Advancements in mass spectrometry for biological samples: Protein chemical cross-linking and metabolite analysis of plant tissues

摘要

This thesis presents work on advancements and applications of methodology for the analysis of biological samples using mass spectrometry. Included in this work are improvements to chemical cross-linking mass spectrometry (CXMS) for the study of protein structures and mass spectrometry imaging and quantitative analysis to study plant metabolites. Applications include using matrix-assisted laser desorption/ionization-mass spectrometry imaging (MALDI-MSI) to further explore metabolic heterogeneity in plant tissues and chemical interactions at the interface between plants and pests. Additional work was focused on developing liquid chromatography-mass spectrometry (LC-MS) methods to investigate metabolites associated with plant-pest interactions.The first chapter includes an introduction into CXMS and MS-based metabolite analysis and the sixth and final chapter includes a brief summary of the work and future directions based on the work presented here.The second chapter discusses the limitations of chemical cross-linking mass spectrometry, and steps taken to overcome these. This includes the incorporation of hydrogen-deuterium exchange mass spectrometry (HDX/MS) to identify the presence of structural distortion caused by the chemical cross-linking reaction. The use of extracted ion chromatograms (XIC) to increase cross-linking detection efficiency is also discussed.The third chapter focuses on the application of MALDI-MSI to investigate seedling maize leaf tissues. Metabolites related to the photosynthetic process are compared at different locations along the leaves of two inbred genotypes. The localization and relative abundances of phosphatidylglycerols (PG) and sulfoquinovosyl diacylglycerols (SQDG) are described in detail.The fourth chapter illustrates how imprinting MSI can be used to study the chemical interface between plants and pests. The interaction between soybean aphids and soybean leaves was studied using MALDI-MSI on imprints of infested leaves. These experiments revealed the spatial distribution of numerous metabolites, including compounds involved in the plant-pest interaction.The fifth chapter discusses the development of a methodology to determine quaternary ammonium compounds (QACs) in plant samples. Hydrophilic interaction liquid chromatography (HILIC) mass spectrometry was used to quantitatively and qualitatively analyze common bean seed exudates. This work was focused on QACs that bacteria are known to uptake for osmoprotection and nutritional benefits.