Extending the Depth of Human Plasma Proteome Coverage Using Simple Fractionation Techniques

Area of Science:

• Biochemistry
• Proteomics
• Clinical Diagnostics

Background:

• Human plasma is vital for clinical analysis, but its proteomic complexity and wide dynamic range challenge biomarker discovery.
• Existing plasma proteomics workflows often lack throughput and sensitivity, hindering clinical applications.
• Efficient workflows are needed to achieve deep plasma proteome coverage with simplified sample preparation and high throughput.

Purpose of the Study:

• To compare different prefractionation techniques for human plasma proteomics.
• To identify workflows that balance proteome coverage, sample complexity, and throughput for clinical sample analysis.
• To evaluate the suitability of affinity depletion and prefractionation methods for large-scale clinical projects.

Main Methods:

• Intensive depletion of high-abundance proteins and enrichment of low-abundance proteins using Agilent multiple affinity removal LC columns (Hu6, Hu14) and ProteoMiner.
• Comparison of these methods with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and C18 prefractionation.
• Evaluation of proteome coverage, sample processing time, and parallel processing capabilities.

Main Results:

• One-dimensional gel-based prefractionation (1D-PAGE) enables parallel sample processing, maintaining proteome coverage compared to serial chromatography.
• 1D-PAGE significantly accelerates analysis time, which is critical for large clinical projects.
• Multiple methodologies can achieve high plasma proteome coverage, offering flexibility in method selection.

Conclusions:

• 1D-PAGE is an effective strategy for accelerating plasma proteomics analysis in clinical settings.
• Methodological flexibility exists for achieving high plasma proteome coverage, catering to specific project needs.
• Optimized plasma proteomics workflows are essential for advancing accurate diagnostics and population health screening.