Assessing Surface Binding Events on Golgi Microsomes Isolated from Cancer Cells by Capillary Electrophoresis

Area of Science:

• Biochemistry
• Molecular Biology
• Pharmacology

Background:

• Intracellular membrane proteins are vital drug targets, but studying them in their native cancer cell environment is difficult.
• Existing methods for analyzing membrane proteins are often complex and require specific conditions.

Purpose of the Study:

• To develop a novel method for direct affinity measurements of intracellular membrane proteins on cancer cell-derived Golgi microsomes.
• To overcome limitations of current techniques, enabling analysis without target modification or labeled probes.

Main Methods:

• Integration of affinity capillary electrophoresis (ACE) with a new mathematical model.
• Utilizing a microfluidic format for minimal sample consumption and flexible detection.
• Application to UDP-glucose ceramide glucuronosyltransferase (UGCG), a cancer-relevant Golgi protein.

Main Results:

• Achieved the first direct affinity measurements on native Golgi microsomes from cancer cells.
• Demonstrated quantitative ligand-binding assays on vesicular fractions preserving cellular architecture.
• Validated the method's applicability to impure compounds and ligands lacking charge or UV absorbance.

Conclusions:

• Established a robust and versatile platform for target engagement studies in complex biological systems.
• ACE shows significant potential for accelerating drug discovery for intracellular and extracellular membrane-bound targets.
• This method facilitates the characterization of novel therapeutic targets within their native cellular context.