High Throughput Abundant Protein Depletion for Plasma Proteomics

Importance of the Topic

Biological fluids such as plasma contain proteins spanning over ten orders of magnitude in concentration. High-abundance proteins like albumin and immunoglobulins mask low-abundance species, posing a major barrier to comprehensive biomarker discovery and quantitative proteomics.

Objectives and Study Overview

This study presents a high-throughput, 96-well filter plate format of an antibody-based depletion resin targeting the top 14 abundant plasma proteins. The performance of the new format is directly compared with conventional spin column depletion, and its applicability is evaluated across different biofluids and mass spectrometry workflows.

Methods and Instrumentation

• Depletion Resin: Thermo Scientific High-Select Top14 Abundant Protein Depletion Resin in a 96-well plate format.
• Sample Preparation: Thermo Scientific EasyPep 96 Sample Prep Kit for rapid digestion.
• Chromatography: Vanquish Neo UPLC system with 50 cm C18 EASY-Spray PepMap Neo column (3–28% acetonitrile over 85 min, 28–45% over 30 min at 300 nL/min).
• Mass Spectrometry: Orbitrap Eclipse Tribrid, Exploris 480, or Astral mass spectrometers.
• Data Analysis: Proteome Discoverer 3.0 (SEQUEST HT, 1% FDR) and DIA searches with CHIMERYS in Proteome Discoverer 3.1.

Main Results and Discussion

• The 96-well format achieved ≥ 90% depletion of the top 14 abundant proteins with < 5% CV, matching spin column performance.
• Protein and peptide identifications were nearly identical between plate and column formats in depleted plasma.
• Application to cerebrospinal fluid and human serum delivered 37–39% more protein IDs, demonstrating broad fluid compatibility.
• Label-free DIA analysis of 32 normal versus lung cancer plasma samples revealed key disease biomarkers: Mucin-5B (3× increase), Cadherin-5 (2× increase), and CD44 (1.5× increase), along with additional cardiovascular and oncology candidates.

Benefits and Practical Applications

• High throughput processing of 96 samples in parallel reduces hands-on time and accelerates large-cohort studies.
• Efficient depletion of abundant proteins enhances detection of low-abundance biomarkers.
• Versatile workflow compatible with plasma, serum, and CSF supports diverse proteomic applications in research and clinical laboratories.

Future Trends and Opportunities

Integration with automated liquid-handling platforms and expansion to additional biofluids will further streamline workflows. Coupling with multiplexed quantitative techniques and larger clinical cohorts will advance biomarker validation and translational applications.

Conclusion

The 96-well abundant protein depletion format delivers robust, high-throughput performance equivalent to spin columns while significantly increasing sample capacity and reproducibility. It represents a powerful tool for comprehensive plasma proteomics and efficient biomarker discovery.