Evans, Adam
(2015)
Multiplexing Techniques in Quantitative Proteomics to Study Disease.
Doctoral Dissertation, University of Pittsburgh.
(Unpublished)
Abstract
Proteomics is the large scale study of a set of proteins from a biological species to understand protein expression, post translation modifications, and protein-protein interactions. Mass spectrometry (MS)-based proteomics allows large scale protein identification and quantitation in the same experiment. Quantitative proteomics reports abundance changes between multiple protein samples, which principally reflect a biological process or disease state. Relative quantitation can be achieved using stable isotope chemical labeling of proteins or peptides using MS or tandem mass spectrometry (MS/MS). Sample throughput is limited by the chemical tag and technique. MS-based protein quantitation employs methods which generate a mass shift in MS to compare abundance changes between two to three samples. An example of precursor quantitation is described using acetylation to study the spleen proteome of mice treated with Adriamycin. Isobaric tags, such as tandem mass tags (TMT), achieve relative quantitation of up to ten samples in MS/MS. In order to analyze additional biological samples beyond ten, multiple experiments must be performed separately, which leads to increased instrument time, higher cost, and variation due to additional preparation steps, sample handling, and MS injections.
Herein, novel methods which enhance multiplexing in quantitative proteomics beyond the current limitations are presented. Enhanced multiplexing is achieved by combining precursor MS labeling with isobaric tags, which is termed “combined precursor isotopic labeling and isobaric tagging” (cPILOT). Initially, cPILOT is used to identify and quantify 3-nitrotyrosine (3NT) containing proteins. The biological significance and proteomic techniques employed to study 3NT are reviewed. cPILOT is expanded to a global strategy by coupling low pH dimethylation with TMT. Global cPILOT was applied to study the liver proteome of an Alzheimer’s disease mouse model, which revealed alterations in metabolism. Finally, the versatility of cPILOT is demonstrated by incorporating N,N-dimethyl Leucine isobaric tags. Overall, the work presented throughout the dissertation highlights novel strategies to enhance multiplexing in quantitative proteomics which are applied to study various diseases.
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Details
| Item Type: |
University of Pittsburgh ETD
|
| Status: |
Unpublished |
| Creators/Authors: |
|
| ETD Committee: |
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| Date: |
13 January 2015 |
| Date Type: |
Publication |
| Defense Date: |
1 December 2014 |
| Approval Date: |
13 January 2015 |
| Submission Date: |
3 December 2014 |
| Access Restriction: |
No restriction; Release the ETD for access worldwide immediately. |
| Number of Pages: |
204 |
| 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, cPILOT, multiplexing, Alzheimer's Disease |
| Additional Information: |
Supplemental tables will be uploaded with the final draft |
| Date Deposited: |
13 Jan 2015 15:06 |
| Last Modified: |
15 Nov 2016 14:25 |
| URI: |
http://d-scholarship.pitt.edu/id/eprint/23755 |
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