Quantifying Biomolecular Interactions in High-Conductivity Samples With Capillary Electrophoresis

Area of Science:

• Analytical Chemistry
• Biophysical Chemistry
• Biomolecular Interactions

Background:

• Capillary electrophoresis (CE) is a versatile technique for biomolecular interaction studies.
• High sample buffer conductivity relative to background electrolyte (BGE) causes peak distortions, impacting accuracy in binding assays.
• Challenges include affinity probe CE and nonequilibrium CE of equilibrium mixtures.

Purpose of the Study:

• To investigate the impact of conductivity mismatches on CE separation of aptamer-protein interactions.
• To develop strategies for accurate quantification under nonideal conductivity conditions.
• To propose an alternative quantification method when traditional CE separation fails.

Main Methods:

• Combination of simulation and experimental approaches.
• Focus on aptamer-protein interactions, specifically conductivity mismatches.
• Analysis of peak distortions (splitting, broadening) and development of artifact exclusion.
• Proposal and validation of a "de-stacked" fraction quantification method.

Main Results:

• Moderate conductivity mismatches caused peak splitting; large mismatches resulted in broad, indistinct peaks.
• Simulations indicated analyte ions trapped in high-conductivity plugs exacerbate artifacts, especially with longer injections.
• Reducing plug length and excluding artifact peaks improved quantification.
• The alternative method yielded dissociation constant (K_d) and Hill coefficient (n) values comparable to fluorescence anisotropy.

Conclusions:

• Practical guidelines and analytical strategies are provided for accurate quantification in CE under nonideal conductivity.
• The proposed "de-stacked" fraction method offers a viable alternative when traditional CE fails.
• This work expands the utility of CE for bioanalytical research involving biomolecular binding.