A structure based plasma protein pre-fractionation using conjoint immobilized metal/chelate affinity (IMA) system

Area of Science:

• Biochemistry
• Analytical Chemistry
• Proteomics

Background:

• Plasma protein analysis is complex due to high abundance proteins.
• Existing pre-fractionation methods can alter protein native states.
• Liquid chromatography-mass spectrometry (LC-MS) requires simplified samples for deeper proteome coverage.

Purpose of the Study:

• To investigate the potential of a continuous, conjoint immobilized metal/chelate affinity (IMA) approach for pre-fractionating native plasma proteins.
• To assess the efficacy of sequential metal-chelate disks in fractionating proteins based on surface histidine topography.
• To evaluate the combined IMA strategy for reducing plasma complexity prior to LC-MS analysis.

Main Methods:

• Developed a conjoint IMA system using iminodiacetic acid (IDA) coated Convective Interaction Media (CIM) disks with sequential transition metal ions (Co(II), Zn(II), Ni(II), Cu(II)).
• Ordered the IDA-metal disks based on hypothesized differential binding affinities related to histidine topography.
• Tested the system using four human plasma proteins (fibrinogen, IgG, transferrin, albumin) and whole human plasma.

Main Results:

• Successfully demonstrated structural pre-fractionation of plasma proteins based on native surface histidine topography.
• The conjoint IMA strategy effectively fractionated proteins in their native state.
• Identified 157 proteins from human plasma using the developed pre-fractionation method.
• Achieved protein fractionation and complexity reduction in a single step using a single buffer system.

Conclusions:

• The conjoint IMA approach offers a powerful tool for native protein pre-fractionation.
• This method significantly reduces plasma complexity, enabling deeper proteomic insights via LC-MS.
• The strategy preserves protein integrity, making it suitable for analyzing native protein structures and interactions.