Date of Award

2015

Embargo Period

8-1-2024

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Cell and Molecular Pharmacology and Experimental Therapeutics

College

College of Graduate Studies

First Advisor

Richard Drake

Second Advisor

Lauren Ball

Third Advisor

Robert Gemmill

Fourth Advisor

Jennifer Isaacs

Fifth Advisor

Michael Janech

Abstract

Non-small cell lung cancer represents 85% of all lung cancers with an average 5 year life expectancy of 15-20%. A wealth of data suggests that altered glycosylation contributes to the progression of these tumors and efforts to improve the specificity of biomarkers have logically shifted towards glycoproteomic investigations. For this purpose, recent innovations in experimental strategies and analytical techniques could be combined to provide a more detailed characterization of glycans than previously achievable. These new methods have not yet been assessed for feasibility in experimental procedures useful for discovery phase efforts. Therefore, we aimed to investigate the utility of novel glycoproteomic and glycomic approaches for defining alterations in glycosylation which may accompany disease progression. To this end, we implemented an azido-sugar metabolic labeling, alkyne-agarose bead enrichment, and liquid chromatography-tandem mass spectrometry for profiling glycoproteins in a cell model of lung cancer induced to express transforming growth factor beta ligand-1. This approach identified putative changes in the sialylation of glycoproteins related to metabolic, cell adhesion, glycan biosynthesis, and extracellular matrix-related proteins. The application of a secondary digest to glycopeptide-bound beads, using peptide-N-glycosidase-F, was useful for verifying N-glycan sites as well as exposing previously undetected glycoproteins. A stable isotope labeling of amino acids in cell culture approach was used to gauge if altered protein expression was contributing to differential capture, however this method provided limited information due to a low overlap of proteins identified from enriched vs. unenriched fractions. In another set of experiments, we applied a novel derivatization strategy combined with high resolution/high mass accuracy mass spectrometry for discerning glycan structures in human lung cancer proximal fluids. This procedure effectively defined the sialic acid anomeric configuration of several prevalent species and identified preliminary trends in the expression of oligomannose and complex glycans in clinically-relevant materials. Finally, matrix-assisted laser desorption ionization imaging mass spectrometry was used to spatially resolve the distribution of N-glycans in lung tissues matched to the proximal fluids. Histological assessment of these tissues facilitated cross-reference of acquired glycan species to regions of interest and provided a direct means for assessing how these trends correlated in the proximal fluids.

Rights

All rights reserved. Copyright is held by the author.

Download

Share

COinS