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Method development for the identification of alternatively sialylated glycoproteins in non-small...

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Method development for the identification of alternatively sialylated glycoproteins in non-small cell lung cancer

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Title	Method development for the identification of alternatively sialylated glycoproteins in non-small cell lung cancer
Author	Roper, Stephen Matthew
Date	2015
Extent	xii, 195 leaves : ill. 29 cm
Subject.MeSH	Academic Dissertations--South Carolina Carcinoma, Non-Small-Cell Lung--metabolism Glycosylation Academic Dissertations Publication Type
Collection	MUSC Theses and Dissertations
Contributing Institution	Waring Historical Library (MUSC)
Digitization Specifications	This work was born digital.
Date Digital	2015
Rights	Copyright is held by the author.
Language	English
SC County	Charleston County (S.C.)
Type	Text
Format	application/pdf
Media Type	Manuscripts
Resource Identifier	mtd2015_roper_sm
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.

Title	Method development for the identification of alternatively sialylated glycoproteins in non-small cell lung cancer, dissertation
Resource Identifier	mtd2015_roper_sm

Method development for the identification of alternatively sialylated glycoproteins in non-small cell lung cancer

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Method development for the identification of alternatively...

Method development for the identification of alternatively...

Method development for the identification of alternatively sialylated glycoproteins in non-small cell lung cancer

Method development for the identification of alternatively sialylated glycoproteins in non-small cell lung cancer, dissertation

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Method development for the identification of alternatively...

Method development for the identification of alternatively...