Mixed-mode ion exchange-based integrated proteomics technology for fast and deep plasma proteome profiling.

Area of Science:

• Proteomics
• Biomarker Discovery
• Analytical Chemistry

Background:

• Plasma proteome profiling is crucial for biomarker discovery using blood liquid biopsy.
• Conventional multi-step sample preparation can lead to protein degradation and analysis variability.
• Integrated sample preparation offers a promising solution to overcome these limitations.

Purpose of the Study:

• To develop a fully integrated proteomics sample preparation technology for rapid and deep plasma proteome profiling.
• To maintain native pH conditions throughout the sample preparation process to minimize protein degradation.
• To enhance efficiency and reproducibility in plasma biomarker analysis.

Main Methods:

• Developed a novel spintip device integrating all sample preparation steps: protein digestion and 2D fractionation (mixed-mode ion exchange and high-pH reversed phase).
• Utilized mixed-mode ion exchange beads for neutral pH sample loading and rapid (30 min) protein digestion.
• Processed 1 μL of plasma with high-abundance protein depletion, generating 12 fractions for LC-MS analysis with a 12-hour gradient.

Main Results:

• Achieved identification of 862 proteins from 1 μL plasma using the integrated technology.
• Demonstrated fast plasma proteome profiling in 2 hours with high identification overlap and quantification precision using Mixed-mode-SISPROT and data-independent MS.
• Successfully performed a proof-of-concept study on samples from 5 healthy donors.

Conclusions:

• The developed integrated technology enables fast and deep plasma proteome profiling while preserving sample integrity.
• Mixed-mode-SISPROT minimizes sample loss and protein degradation by avoiding pH changes.
• This technology is expected to become a broadly applicable tool for clinical plasma proteome profiling and biomarker discovery.