A complete and automated end-to-end sample preparation strategy for high-throughput and standardized proteomics with high sensitivity

Importance of the Topic

As proteomics moves towards large cohort analyses, robust and sensitive sample preparation is critical to ensure data quality, reproducibility, and cost efficiency.

Objectives and Study Overview

The study aimed to develop and validate a fully automated end-to-end sample preparation protocol on the Opentrons OT-2 system, integrating protein aggregation capture and direct peptide loading onto Evotips, optimized for low input amounts and high throughput.

Methodology and Instrumentation

• Opentrons OT-2 robotic platform for automated liquid handling
• Protein aggregation capture on magnetic microparticles
• Digestion at ambient temperature for variable times (1 to 4 hours versus overnight)
• Evotip-based peptide cleanup and loading
• Analysis using 30 SPD and Whisper 40 SPD mass spectrometry methods
• Online HTML form for customizable protocol generation

Main Results and Discussion

• Optimal digestion time: 4 hours balanced speed and cleavage efficiency with minimal missed cleavages
• Proteome depth: identification of over 30,000 precursors from 1 µg digests; more than 7,000 proteins from 125 ng samples
• Reproducibility: median CVs of 22% at precursor level and 13% at protein group level
• Peptide stability: Evotip-loaded samples remained over 80% recovered after 10 days at 4 °C
• Throughput capacity: processing of 192 samples in under eight hours, enabling up to 384 samples per working day

Benefits and Practical Applications

• High sensitivity workflow suitable for limited sample inputs down to 1 ng
• Standardized hands-off processing reduces operator variability
• Cost-effective use of reagents and consumables
• Scalable solution for large-scale proteomic studies and QA/QC applications
• Customizable via user-friendly HTML protocol generator

Future Trends and Opportunities

Emerging mass spectrometry platforms and advanced data analysis algorithms will further enhance throughput and sensitivity. Integration of AI-driven quality control and miniaturized workflows may expand applications to single-cell proteomics and clinical diagnostics.

Conclusion

The automated end-to-end proteomic sample preparation protocol on the Opentrons OT-2, combined with Evotip integration, delivers rapid, reproducible, and highly sensitive analyses across wide sample inputs, facilitating standardized workflows for large-scale proteomics.