Multi-faceted mass spectrometric approaches for the analysis of neuropeptides in crustacean: Toward functional discovery

In this thesis, an array of biological mass spectrometry (MS) based methods were developed and employed to enable extended characterization of neuropeptides. Neuropeptides encompass the largest and most diverse group of signaling molecules in the nervous system. They are necessary for the initiation and regulation of numerous physiological processes such as feeding, reproduction and development. Due to the complexity and inaccessibility of human systems, many invertebrate model nervous systems have been explored. Of these, the decapod crustacean stomatogastric nervous system (STNS) provides an excellent test bed to facilitate technology development and investigate the functional roles of neuropeptides. A review of the current status and progress in the field of invertebrate neuropeptide analysis is included in this thesis. The dissertation research presented in this thesis describes new analytical methodology and provides novel insights into the possible functionality of certain peptides by identifying neuropeptides found to be differentially expressed in correlation with food intake and changes of environmental conditions. This comparative peptidomic study utilized direct tissue analysis, mass spectral imaging (MSI) and stable isotopic labeling to provide comprehensive information regarding to peptide expression level changes under different physiological states. In addition, sample preparation method was developed to detect trace-level of neuropeptides from crustacean circulating fluid hemolymph. Furthermore, imaging mass spectrometry for the localization of neuropeptides in tissues in three dimensions was developed and demonstrated. Finally, using established techniques, novel neuropeptides were identified from the neuroendocrine organs in the lobster H. americanus , and their distribution was also investigated. Collectively, this body of work extends the capability of mass spectrometry as a bioanalytical tool for neuropeptide analysis and advances the basic understanding of neuronal signaling. Preliminary results of two on-going projects were also presented: (I) study of neuropeptide expression and localization within the STNS; (2) mass spectral imaging of metabolites from Medicago roots that are related with nitrogen fixation.