Date of Completion

12-12-2016

Embargo Period

12-5-2018

Keywords

Chemistry, Analytical Chemistry, Biological Chemistry, Mass Spectrometry, Lipids, Biomarker Discovery, Atherosclerosis, Phospholipase A2, Lipid 654, 18O-labeling

Major Advisor

Xudong Yao

Associate Advisor

Frank C Nichols

Associate Advisor

Christian Bruckner

Associate Advisor

Alfredo Angeles-Boza

Associate Advisor

Edward Neth

Field of Study

Chemistry

Degree

Doctor of Philosophy

Open Access

Open Access

Abstract

Diseases such as periodontal disease, chronic inflammatory bowel disease and autoimmune disease are reported to be associated with changes in the bacteria populations in specific locations of the body, particularly the gastrointestinal tract and the oral cavity. Because oral and gastrointestinal microbiomes do not typically invade tissues in the host with chronic inflammatory disease, considerable work has instead focused on virulence factors produced by these organisms and their role in triggering innate immune responses that may promote chronic inflammatory diseases. Toll-like receptors (TLRs) are innate immune receptors that recognize specific structural moieties shared among microbes of different classes. My work has concentrated on developing mass spectrometry (MS)-based technologies to study the structure-function characteristics of a subclass of complex lipids called lipopeptides that engage TLR2. A significant structure-activity relationship is controlling TLR2 binding for lipopeptides and the resulting activation of immune responses. Separation power of chiral liquid chromatography (cLC) and a diastereomeric mixture of isotopically labeled internal standards were utilized to develop a novel method of cLC stable isotope dilution multiple reaction monitoring MS (cLC-SID-MRM MS) to simultaneously determine structure and amount of stereoisomers of lipopeptides. In the second part of the study, similar MS-based approaches were applied to study the relationship between enzymatic hydrolysis of these lipopeptides and human disease. This work identified a novel function for phospholipase A2 (PLA2) enzymes. The combination of 18O-labeling and MS was used to investigate the hydrolysis reaction. Furthermore, cLC-MRM MS was utilized to investigate stereoselectivity of PLA2 enzymes for the hydrolysis of a specific class of lipopeptides. Overall, this work provides novel methods for structure elucidation and quantitation of bacteria-derived lipopeptides as well as their expression in specific human inflammatory diseases.

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