High-Efficient and Controllable Purification of Protein by Cyclic Injection in an Orthogonal Micro-Free-Flow...

Area of Science:

• Biotechnology
• Analytical Chemistry
• Biochemistry

Background:

• High-abundance proteins (HAPs) in plasma pose significant challenges for purifying low-abundance proteins (LAPs).
• Existing methods often struggle with efficient separation and purification of target proteins from complex biological matrices.
• Developing advanced separation techniques is crucial for proteomics and diagnostics.

Purpose of the Study:

• To develop and validate a novel orthogonality microfluidic free-flow electrophoresis (OMFFE) platform for selective protein purification.
• To demonstrate the platform's capability in removing HAPs and recovering LAPs from plasma.
• To assess the efficiency and purity achieved by the OMFFE device.

Main Methods:

• A custom-built OMFFE device was designed, incorporating focusing and orthogonal separation channels with voltage-controlled electrodes.
• Scan voltage mode was used to determine optimal separation voltages based on protein deflection angles.
• Selective voltage mode was employed for targeted deflection and separation of proteins based on mass-to-charge ratio.
• Cyclic injection was utilized for continuous protein purification.

Main Results:

• The OMFFE device achieved optimal resolution (Rs = 0.37) between HAPs (HSA, IgG) and LAP (GFP) at 75 V.
• High removal rates of 94.7 ± 4.1% for HSA and IgG, and a recovery rate of 95.3 ± 3.7% for GFP were obtained.
• GFP purity increased approximately 32-fold, with in-plasma removal rates of 83.2 ± 3.9% for HAPs and recovery rates of 76.3 ± 7.5% for LAP.
• The device demonstrated effective HAP removal and LAP purification in human plasma.

Conclusions:

• The OMFFE platform offers a unique and effective solution for separating and purifying proteins from complex samples.
• The device demonstrates high efficiency in removing interfering HAPs and recovering target LAPs.
• This technology shows significant potential for applications in proteomics, protein function analysis, and medical diagnostics.