University of Illinois Urbana-Champaign

Mass spectrometry-based identification and quantitation of endogenous peptides in complex biological matrices

Dowd, Sarah E

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DOWD-DISSERTATION-2015.pdf

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https://hdl.handle.net/2142/89278

Description

Title
Mass spectrometry-based identification and quantitation of endogenous peptides in complex biological matrices
Author(s)
Dowd, Sarah E
Issue Date
2015-10-14
Director of Research (if dissertation) or Advisor (if thesis)
Sweedler, Jonathan V
Doctoral Committee Chair(s)
Sweedler, Jonathan V
Committee Member(s)
  • Bailey, Ryan C
  • Rhodes, Justin S
  • Rodríguez-López, Joaquín
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
2016-03-08T17:21:47Z
Keyword(s)
  • mass spectrometry
  • peptides
  • neuropeptides
  • quantitative mass spectrometry
Abstract
In the brain, chemical signaling molecules transfer information from cell-to-cell to modulate mood, behavior, and physiological functions like metabolism and circadian rhythm. The research presented in this dissertation is focused on characterizing a specific class of signaling molecules, endogenous signaling peptides. Despite being gene products, this class of signaling molecules is complex and diverse. Cell to cell signaling peptides are differentially processed, modified, and distributed depending on time and spatial location. The detection of the peptide complement in a biological sample is an analytical challenge due to the broad range of concentrations and diverse chemical properties of endogenous peptides. The presence of other classes of molecules, like salts and lipids, add to sample complexity and further complicates the characterization of signaling peptides. The ability to characterize endogenous peptides is dependent on the selection of appropriate, information-rich tools. In this dissertation, a set of mass spectrometry approaches coupled with optimized liquid chromatography separations and appropriate sample preparation were successfully utilized to measure chemical information for peptides in biological samples. Multiple MS platforms, each with distinct advantages and limitations, were utilized for identification and quantitation. To gain insight into the role of signaling peptides in a specific behavior, relative quantitation measurements with MS were performed in a non-targeted approach. Specifically, measurements were made for peptide level changes in the hippocampus and amygdala of mice to study the extinction of conditioned place preference (CPP) for cocaine in mice that have access to a running wheel after CPP training. MS measurements have yielded both known and novel candidate peptides that show relative abundance changes with exercise and context re-exposure, which yields insight into the biochemical mechanisms driving the observed extinguishment of CPP with running. Successful MS measurements of endogenous peptide measurements are dependent on appropriate and optimized sample preparation procedures. An optimized sampling procedure was developed and applied to detect the novel peptide alarin in the supernatant of a transfected cell line. While a synthetic version of the peptide spiked into the medium was detected by MS, the endogenous form of the peptide expressed by the cell line could not be unambiguously identified. Additionally, MS measurements were employed to evaluate the effects of different sample handling treatments on the detection of peptides in the mouse striatum and hypothalamus. The results of this preliminary study indicate for the sample treatments used, each has its limitations and the selection of an appropriate sample treatment in biological studies is dependent on the study goals and resources. This demonstrates the capabilities of mass spectrometric approaches to investigate peptide identities and dynamics, which can be utilized to enhance our understanding of the role of peptides in cell-to-cell signaling in the nervous system.
Graduation Semester
2015-12
Type of Resource
text
Permalink
http://hdl.handle.net/2142/89278
Copyright and License Information
Copyright 2015 Sarah Dowd

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