Date of Completion

5-26-2015

Embargo Period

5-25-2017

Keywords

mass spectrometry, reversed-footprinting, structural marker peptides, CFTR, lipidomics, Lipid 654

Major Advisor

Xudong Yao

Associate Advisor

Frank C. Nichols

Associate Advisor

C. Vijay Kumar

Associate Advisor

Christian Brϋckner

Associate Advisor

Edward J. Neth

Field of Study

Chemistry

Degree

Doctor of Philosophy

Open Access

Campus Access

Abstract

Mass spectrometry together with fast photochemical oxidation of proteins was utilized to study structure of cystic fibrosis transmembrane conductance regulator (CFTR), an important membrane transporter model in a “reversed-footprinting” approach. Mass spectrometry was also utilized for qualitative and quantitative study of lipids. First, an experiment was designed to covalently tag the structure of CFTR on the cell membrane to footprint the solvent accessible surface area and subsequently measure the degree of covalent oxidative modifications on distinct proteolytic peptides with targeted mass spectrometry on unmodified peptides. This work strives to introduce a method that mitigates difficulties in structural analysis of highly challenging molecules i.e. integrated membrane proteins. In second part, tandem mass spectrometry was utilized as a complimentary method to NMR for the analysis of lipids in two aspects of structural characterization and reliable quantification in complex clinical samples. The pillar of both of the protein and lipid analyses is targeted mass spectrometry that provides a sensitive and selective analysis to achieve these goals.

First, for CFTR, a workflow was designed to conduct the study on cells in order to keep the native conformation of the protein. Merits of fast photochemical oxidation backed with a targeted mass spectrometry quantitation workflow helped to introduce a number of peptides on CFTR as potential “structural markers peptides” that can help for screening the biological and functional states of protein.

In the second part of the dissertation, liquid chromatography and tandem mass spectrometry (LC-MS/MS) was utilized as a complimentary method to NMR and GC-MS to characterize the structure of biologically active lipids from commensal bacteria origin, termed as Lipid 654 and Lipid 430. Screening of Lipid 654 fragmentation pattern using MS/MS helped to facilitate the structural elucidation. Also a targeted stable isotope dilution (SID) MS method was developed to quantify Lipid 654 in processed serum samples collected from patients with multiple sclerosis and Alzheimer’s as well as normal healthy volunteers to investigate for the clinical relevance of Lipid 654 as a potential blood biomarker. Results showed that with a statistical significance, the level of Lipid 654 was lower in samples from multiple sclerosis patients compared to patients with Alzheimer’s or healthy volunteers.

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