Development and Application of Mass Spectrometry-Based Strategies for Structural Elucidation of Heparin Isomers and Metabolic Investigations of Nutrition and Pluripotent Stem Cells

The goals of my dissertation were to develop and apply new MS-based strategies to enhance knowledge and understanding of biochemical phenotypes; in particular, my novel mass spectrometry methods were applied to differentiate pluripotent stem cell types, to understand the metabolic effects of fructose consumption, and to study the role of rare heparin and heparan sulfate structural features in mediating protein binding.;Chapter 1 covers the development and application of methodology employing collision induced dissociation (CID) to effectively identify heparin and heparan sulfate disaccharide sulfation patterns, including forms containing the rare 3-O-sulfate moiety. A method employing two successive CID events was developed and applied to an 11-sulfated heparin octasaccharide structure displaying affinity for chemokine ligand 2 (CCL2) revealing that, in contrast to several other heparin and heparan sulfate binding proteins, CCL2 does not preferentially bind a structure containing 3-O-sulfation.;Chapter 2 explores characterization of currently available instrumentation, chromatography methods, and software tools for construction of a liquid chromatography mass spectrometry (LCMS)-based metabolite profiling method maximizing the capabilities of current technologies. Six different chromatography columns with twelve different mobile phase combinations were evaluated with a series of standards to elucidate an effective chromatography method. This series of characterization and evaluation experiments enabled selection and integration of individual components to construct a hydrophilic interaction chromatography (HILIC)-QTOF MS metabolite profiling workflow which maximizes the capabilities of tested technologies and possesses the potential to expand understanding of biological systems across many research projects.;Chapter 3 investigates the metabolic relationships of induced pluripotent stem cells (iPSCs), iPSC parental embryonic fibroblasts, and embryonic stem cells (ESC). The HILIC-QTOF workflow developed in chapter 2 and an established gas chromatography time of flight (GC-TOF) method were used for metabolite profiling of all three cell types. Results indicate that iPSCs display greater metabolic similarity to genuine ESCs than the iPSC parental embryonic fibroblasts. However, iPSCs possess clear differences from ESCs in complex lipid structures, essential and non-essential amino acids, and metabolites involved in polyamine biosynthesis.;Chapter 4 explores the metabolic effects of fructose with a human HepG2 liver cell model. Targeted methodologies were applied to test the hypothesis that high fructose exposure would increase hexosamine generation, and that the increase in hexosamine generation would be associated with greater lipogenic gene expression in the HepG2 model system. Application of the targeted metabolite analysis strategies in combination with lipogenic gene expression and enzyme abundance analysis revealed that fructose exposure does not result in increased levels of hexosamine biosynthesis pathway metabolites or, contrary to rodent models, cause increased expression of lipogenic enzymes in the HepG2 model system. Should this effect translate to the human liver in vivo, it would suggest that increased lipogenesis caused by high fructose consumption occurs primarily through means other than lipogenic gene expression.;Chapter 5 explores the metabolic effects of fructose with a human HepG2 liver cell model. However, untargeted metabolite profiling methods were applied to generate a more broad investigation of fructose effects on liver cell metabolism. The HILIC-QTOF workflow developed in chapter 2 and an established GC-TOF method were used for metabolite profiling of HepG2 metabolite extracts from cells incubated in media conditions identical to the previous targeted analysis. Results indicate that metabolite profiles of HepG2 liver cells grown in all three media conditions are greatly similar. However, several distinct changes in metabolite abundance were observed based on both fructose addition and hexose concentration. Fructose dependent effects were observed in long-chain acylcarnitine structures and amino acids involved in folate metabolism. Carbohydrate concentration dependent effects were observed with acylcarnitine and complex lipid structures. (Abstract shortened by UMI.).