Date of Graduation
Eberly College of Arts and Sciences
Lisa A. Holland
Harry O. Finklea
Harry O. Finklea
Justin A. Legleiter
Stephen J. Valentine
Protein glycosylation is one of the most prevalent post-translational modifications which plays an important role in determining the structure, stability, and function of proteins. Changes in glycosylation patterns are a hallmark of cancer and other diseases. Characterizing glycosylation is difficult because these structures vary in their linkage and monomer sequence, which gives rise to vast microheterogenity that could affect the function of the molecule. A novel method using capillary gel electrophoresis by integrating microscale enzymes and lectin reactions in the capillary was developed to verify glycan structure by analyzing monosaccharides. Capillary electrophoresis (CE) improves upon conventional methods of glycan structure characterization by reducing the amount of enzyme or lectins from milliliters to nanoliters and incubation times needed for enzyme reactions from hours to minutes. The significant advancement that was found is to use nanoliter volumes of enzymes costing about $ 0.01 for a single analysis and shorter incubation times.
Characterizing glycosylation for complex glycans was demonstrated using enzymes alone or a combination of enzymes and lectins with aid of a nanogel to determine specific signatures in glycoproteins. Nanogels are used to increase enzyme stability, and non-covalently pattern enzymes in the capillary, enabling more cost-effective use of biological reagents that provide insight into glycan structures. This advancement allows for in-capillary electrophoretic mixing of a substrate and an enzyme to achieve similar rates irrespective of incubation times. These findings are important to study the Michaelis-Menten constants of two enzymes obtaining different catalysis rates. With the knowledge of rates, a cheaper enzyme was used instead of an expensive enzyme to obtain similar information, deciphering glycan linkage and composition in a cost-effective manner. The identification of type-1-LacNac signature in glycoproteins was demonstrated with the use of an enzyme and lectin combination. The enzymes and lectins can be used individually, or in combination, to identify the monomer and linkage information in glycans.
Gattu, Srikanth, "INTEGRATING MICROSCALE ENZYME AND LECTIN REACTIONS USING NANOGEL ASSISTED CAPILLARY ELECTROPHORESIS" (2018). Graduate Theses, Dissertations, and Problem Reports. 3693.