveness for large-scale proteomic analysis.
Main Methods:
- A novel automated workflow integrating sample digestion, cleanup, and Evotip loading was developed on the Opentrons OT-2 robot.
- High-throughput analysis was performed using Evosep One coupled with Orbitrap Astral mass spectrometry with narrow-window data-independent acquisition (nDIA).
- An automated phosphopeptide enrichment step using magnetic titanium-immobilized metal ion affinity chromatography beads was incorporated.
Main Results:
- The automated workflow processed up to 192 samples in 6 hours, achieving a throughput of 100 samples per day.
- Consistent identification of ~8000 protein groups and ~130,000 peptide precursors per sample.
- Demonstrated high sensitivity, reproducibility, and stability with low sample input, optimizing reagent costs.
- Successful application in evaluating cancer immune therapy effects on plasma proteome in metastatic melanoma patients.
Conclusions:
- The automated workflow offers a simple, efficient, and scalable solution for high-throughput proteomic sample preparation.
- This method significantly reduces bottlenecks in sample preparation, enabling large cohort studies and biomarker discovery.
- The integrated workflow provides high sensitivity for both proteome and phosphoproteome analysis, applicable to clinical research.
