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Title:Neuropeptidomics: MS-base peptide identification and quantitation study
Author(s):Yin, Ping
Director of Research:Sweedler, Jonathan V.
Doctoral Committee Chair(s):Sweedler, Jonathan V.
Doctoral Committee Member(s):Kelleher, Neil L.; Scheeline, Alexander; Yau, Peter M.
Department / Program:Chemistry
Degree Granting Institution:University of Illinois at Urbana-Champaign
liquid chromatography coupled to mass spectrometry (LC-MS)
Peptide characterization
Stable isotope labeling
Label-free quantitation
Abstract:Neuropeptidomics refers to a global characterization approach for the investigation of neuropeptides, often under specific physiological conditions. Neuropeptides comprise a complex set of cell-to-cell signaling molecules that are important in regulatory functions and behavior in the nervous system. Neuropeptide production typically involves endoproteolytic prohormone cleavage, exoproteolytic cleavage, and post-translational modifications by a variety of enzymes. Neuropeptides are packaged into and eventually released from the large dense-core vesicles in a regulated secretory pathway. Neuropeptidomics is inherently challenging because neuropeptides are spatially, temporally and chemically heterogeneous in the brain. In addition, the unconventional peptide precursor cleavages and various post-translational modifications require the unbiased measurements of actual peptide forms. Mass spectrometry (MS), coupled with optimized liquid chromatography separation and aided by appropriate sample preparation, has been successfully applied for peptide identification and quantitation. Neuropeptide production regulated by DIMM-dependent genetic mechanism and post-translational processing through alpha-amidation has been studied with different MS-based approaches such as matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS and electrospray ionization ion-trap (ESI-IT) MS. DIMM, a transcription factor in Drosophila, appears able to make non-peptidergic cells into peptidergic neurons. Qualitative MS approaches allowed us to observe the fully processed ectopic peptide MII in Drosophila non-peptidergic cells only when DIMM was present. Quantitative MS approaches, together with molecular biological methods, revealed DIMM enabled this complete and efficient ectopic peptide processing through regulating gene CG13248. In addition, an important post-translational modification, peptide alpha-amidation, was studied with mice of different genotypes on different copper diets. The results on the peptide level changes of mature peptides and their immature intermediate forms facilitate our understanding on peptide amidation reaction and its dedicated enzyme peptidylglycine alpha-amidating monooxygenase (PAM). Neurons and glia are two main types of cells in the central nervous system. Analogous to neuropeptidome, the knowledge of the gliopeptidome would aid our understanding of the interaction between glia and neurons. Therefore, the peptides in and regulatedly released from glia were examined. Peptides derived from proteins or putatively from neuropeptide precursors were identified with liquid chromatography coupled to mass spectrometry (LC-MS). Future studies would focus on the confirmation of the putatively identified peptides and the functions of identified peptides in glia. This dissertation demonstrates MS-based approaches are useful tools for probing peptides qualitatively and quantitatively, which would greatly expand our understanding of cell-to-cell communication in the nervous system.
Issue Date:2011-01-21
Rights Information:Copyright 2010 Ping Yin
Date Available in IDEALS:2011-01-21
Date Deposited:2010-12

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