A Simplified Perchloric Acid Workflow With Neutralization (PCA N) for Democratizing Deep Plasma Proteomics at...

Area of Science:

• Biochemistry
• Proteomics
• Biomarker Discovery

Background:

• Large-scale plasma proteomics is crucial for biomarker discovery but faces challenges in depth, throughput, and cost.
• Existing methods struggle to balance analytical capabilities with economic feasibility for population-level studies.

Purpose of the Study:

• To present an optimized perchloric acid-based workflow (PCA-N) for plasma proteomics.
• To address limitations in depth, throughput, and cost-effectiveness for large-scale studies.
• To enable deep plasma proteome coverage with minimal sample volume.

Main Methods:

• Developed a perchloric acid-based workflow with a neutralization step (PCA-N) after protein precipitation.
• Enabled direct enzymatic digestion without additional purification, reducing plasma input to 5 μl.
• Validated the workflow using CLSI C64 guidelines and analyzed over 40,000 samples.

Main Results:

• PCA-N achieved deep plasma proteome coverage with only 5 μl of plasma.
• The workflow demonstrated high throughput (over 10,000 samples/day) at costs comparable to NEAT.
• PCA-N doubled proteomic depth compared to NEAT while maintaining excellent biological resolution and reproducibility.
• Workflow stability was confirmed over 353 days with consistent performance across instruments and batches.

Conclusions:

• PCA-N offers an optimized, cost-effective, and scalable solution for large-scale plasma proteomics.
• The workflow democratizes access to deep proteomic analysis for population studies.
• Minimal sample requirements and operational simplicity make PCA-N highly attractive for biomarker discovery.