Semester

Spring

Date of Graduation

2022

Document Type

Dissertation

Degree Type

PhD

College

Eberly College of Arts and Sciences

Department

Chemistry

Committee Chair

Lisa Holland

Committee Co-Chair

Stephen Valentine

Committee Member

Stephen Valentine

Committee Member

Peng Li

Committee Member

Harry Finklea

Committee Member

Werner Geldenhuys

Abstract

Abstract

Capillary Electrophoresis: Method Development and Application in Biomolecule Analysis

Lindsay Veltri

Biomolecule analysis is essential in disease diagnostics and the development of therapeutic agents. There is a vast array of methods available for biomolecule analysis, however, capillary electrophoresis is well-suited due to minimal sample consumption (~nanoliters of solution) and fast analysis times. In this report, capillary electrophoresis was used to analyze glycosylation of therapeutic proteins. Additionally, a capillary electrophoresis-mass spectrometry method is reported for the analysis of proteins.

Post-translation protein modifications like glycosylation, play an important in protein structure and function. Modifications of protein glycosylation can lead to improved therapeutic efficacy. Therefore, significant efforts are made into the development of biosimilars via glycoengineering. A capillary electrophoresis method is reported for the determination of the branch specificity of a transferase enzyme used in glycoengineering, β-1,4-galactosyltransferase. Enzyme specificity was studied using both a native and non-native sugar as the donor molecule. Identification of positional isomers was enabled using a semi-permanent nanogel capillary coating. This enabled analysis of rate constants for each step in the reaction.

Capillary electrophoresis-mass spectrometry is a powerful analytical tool for the analysis of biomolecules. However, coupling of the two techniques is not straightforward. Here, a novel, voltage-free interface for coupling capillary electrophoresis and mass spectrometry for the analysis of proteins is reported. Through the incorporation of a semi-permanent nanogel capillary coating, the limitations of bare-fused silica separations were overcome, and analyses of cationic proteins were performed at biological relevant pH values. The resulting charge state distributions determined proteins were not denatured, suggesting the method is an alternative method for native analyses.

Embargo Reason

Patent Pending

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