Date of Award

2020

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

Anand Mehta

Third Advisor

Peggi Angel

Fourth Advisor

Patrick Woster

Fifth Advisor

Robin Muise-Helmericks

Abstract

With hepatocellular carcinoma (HCC) remaining as the fifth most common cancer in the world, causing more than 700,000 deaths annually, the need for reliable, early stage diagnoses and preventive treatments is crucial. While serum glycoproteins are hepatic in origin, making them excellent targets for HCC biomarkers, they can originate from both cancerous and non-cancerous regions and direct analysis of cancerous tissue itself is lacking. To counteract this, I hypothesized that direct tissue analysis combined with proteomic analysis could be utilized to identify more potential targets specific to HCC for early detection. This was done with a primary focus on glycosylation—as most clinically approved biomarkers are glycoproteins—and examined direct tissue glycomics in conjunction with glycoproteomic techniques through two specific aims: 1) Determining patterns of N-linked glycan changes in HCC tissue using MALDI imaging mass spectrometry to compare to previously published serum changes and 2) identifying glycopeptides containing changes in observed patterns of N- linked glycans in HCC samples using a targeted glycoproteomic approach. In Aim 1, HCC tissue was examined using MALDI imaging mass spectrometry to v verify changes in glycosylation via direct tissue analysis. Here, it was found that increased branching and fucosylation were directly associated with the cancerous tissue when compared to normal or cirrhotic. To further identify changes in glycosylation, two methods (one novel and one adapted for imaging) were implemented on tissue to further classify N-linked glycan isoforms through linkage analysis, specifically for sialic acids and core fucose. Again, it was shown that core fucose is most directly related to HCC tissue, thus confirming serum findings in the literature. For Aim 2, the novel method of determining core fucosylation was used in conjunction with glycoproteomic techniques to further elucidate the core fucosylated glycoproteins of interest. With the tag left behind following the enzymatic cleavage, targeted glycoproteomics was used to determine glycoproteins of interest while eliminating some biases inherent in the method, such as low ionization efficiencies for more complex N-glycans. This work outlines the first in-depth analysis of HCC tissue specifically regarding N- glycan changes, a novel application to determine N-glycan isoforms, and the application of these methods for glycoproteomic enhancement. With these findings, new trends in glycosylation related to the disease state could be further uncovered, as well as provide new biomarker candidates or therapeutic targets for future studies.

Rights

All rights reserved. Copyright is held by the author.

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