Identifying crustacean neuropeptides and precursor-related peptides by LC/MS: An investigation of strategies for extraction and orthogonal separations
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Restricted Access Thesis
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Elizabeth A. Stemmler
Crustaceans, including the lobster, Homarus americanus, are excellent model organisms used to study nervous system function. This is especially true because of the role neuropeptides play in modulating rhythmic motor pattern generators. However, the identification of neuropeptides and precursor-related peptides in these organisms is challenging because of sample complexity– the large number of peptides found in a tissue matrix containing compounds that include larger proteins, phospholipids, and salts. Furthermore, the peptides exhibit a range of analyte properties (size, charge, hydrophobicity) that are impacted by post-translational modifications (amidation, pyroglutamation, sulfation, and disulfide bond formation). The goal of this study is to compare techniques for neuropeptide extraction (including extraction with 8 M urea vs acidified 65% methanol) and neuropeptide fractionation (offline or online liquid chromatography, in connection with reversed-phase (RP) or hydrophilic interaction chromatography (HILIC) stationary phases) using nanoLC-Q-TOF/MS/MS for neuropeptide identification. Techniques were tested using the analysis of brain neuropeptides from H. americanus in combination with the prior identification of over 100 neuropeptides and over 100 precursor-related peptides. This study will report a comparison of these techniques for better separating neuropeptides to further research in the field of neuropeptide identification. With respect to neuropeptide extraction, the 8 M urea extraction solvent was not advantageous over acidified 65% methanol. With respect to neuropeptide fractionation, the HILIC stationary phase did not demonstrate improved separation of neuropeptides; however, the data collected in this research allowed for new discoveries about polar compounds in H. americanus brain samples.
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