Characterization and spatial mapping of isomerized residues in neuropeptides
Okyem, Samuel
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https://hdl.handle.net/2142/130090
Description
Title
Characterization and spatial mapping of isomerized residues in neuropeptides
Author(s)
Okyem, Samuel
Issue Date
2025-07-10
Director of Research (if dissertation) or Advisor (if thesis)
Sweedler, Jonathan V
Doctoral Committee Chair(s)
Sweedler, Jonathan V
Committee Member(s)
Gillette, Martha
Shen, Mei
Romanova, Elena V
Department of Study
Chemistry
Discipline
Chemistry
Degree Granting Institution
University of Illinois Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Neuropeptides, Isomers, Mass spectrometry, Galanin, Isoaspartate
Abstract
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.
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