Method development for investigating chemical distributions in the nervous system using mass spectrometry imaging

Chemical heterogeneity is a fundamental property of multicellular organisms. Multiple classes of biomolecules exhibit complex spatial and temporal variations and interactions during different biological processes. One way to investigate this chemical diversity is to visualize biomolecule localization in an organism, which may then translate into insight on how chemicals participate in organism functions and states. In this dissertation, multiple methodologies are applied using mass spectrometry imaging (MSI) toward analyzing peptide distributions within the nervous system. The nervous system contains a variety of signaling molecules that are used for cell-to-cell communication, such as small molecule neurotransmitters and neuropeptides. MSI is a multiplex and non-targeted chemical imaging technique that can reveal the localization of many compounds in one experiment without having to pre-select a specific analyte. It is compatible with multiple compound classes, covering metabolites, lipids, peptides, and proteins. These properties make MSI particularly useful for de novo analysis of complex biological systems, generating a comprehensive data set of chemical distributions for data mining and hypothesis generation. This dissertation applies MSI to a variety of biological samples ranging from tissues to dissociated cell populations, demonstrating the applicability of the approach for analyzing many systems. Sample preparation protocols are developed to prepare tissue sections for MSI. Multiple approaches are explored to improve ion image quality, enhance signal, and preserve tissue section integrity during sample processing. A couple of these include comparing spraying and sublimation for applying MALDI matrix, and different tissue rinsing and drying techniques. A variety of statistical methods are also applied on the collected data sets for visualizing chemical trends and for determining chemical markers. The developed MSI protocols are used to study different biological systems. One study reveals peptide changes in the spinal cord in response to a painful stimulus from the peripheral nervous system applied via the formalin test. Another study visualizes chemical distribution trends during planarian nervous system regeneration, revealing complex patterns in peptide content and distribution at different regeneration time points. In addition to traditional MSI, a high-throughput single cell MS analysis approach is developed to classify cell types and reveal rare cells based on their characteristic peptide markers. This approach modifies how MSI is typically conducted in order to specifically target features of interests on a substrate (e.g. cells), reducing analysis time and instrument wear. The improved efficiency enables hundreds to thousands of cells to be analyzed per experiment. Along with protocol developments are also instrument advances. Software are created for a modified mass spectrometer that has dual MALDI and SIMS capabilities. The software provides automatic control of parts of the instrument, enabling new functionalities, such as depth profiling, to be conducted. The development of these new methodologies and software tools allows new measurements to be obtained via MSI, generating insight into the chemical and spatial relationship involved in different biological processes