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Multiplexing Techniques in Quantitative Proteomics to Study Disease

Evans, Adam (2015) Multiplexing Techniques in Quantitative Proteomics to Study Disease. Doctoral Dissertation, University of Pittsburgh. (Unpublished)

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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:
CreatorsEmailPitt UsernameORCID
Evans, Adamare11@pitt.eduARE11
ETD Committee:
TitleMemberEmail AddressPitt UsernameORCID
Committee ChairRobinson, Renãrena@pitt.eduRENA
Committee MemberWeber, Stephen Gsweber@pitt.eduSWEBER
Committee MemberMichael, Adrianamichael@pitt.eduAMICHAEL
Committee MemberFreeman, Bruce A.freerad@pitt.eduFREERAD
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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