High throughput abundant protein depletion for plasma proteomics

Significance of the topic

Plasma proteomics is essential for disease biomarker discovery but is challenged by high-abundance proteins that mask low-level targets. Effective depletion improves proteome coverage, quantitation and reproducibility in large-scale studies.

Objectives and Overview of the Study

This work introduces a 96-well filter plate format using an antibody-based resin for high-throughput depletion of the top 14 abundant plasma proteins. It benchmarks performance against conventional spin columns and acid precipitation, and demonstrates applicability to serum, CSF and clinical plasma samples.

Methodology and Instrumentation

• Sample preparation with Thermo Scientific High-Select Top14 Abundant Protein Depletion Resin in 96-well format combined with EasyPep 96 sample prep.
• Biofluids tested: human plasma, serum and cerebrospinal fluid (CSF).
• Depletion efficiency ≥90% for target proteins across 96 samples with CVs <5%.
• LC-MS analysis using Vanquish Neo UPLC (50 cm C18 PepMap Neo column), Orbitrap Eclipse Tribrid, Exploris 480 or Astral mass spectrometers.
• Label-free DIA with ~250 ng peptide load per run.
• Data processed in Proteome Discoverer (SEQUEST HT, CHIMERYS), filtered at 1% protein FDR.

Main Results and Discussion

• Comparable performance between 96-well and spin-column formats: ~1,200 proteins and >10,000 peptides identified.
• Consistent depletion of high-abundance proteins (90–95%) with inter-plate reproducibility (CVs <10%).
• Extension to CSF and serum yielded 37% and 39% increases in protein identifications, respectively.
• Analysis of 32 clinical plasma samples (normal vs lung cancer) revealed significant biomarker changes (Mucin 5B, Cadherin-5, CD44).

Benefits and Practical Applications

• Enables processing of 96 samples in 7–8 hours, reducing hands-on time and labor.
• Supports high-throughput biomarker discovery across plasma, serum, CSF and clinical cohorts.
• Enhances detection of low-abundance proteins for translational research in oncology, neurology and industrial QA/QC.

Future Trends and Opportunities

Advances in automation, multiplexed acquisition and targeted proteomics will further increase throughput and depth. Development of expanded depletion panels and integration with clinical workflows will drive personalized medicine and large-cohort studies.

Conclusion

The 96-well antibody-based depletion platform delivers robust, efficient and scalable reduction of high-abundance proteins, outperforming traditional methods and supporting diverse biofluid analyses for biomarker discovery.

Reference

• Flora A., Klenke A., Jarrett N., Patel B., Bomgarden R. High throughput abundant protein depletion for plasma proteomics. Thermo Fisher Scientific, Poster P-II-0399, 2024.