LFQ HR-DIA WORKFLOW FOR PLASMA PROTEOMICS ON AN AUTOMATED PLATFORM

Significance of the Topic

A reliable and high throughput workflow for plasma proteomics is critical due to the complexity and wide dynamic range of plasma proteins. Label free quantitation combined with high resolution data independent acquisition enables comprehensive profiling of plasma proteins, supporting biomarker discovery, disease monitoring and personalized medicine applications.

Objectives and Overview

This study presents an automated LFQ HR DIA workflow for plasma proteome analysis. The aim is to demonstrate consistent sample processing, robust protein identification and accurate quantitation using the Thermo Scientific AccelerOme platform paired with Vanquish Neo UHPLC and Orbitrap Exploris 480 MS.

Methodology and Instrumentation

The workflow integrates standardized procedures for protein extraction, reduction, alkylation and digestion on the AccelerOme automated platform. Peptides are separated on a Vanquish Neo UHPLC system equipped with a 50 cm EASY Spray PepMap C18 column at 60 deg C and analyzed on an Orbitrap Exploris 480 mass spectrometer using a data independent acquisition scheme. Data processing and quantification are performed with Spectronaut DirectDIA against the Human UniProt database.

Main Results and Discussion

The workflow achieved consistent peptide recovery and low variation across 20 samples from multiple operators. Overlapping base peak chromatograms demonstrated reproducible chromatographic performance. High resolution DIA enabled identification of hundreds of plasma proteins with a 1 percent protein level false discovery rate. Group specific coefficients of variation remained below acceptable thresholds, confirming quantitation precision.

Benefits and Practical Applications

• Automated sample preparation reduces hands on time and operator to operator variability
• High throughput processing suitable for large cohort studies
• Robust identification and quantitation of plasma proteins without depletion steps
• Compatibility with existing LC MS infrastructure in proteomics laboratories

Future Trends and Possibilities

Further development may include integration of high abundance protein depletion to expand dynamic range, adoption of machine learning approaches for advanced data analysis, and extension to other biofluids or tissue digests. Hybrid workflows combining DIA with emerging ion mobility separation could enhance proteome coverage.

Conclusion

The automated LFQ HR DIA workflow on the AccelerOme platform provides a streamlined and reproducible solution for plasma proteomics, delivering consistent sample quality, high proteome coverage and precise quantitation essential for clinical and research applications.

References

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