Date of Graduation
Eberly College of Arts and Sciences
Lisa A Holland
Protein glycosylation is of interest in a number of emerging fields, and plays important functional roles in cellular biology. The analysis of these molecules is nontrivial because they exhibit great complexity in both monomer makeup and linkages of these monomers. As a result, a common challenge that analytical techniques face is the separation of linkage glycan isomers. This dissertation is based on the research leading to the development of a phospholipid-capillary electrophoresis method that allows for highly efficient separations and the non-covalent incorporation of enzymes and lectins for in-capillary interactions. Glycans were removed from glycoproteins through enzymatic means, then labeled with the fluorescent tag 9- aminopyrene-1,3,6-trisulfonic acid. This label provides charge to the otherwise neutral glycan, as well as the very low limits of detection (15fM) that laser induced fluorescence detection affords. Separations efficiencies, based on hydrodynamic volume, were as high as 640,000 theoretical plates. These methods not only provide superior separations efficiencies, but also crucial structural information about the glycans themselves. This method is amenable for a wide variety of lectins/enzymes for use, and has been conducted in a number of different capillary inner diameters. The method was used to probe glycans solutions taken from MCF7 immortalized breast cancer cells, as well as glycans from the therapeutic antibody, Trastuzumab. Only microgram amounts of these proteins were need to provide the glycans necessary for analysis. These samples display a number of high and low abundance glycans that were well resolved. The identity of these glycans were confirmed with the use of glycan standards as well as a number of monomer and linkage-specific enzymes and lectins. Multiple enzymes may be used singly, or in tandem, to systematically remove glycan monomers and discern layers of information about the glycans in use.
Archer-Hartmann, Stephanie A., "Glycan characterization and manipulation using phospholipid additives for capillary electrophoresis" (2012). Graduate Theses, Dissertations, and Problem Reports. 4826.