Development And Application Of Mass Spectrometry Based Strategies For Structural Elucidation Of Heparin Isomers And Metabolic Investigations Of Nutrition And Pluripotent Stem Cells
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Development and Application of Mass Spectrometry-based Strategies for Structural Elucidation of Heparin Isomers and Metabolic Investigations of Nutrition and Pluripotent Stem Cells
Mass spectrometry (MS) is a powerful analytical tool applied in modern biological sciences research. Its capacity for accurate quantitation of molecular structures, ability to determine molecular mass with millidalton accuracy, and capability to fragment ions into constituent structures provide scientists with the means necessary to effectively explore complex biological systems. The goals of my dissertation work 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. Heparin and heparin sulfate possess a large number of structural isomers due to variable sulfation patterns spanning numerous positions within a repeating disaccharide subunit, and a single CID event was inadequate to generate fragment ions differentiating 3-O-sulfated species from other structural isomers. However, application of two successive CID events enabled cross-ring dissociation generating unique fragment ions for 3-O-sulfated structures. 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. Following completion of the heparin/heparan sulfate project, the focus of the dissertation work shifted toward metabolomics. Metabolomics, the identification and quantification of all metabolites in a system under a given set of conditions, is a growing discipline in biological research and can enhance our understanding of biochemical response in complex biological systems to disease state, environmental stress, nutrition, and many other factors. 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. The mass accuracy and isotope abundance error of the Agilent 6530 quadrupole time of flight (QTOF) and Leco Citius liquid chromatography high resolution time of flight mass spectrometers were determined with known plasma sample metabolites to identify the limitations of each platform. The capabilities and limitations of four untargeted data processing tools, including MZmine and Genedata Refiner MS, were evaluated based on their ability to accurately report intensity of known metabolites in plasma samples with data sets from both instruments. 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. GC-TOF data processing provided 111 identified metabolites including glycolysis, pentose phosphate pathway, and citric acid cycle metabolites as well as amino acids, free fatty acids, and sugar alcohols. HILIC-QTOF data processing yielded 55 annotated metabolites including many complex lipids, acylcarnitines, amino acids, and purine structures. Annotated structures were integrated into MetaMapp metabolite networks to facilitate identification of compound classes and metabolite pathways displaying statistically significant differences between cell lines. 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. Lipogenic enzyme abundance and expression levels were determined for HepG2 cells incubated in media containing 5.5 mM glucose, 5.5 mM glucose + 5.0 mM fructose, and 10.5 mM glucose. Hexosamine biosynthesis pathway (HBP) metabolite levels were determined with targeted LC-QTOF and GC-TOF strategies for each hexose condition. 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. Similar to chapter 4, 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. Analysis of cell extracts with the GC-TOF system enabled identification of 112 different metabolites, and analysis with the HILIC-QTOF system enabled annotation of 54 different metabolites. Integration of GC-TOF and HILIC-QTOF data sets yielded 156 unique annotations representing a wide array of structural classes and enzymatic pathways. 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.
Advanced Mass Spectrometry-based Analytical Separation Techniques for Probing the Polar Metabolome
Author: Rawi Ramautar
language: en
Publisher: Royal Society of Chemistry
Release Date: 2021-07-13
The efficient analysis of polar and charged metabolites in biological samples remains a huge challenge in the field of metabolomics. Over the past years, novel mass spectrometry-based analytical tools have been developed to enable the sensitive and efficient profiling of polar ionogenic metabolites in various biological samples. This book gives the reader a comprehensive overview of these recent technological developments. Topics covered include the use of chemical labelling strategies for allowing the analysis of polar metabolites using reversed-phase liquid chromatography–mass spectrometry (RPLC-MS) and the latest methodological developments in RPLC-MS, hydrophilic interaction liquid chromatography (HILIC)-MS and ion-pair LC-MS approaches. Attention is also paid to developments in nano-LC-MS and capillary electrophoresis–mass spectrometry methods specifically for profiling polar metabolites in small volume biological samples. The utility of ion-mobility MS and NMR spectroscopy will also be outlined. Sample preparation is the key part in the analytical workflow employed for metabolomics. Therefore, ample emphasis will be given on recent solid-phase extraction and solid-phase micro-extraction methods. Finally, analytical techniques for chiral metabolic profiling will also be considered. Discussing the state-of-the-art of the proposed topics in one single book for probing the polar metabolome, using relevant examples, is unique and needed in the metabolomics field. This book has relevance and appeal to an international audience of analytical and biomedical researchers in industry and academia.
Mass Spectrometry-based Strategies for Biomolecular Structure Analysis
Mass spectrometry is an important method for studying the structure of both small molecules and large biomolecules (e.g., proteins). The majority of the applications prior to 1970 were focused on small molecules, owing to the limited ionization methods which posed difficulties in producing gas-phase ions for large biomolecules then. Beginning in the 1980's, with the introduction of new ionization methods (ESI and MALDI), the applications have gradually switched to biological science measuring large bioorganic molecules. Today, with the developing interest in metabolomics and proteomics, and ongoing improvement in MS-based techniques, mass spectrometry is extensively applied in the study of both small and large molecules. The research presented in this thesis falls into two main parts, which focus on the application of MS in (1) structural analysis of steroid metabolites and (2) characterization of protein-protein interactions. In the first part, combinations of different MS methods are adopted and used to solve the structures of unknown steroid metabolites, which are the pheromones responsible for mouse communication in mouse urine. This part includes three chapters, the first two of which discuss the method development of using MS to study the structure of steroid metabolites; and the third chapter presents the application of the MS methods in solving a newly discovered steroid pheromone, which is determined as a sex-specific hormone. In the second part, two MS-based strategies, namely, hydrogen-deuterium exchange (HDX) and fast photochemical oxidation of proteins (FPOP), are applied in two studies of protein-protein interactions, including: (1) dimerization of SecA, which is a motor protein in bacteria translocation pathway; and (2) interface mapping of EGFR binding to Adnectin1. In the first chapter in Part 2, we used HDX MS to characterize the dimer interface of SecA, and, meanwhile, detected a conformational change from open to closed forms at the pre-protein binding domain upon dimerization. This conformational change provided leads for the active form of SecA. In the second chapter in Part 2, we applied FPOP, which is modified to suit therapeutic protein formulation conditions, to map the epitope of Adnectin1-EGFR interaction at amino acid residue level. The epitope identified agrees with that from both HDX study and crystallography results, presenting more evidence of the capability of FPOP in epitope mapping. These five studies on characterization of steroid metabolites and protein-protein interactions show the successful application of mass spectrometry in the structural study of both small molecules and large proteins. Furthermore, there's a great potential for study of more complex systems.