Methods for the stereochemical analyses of endogenous cell-cell signaling peptides in animals

Mast, David Hamilton

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https://hdl.handle.net/2142/113949 Copy

Description

Title

Methods for the stereochemical analyses of endogenous cell-cell signaling peptides in animals

Author(s)

Mast, David Hamilton

Issue Date

2021-09-03

Director of Research (if dissertation) or Advisor (if thesis)

Sweedler, Jonathan` V

Doctoral Committee Chair(s)

Sweedler, Jonathan` V

Committee Member(s)

• Kraft, Mary L
• Leckband, Deborah E
• Yau, Peter M

Department of Study

Chemistry

Discipline

Chemistry

Degree Granting Institution

University of Illinois at Urbana-Champaign

Degree Name

Ph.D.

Degree Level

Dissertation

Date of Ingest

2022-04-29T21:42:53Z

Keyword(s)

Neuropeptides, Peptides, Isomerization, D-amino acids, Peptidomics, Stereochemistry, Mass spectrometry, liquid chromatography, chiral analysis

Abstract

D-amino acid containing peptides (DAACPs) represent an unusual class of signaling molecules in animals. Characterized by the presence of a non-proteinogenic D-amino acid residue in the backbone of an otherwise all-L residue peptide, DAACPs have fundamentally challenged the current understanding of animal protein chirality. DAACPs are considered uncommon because relatively few have been discovered in just a handful of species. However, it is possible many animal DAACPs have gone unidentified because modern peptide characterization studies do not routinely evaluate peptide stereochemistry. With increasing availability of genome/transcriptome-derived protein databases, and the widespread adaptation of liquid (LC)-chromatography-tandem mass spectrometry (MS/MS) sequencing, it is possible for hundreds to thousands of endogenous peptides to be identified in a single experiment. The reporting of endogenous peptide sequences without any stereochemical characterization has become widely accepted. The justification for this practice is premised on the overwhelming empirical evidence for a highly stereoselective ribosomal protein synthesis pathway in animals. D-amino acid residues can be incorporated into animal cell-cell peptides enzymatically, by a post-translational modification (PTM) known as L- to D-residue isomerization. Like most PTMs, predicting which substrates will be modified by L- to D-residue isomerization based solely on genomic information is challenging and therefore, the PTM must be measured directly. However, unlike most other PTMs, L- to D-residue isomerization is a zero Dalton mass change PTM that is not accompanied by the addition or subtraction of a function group, or a change in amino acid sequence. Consequently, peptides that undergo L- to D-residue isomerization can easily go undetected during LC-MS/MS sequencing because MS/MS on its own cannot be used for the ab initio determination of amino acid residue chirality. This dissertation asserts that the prevalence of post-translational L- to D-residue isomerization in animals has been underestimated and that many cell-cell signaling DAACPs in animals may remain undiscovered or have been overlooked by previous studies. An analytical framework was applied to search for cell-cell signaling DAACPs in Aplysia californica and Rattus norvegicus tissues. This dissertation describes the development and application of non-targeted methods to discover DAACPs among singling peptides in animals. These methods were used to discover eight enzyme derived cell-cell signaling DAACPs in the A. californica central nervous system. These DAACPs were encoded by protein precursors that had previously not been identified as encoding isomerized peptides. The tissue specific, and single cell localization of DAACPs was also investigated in A. californica to reveal important insights into the biosynthesis of DAACPs. In addition, a preliminary search for DAACPs, and other isomeric peptides, was performed on Rattus norvegicus neuro-endocrine tissues. The findings from screening R. norvegicus tissues for DAACPs were compared to the findings from A. californica, and the implications they have on future DAACP discovery efforts in animals are discussed.

Graduation Semester

2021-12

Type of Resource

Thesis

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http://hdl.handle.net/2142/113949

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Copyright 2021 David Mast

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