Date of Graduation

12-2018

Document Type

Dissertation

Degree Name

Doctor of Philosophy in Cell & Molecular Biology (PhD)

Degree Level

Graduate

Department

Biological Sciences

Advisor

Wesley E. Stites

Committee Member

Suresh K. Thallapuranam

Second Committee Member

Joshua Sakon

Third Committee Member

Ralph L. Henry

Keywords

Mass spectrometry, Proteome, Smoker

Abstract

Reactive oxygen species are naturally generated within the human body and they are known to modulate signaling pathway and mediate other physiological activities. However, excessive generation of ROS and the inability of body defense system in detoxifying them results in the so called “oxidative stress”. Methionine has powerful antioxidant properties due to the presence of electronegative sulfur in its structure. Therefore, Met is readily oxidized, and methionine sulfoxide has been linked to several pathological conditions.

The urinary proteome is an attractive candidate for the discovery of biomarkers to diagnose and classify health conditions because of the non-invasive collection procedure. However, protocols developed for the analysis of the urinary proteome have limitations both in identification of proteins and eliminating false positives.

Urine samples were collected from smoker and non-smokers participants. Proteins were concentrated and concentrates then were subjected to separation via SDS-PAGE gel electrophoresis technique. Gel was cut out into five sections including the band representing intact HSA. Gel sections then were digested with trypsin. Total proteins and levels of methionine sulfoxide in the resulting peptides were assessed by mass spectrometry. Data analysis was performed using Scaffold and Skyline software packages.

Correspondence with predicted retention times, MS/MS sequence information, and a minimum of two tryptic peptides were required to reduce false positives. Proteins were matched to 1023 UniProtKB identification numbers, 467 of which have not been previously reported in the human urinary proteome. Analysis of methionine sulfoxide proteome revealed 392 peptides mapped to 198 proteins that were detected with a significant fold change in smokers. Regions of gels with proteins of lower mass showed significant higher levels of oxidation in smokers compared to control, suggesting an association between oxidative caused by smoking and fragmentation of proteins.

Available for download on Thursday, July 11, 2019

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