The analysis of proteins in biological fluids by capillary electrophoresis (CE) is of interest in clinical chemistry. However, due to low analyte concentrations and poor concentration limits of detection (CLOD), protein analysis by this technique is frequently challenging. Coupling preconcentration techniques with CE greatly improves the CLOD. An on-line preconcentration-CE method that can selectively preconcentrate any protein for which an antibody is available would be very useful for the analysis of low abundance proteins and would establish CE as a major tool in biomarker discovery. To accomplish this, an on-line protein G monolithic preconcentrator CE system for enrichment and separation of proteins was developed. This system proved effective for on-line sample extraction, clean-up, preconcentration, and CE of IgG in human serum. IgG from diluted (500 and 65,000 times) human serum samples was successfully analyzed using this system. The approach can be applied to the on-line preconcentration and analysis of any protein for which an antibody is available. The desire to separate all proteins present in human tissues, cells and biological fluids has challenged the separation research community for many years. The difficulty of this task resides in the complexity of the sample. Blood serum, for instance, may express up to 10,000 proteins with an estimated dynamic range of 9 orders of magnitude. Additionally, most of these proteins are present at very low concentrations (ng/mL). Identification and quantification of low abundance proteins is hindered by the presence of high abundance proteins, such as human serum albumin (HSA) and immunoglobulins (IgG). Therefore, in most cases, removal of the high abundance proteins or enrichment of low abundance proteins is necessary prior to the analysis of low abundance proteins. To address this, a coupled affinity-hydropobic monolithic column for the simultaneous removal of IgG, preconcentration of low abundance proteins, and separation by capillary zone electrophoresis was designed. The system proved to be very reproducible. The run-to-run %RSD values for migration time and peak area were less than 5%, which is typical of CE. Finally, a new method was developed to prepare monoliths with anion exchange functionality. Polymer monoliths were prepared by in situ polymerization of methacrylate monomers. The monoliths were coated with a water soluble polymer and used for the analysis of proteins. Using this approach, a model monolith was prepared. Subsequent coating yielded a monolith with quaternary ammonium groups on the surface, which was confirmed by strong anodic electroosmotic flow. Analysis of standard proteins by ion exchange LC and CEC was demonstrated. This simple and rapid method for surface modification opened new avenues for the preparation of monoliths with a broad range of functionalities.
College and Department
Physical and Mathematical Sciences; Chemistry and Biochemistry
BYU ScholarsArchive Citation
Armenta Blanco, Jenny Marcela, "Capillary Electrophoresis of Proteins with Selective On-line Affinity Monoliths" (2006). All Theses and Dissertations. 819.
Monolith, SPE, Preconcentration, Capillary electrophoresis, Protein G, IgG, Proteins