Characterization and spatial mapping of isomerized residues in neuropeptides

The discovery and characterization of post-translational modifications (PTMs) in neuroendocrine peptides, particularly those that result in peptide diastereomers and positional isomers, such as D-amino acid incorporation, isoaspartate (isoAsp) formation, and differential covalent modifications, remain significantly limited by the lack of comprehensive analytical and -computational methodologies. Neuroendocrine peptides regulate a wide range of physiological processes, including neuromodulation, energy balance, and homeostasis. Consequently, even subtle chemical alterations can profoundly impact their stability, bioavailability, and function. For instance, the presence of D-amino acid residues is known to enhance peptide stability and receptor potency. At the same time, the formation of isoAsp has been associated with increased fibrillization, potentially facilitating peptide storage or contributing to cytotoxicity. To advance our understanding of these structural variants, often referred to as “zero-Dalton” modifications due to their mass equivalence, it is essential to develop robust tools for their detection and characterization. This dissertation presents a comprehensive suite of bioanalytical, computational, and biochemical techniques for the untargeted discovery and site-specific characterization of D-amino acid- and IsoAsp-containing neuropeptides. Additionally, a mass spectrometry imaging (MSI) method based on matrix-assisted laser desorption/ionization trapped ion mobility spectrometry (MALDI-TIMS-MSI) is introduced, enabling spatial mapping of neuropeptides and their isomers at single-cell resolution. Application of these integrated methods led to the discovery of IsoAsp-containing forms in multiple hypothalamic peptides from rat brain, including galanin, and peptides derived from proenkephalin, melanin-concentrating hormone, secretogranin, and pituitary adenylate cyclase-activating peptide (PACAP) prohormones. Furthermore, three novel D-amino acid-containing peptides were identified from the nervous system of A. californica. These methods also enabled the identification and structural characterization of diacetylated and triacetylated forms of α-melanocyte-stimulating hormone (α-MSH) in both rat and human pituitary tissue. These acetylation variants were found to exhibit species-specific patterns and region-specific localization, offering new insights into their potential physiological roles. The work presented in this dissertation advances the field of peptidomics by providing comprehensive tools for probing subtle yet functionally important modifications.

Name thesis:degree_name

Ph.D.

Level thesis:degree_level

Dissertation

Discipline thesis:degree_discipline

Chemistry

Grantor

University of Illinois Urbana-Champaign

Year dc:date

2025

Author dc:creator

• Okyem, Samuel

Contributors dc:contributor

• Sweedler, Jonathan V.
• Sweedler, Jonathan V
• Gillette, Martha
• Shen, Mei
• Romanova, Elena V.

• Neuropeptides
• Isomers
• Mass Spectrometry
• Galanin
• Isoaspartate

Statement dc:rights

• Copyright 2025 Samuel Okyem

Language dc:language

en, eng

Handle dc:identifier

https://hdl.handle.net/2142/130090

OAI record oai:identifier

https://www.ideals.illinois.edu/oai-pmh?verb=GetRecord&metadataPrefix=etdms&identifier=oai%3Awww.ideals.illinois.edu%3A2142%2F130090