A fully integrated multi-column system for abundant protein depletion from serum/plasma.

Area of Science:

• Biochemistry and Proteomics
• Analytical Chemistry
• Biotechnology

Background:

• Serum and plasma protein depletion is crucial for deep proteome analysis.
• Conventional methods can be time-consuming and require manual intervention.
• Automated, low-pressure systems offer potential for increased throughput and reproducibility.

Purpose of the Study:

• To detail the modification of a conventional HPLC system into a low back pressure liquid chromatography setup.
• To automate serum/plasma depletion and fractionation processes.
• To validate the performance and reproducibility of the modified system.

Main Methods:

• A Dionex U3000 HPLC system was modified for low back pressure operation (≤125 psi) using larger inner-diameter tubing.
• The system was configured with two immunoaffinity columns (Seppro IgY14 and Seppro SuperMix) for sequential protein depletion.
• An autosampler was utilized for automated injection, flow-through collection, and bound protein fractionation.

Main Results:

• The modified system successfully depleted the top 14 and the next 200-300 most abundant proteins from serum/plasma.
• Automated processing allowed for consecutive depletion of three samples before user intervention, with up to nine samples processed in 24 hours.
• Validation with 90 patient samples showed overall coefficients of variation (CVs) within 95% confidence intervals for 86 samples, indicating high performance and reproducibility.

Conclusions:

• The conversion of a conventional HPLC to a low back pressure system provides an effective automated solution for serum/plasma protein depletion and fractionation.
• The validated system demonstrates excellent performance and reproducibility, suitable for high-throughput proteomic studies.
• This approach enhances efficiency and reliability in preparing biological samples for in-depth proteomic analysis.