Advances in Islet Metabolomics using Hydrophilic Interaction Chromatography-Mass Spectrometry

Abstract

Metabolomics refers to the measurement of small molecules in a biological system with the goal of identifying biochemical mechanisms and markers of cell functions. In many instances the biological tissue of interest is only available in amounts too small to access by conventional sample preparation and analytical techniques. For instance, islets of Langerhans are endocrine microorgans that release hormones like insulin and glucagon to regulate glucose metabolism. Common sample preparation and metabolomics platforms, such as liquid chromatography-mass spectrometry (LC-MS) require pooling hundreds of islets from multiple mice to perform a single metabolomic study. The goal of this thesis was to advance the field of islet metabolomics by developing a platform to analyze minute sample amounts such as single islets or primary α-cells. To begin to understand how glucose alters the intracellular metabolome of α-cells, we investigated a clonal pancreatic alpha cell line. A hydrophilic interaction chromatography-mass spectrometry (HILIC-MS) method was developed to quantify metabolites of glycolysis, tricarboxylic acid cycle, and pentose phosphate pathway, as well as associated nucleotides and cofactors under various glucose conditions to understand glucagon secretion. Our results reveal differences in the level of basal metabolites when utilizing different experimental buffers such as culture media or salt solution. When evaluating the effect of glucose concentration on metabolism, significant differences in the levels of lactate and adenosine diphosphate, were observed at 1 mM glucose, supporting their role in glucagon secretion at low glucose conditions. When measuring the changes in metabolites in response to a decrease in glucose, metabolites involved in lactate oxidation and malate-aspartate shuttle were found to correlate with glucagon secretion. A capillary HILIC method interfaced to negative ion mode nano electrospray MS was developed for the quantification of metabolites in low volume samples. A commercial capillary LC system was used to evaluate the effect of column inner diameter (40-150 μm ID capillary HILIC columns) and injection volume (50-1000 nL) on sensitivity, isomeric separation, and column robustness for 18 metabolites in a sample volume of 1 μL. A post column additive of acetonitrile and 0.2% trifluoroethanol was used to overcome spray instability and electrical discharge in negative mode under highly aqueous conditions of the HILIC analysis. Small volume injections, i.e. 50 nL, result in nearly 10% more variability of peak area than injections at 500 nL or greater. Injections of 500 nL or greater are also preferred as higher signal is observed at all column IDs explored. Peak width increases with increasing injection volumes, thus a decrease in isomeric resolution is observed at larger injection volumes. The utility of this method was demonstrated through the measurement of metabolites extracted from a 1 μL human pooled plasma sample. Through a miniaturized sample preparation procedure in a low volume tube, we were able to quantify five metabolites from a single islet in a sample volume of 2 μL when coupled to capillary HILIC-MS. Multilayer silicon/PDMS and glass/PDMS hybrid microfluidic devices that incorporate pneumatic valve actuation were designed to explore the feasibility of automating cellular sample preparation procedures on a microfluidic device or “chip”. The work presented in this thesis describes the development of improved HILC-MS methods, including volume limited sample preparation for metabolomic analysis of material-limited cell samples. This work opens the possibility of metabolomic analysis of islets or primary cells to better understand stimulus secretion coupling.