Gu, Liqing
(2016)
DEVELOPMENT OF PROTEOMICS APPROACHES TOWARDS CHARACTERIZING OXIDATIVE MODIFICATIONS.
Doctoral Dissertation, University of Pittsburgh.
(Unpublished)
Abstract
Mass spectrometry (MS) is an analytical technique allowing the investigation of a single protein or the entire complement of proteins from biomatrices for understanding attributes such as sequences, modifications, structures, abundances and interactions. Protein oxidative modifications, such as carbonylation and cysteine reversible oxidations, have important roles in physiological processes, including redox signaling, homeostasis, enzymatic catalysis and protein degradation. MS-based redox proteomics can identify and quantify oxidized protein modifications within the proteome. However it is challenging to globally investigate cysteine reversible modifications, due to the low abundance (~ < 1%) and diversity (e.g., S-nitrosylation, S-glutathionylation, sulfenic acid, disulfide bonds) of these modifications. Novel proteomics approaches are needed to better understand cysteine-related redox signaling and oxidative stress in disease.
This dissertation presents studies of protein oxidative modifications using MS-based approaches. First, proteomics methodologies to study protein carbonylation and cysteine reversible modifications are reviewed, including the relevant applications in neurodegenerative disease. Next, a MS-based characterization of a whole protein is described by studying oxidative modifications generated through treatment of a model protein with oxidants. Novel methods towards characterizing endogenous cysteine oxidations in disease are then presented, including inexpensive and high-throughput approaches. The first approach utilizes low-cost isotopic dimethyl peptide labeling for comparing two proteome samples. This methodology has the ability to isolate and quantify total cysteinyl peptides or oxidized cysteinyl peptides from complex samples, and is employed to characterize the liver proteome of an Alzheimer’s disease (AD) mouse model. The second approach is cysteine-selective combined precursor isotopic labeling and isobaric tagging (cysteine-selective cPILOT), which incorporates isobaric tags to achieve 12-plex multiplexing capability. Cysteine-selective cPILOT is used to isolate total cysteinyl peptides from liver proteins and S-nitrosylated peptides from brain proteins of an AD mouse model. Overall the novel proteomics approaches developed herein lower experimental costs and improve the throughput of cysteine redox proteomics studies.
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Details
| Item Type: |
University of Pittsburgh ETD
|
| Status: |
Unpublished |
| Creators/Authors: |
|
| ETD Committee: |
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| Date: |
29 September 2016 |
| Date Type: |
Publication |
| Defense Date: |
18 April 2016 |
| Approval Date: |
29 September 2016 |
| Submission Date: |
20 April 2016 |
| Access Restriction: |
No restriction; Release the ETD for access worldwide immediately. |
| Number of Pages: |
221 |
| Institution: |
University of Pittsburgh |
| Schools and Programs: |
Dietrich School of Arts and Sciences > Chemistry |
| Degree: |
PhD - Doctor of Philosophy |
| Thesis Type: |
Doctoral Dissertation |
| Refereed: |
Yes |
| Uncontrolled Keywords: |
Quantitative Proteomics; Redox Proteomics; Oxidative Modifications |
| Date Deposited: |
30 Sep 2016 01:38 |
| Last Modified: |
15 Nov 2016 14:33 |
| URI: |
http://d-scholarship.pitt.edu/id/eprint/27770 |
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