Neuropeptidomic studies in crustacean via multi-faceted mass spectrometric techniques

Neuropeptides are the largest and the most diverse group of signaling molecules in the nervous system. They are significant for the initiation and regulation of numerous physiological processes such as feeding, reproduction and development. Compared to highly complex mammalian and human nervous system, the crustacean nervous system provides an ideal model system to study the roles of neuropeptides in network functioning. The presence of a manageable number of neurons in the crustacean stomatogastric nervous system (STNS) coupled with a rich repertoire of neuropeptides makes it an attractive preparation for technology development and functional study of neuropeptides. In this thesis, a multi-faceted mass spectrometric approach combining complementary sample preparation techniques, improved labeling strategy, has been employed to study crustacean neuropeptidomes. A new analytical quantitation methodology has been developed and direct tissue quantitation has been realized. In addition, ionic liquid was employed as matrix to generate homogenous crystallization, thus achieving more reliable quantitation. The differential expression of neuropeptides in correlation with food intake and different environmental conditions were investigated in three different crustacean species. This comparative peptidomic study employed small tissue extraction, direct tissue analysis, capillary electrophoresis (CE), mass spectral imaging (MSI) and stable isotopic labeling to provide comprehensive information about peptide expression level changes under different physiological states. Furthermore, quantitative imaging mass spectrometry employing isotopic labeling, internal standard, selected reaction monitor (SRM) was investigated. This research greatly expanded the knowledge of neuropeptides in crustacean model systems and built the foundation for the future functional study of neuropeptides in human nervous system. Collectively, this body of work extends the capability of mass spectrometry as a bioanalytical tool for neuropeptide analysis and advances the basic understanding of signaling.