Mass spectrometric analysis of signaling peptides in single neurons and their classification

Cell-to-cell signaling peptides (called neuropeptides when produced by neurons) are a diverse, multifunctional class of molecules which influence neuronal and nonneuronal tissues, affecting an array of physiological processes including reproduction, water and salt metabolism, temperature regulation, pain, appetitive state, and circadian rhythm. Neuropeptides are the post-translationally processed products of prohormone precursors. Prohormone expression, processed mature peptides, and peptide post-translational modification varies among neurons in the central nervous system (CNS). Adding to the chemical complexity is the vast size of the CNS. The human brain is estimated to contain 1 trillion cells and the rodent brain 80 million. This challenging chemical heterogeneity and vast size has meant that much current research into the CNS’ neuropeptide content is often based on the bulk analysis of cellular or tissue homogenates. These analyses average the chemical content of all cells and potentially dilutes analytes found in a small number of cells to concentrations below the limit of detection. Single cell methods are of interest as they detect analytes in their relatively high concentration in a single cell and reveal cellular populations and unique cells. This dissertation focuses on the development and application of a high-throughput, single cell matrix assisted laser desorption/ionization mass spectrometry (MALDI MS) method to characterize and classify single neurons based on their peptide content. We have sampled greater than 26,000 single neurons from the CNS of the neurobiological model organism Aplysia californica. This number of neurons is on the order of the entire CNS. The single neurons were grouped with a community detection algorithm to identify populations of neurons based on their peptide profiles. A higher mass accuracy and mass resolution MALDI Fourier transform ion cyclotron resonance (FT-ICR) MS method was developed and applied to single Aplysia abdominal neurons, allowing for the interrogation of lower mass-to-charge ratios (m/z) which were previously inaccessible due to isobaric lipid species. Several methods for the targeted analysis of prohormones of interest were further investigated alongside methods to add confidence to the mass matching assignment of mass spectral peaks to peptides. Lastly, we applied the high-throughput MALDI MS method to another neurobiological model organism, Pleurobranchaea californica. Pleurobranchaea is useful for the study the neuronal basis of decision making, such as its predatory behavior, but its neuropeptidome is less characterized than Aplysia. Several putative, homologous prohormones had their predicted peptides colocalize in single neurons, adding confidence to their peptide and prohormone assignment.

Name thesis:degree_name

Ph.D.

Level thesis:degree_level

Dissertation

Discipline thesis:degree_discipline

Chemistry

Grantor

University of Illinois at Urbana-Champaign

Year dc:date

2022

Author dc:creator

• Chan-Andersen, Peter Christian

Contributors dc:contributor

• Sweedler, Jonathan V
• Gewirth, Andrew A
• Gillette, Rhanor
• Kraft, Mary L

• Mass spectrometry
• MALDI
• Aplysia
• single cell
• clustering
• Pleurobranchaea
• neuron
• neuropeptide
• peptide
• signaling peptide
• mass spectrometry imaging

Statement dc:rights

• Copyright 2022 Peter Chan-Andersen

Language dc:language

en, eng

Handle dc:identifier

https://hdl.handle.net/2142/117515

OAI record oai:identifier

https://www.ideals.illinois.edu/oai-pmh?verb=GetRecord&metadataPrefix=etdms&identifier=oai%3Awww.ideals.illinois.edu%3A2142%2F117515