ORCID Identifier(s)

0009-0003-8334-3438

Graduation Semester and Year

Fall 2025

Language

English

Document Type

Dissertation

Degree Name

Doctor of Philosophy in Chemistry

Department

Chemistry and Biochemistry

First Advisor

Daniel W. Armstrong

Second Advisor

Purnendu K. Dasgupta

Third Advisor

Morteza G. Khaledi

Fourth Advisor

Kayunta L. Johnson-Winters

Abstract

Peptide epimerization and isomerization are spontaneous or enzymatically catalyzed post-translational modifications that alter stereochemistry without changing mass, making them analytically elusive yet biologically consequential. This dissertation investigates the analytical, structural, and biological consequences of peptide stereochemical modifications, using amyloid-β (Aβ) peptides, implicated in Alzheimer’s disease (AD), as a model system.

First, a basic ammonium hydroxide liquid chromatography method was developed, demonstrating enhanced separation and altered retention order of peptide epimers and isomers compared to traditional acidic systems, with significantly improved mass spectrometric sensitivity. Subsequent studies revealed that stereochemical variations in Aβ tryptic fragments profoundly affect peptide ionization efficiency and fragmentation behavior, underscoring potential quantitative biases in mass spectrometric analyses that overlook stereochemistry.

To understand biological persistence, carboxypeptidase Y kinetics were examined with L- and D-amino-acid-containing peptides. The enzyme exhibited strong stereoselectivity, hydrolyzing D-amino-acid peptides several orders of magnitude slower than their all-L counterparts, suggesting a mechanism for the age-related accumulation of D-amino-acid-containing peptides. Structural studies using circular dichroism (CD) and dynamic light scattering (DLS) revealed that Aβ1–42 epimers and isomers follow distinct secondary structure and aggregation kinetics, ranging from rapid first-order to delayed sigmoidal and zero-order behaviors. Specific modifications, such as D-Ser8 and L-isoAsp7, accelerated β-sheet formation, while others, like D-Ser26 and D-isoAsp23, inhibited β-sheet formation entirely.

Finally, immunoprecipitation–mass spectrometry assays showed that common anti-Aβ antibodies display variable binding affinities toward modified Aβ variants, with N-terminal epimerization often reducing recognition. These findings have critical implications for diagnostic assays and immunotherapeutic design.

Together, this work integrates analytical and bioanalytical techniques to reveal how epimerization and isomerization modulate peptide behavior across scales, from chromatographic and mass spectrometric detectability to enzymatic degradation, aggregation kinetics, and antibody binding. The results establish a mechanistic framework linking molecular chirality to peptide stability, folding, and therapeutic targeting in Alzheimer’s disease.

Keywords

Peptide, Alzheimer's Disease, Epimer, Isomer, Beta Amyloid, Mass Spectrometery, Circular Dichroism, Liquid Chromatography, Antibody

Disciplines

Amino Acids, Peptides, and Proteins | Analytical Chemistry | Biochemistry | Enzymes and Coenzymes

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