Mass Spectrometric Studies of Neuropeptides in Feeding

描述（由申请人提供）：摄食行为对动物生存至关重要，也是能量稳态的一个基本方面。越来越多的饮食失调发病率及其相关的健康成本导致了深入的研究工作，旨在了解控制和调节食物摄入和能量稳态的机制和信号通路。这一过程似乎是由涉及大量神经肽的高度复杂的神经内分泌系统调节的。然而，鉴于神经肽的高化学复杂性和广泛分布，在细胞和网络水平上的精确分子机制仍然是难以捉摸的。这在很大程度上是由于缺乏在复杂微环境中测量和识别这些低丰度内源性信号分子的分析能力。显然，开发高灵敏度和选择性的神经肽鉴定和定量分析工具的需求很大。本项目旨在通过构建和实施一个独特的分析测量平台，并开发改进的基于质谱（MS）的方法来探测饲料中的肽能信号，从而提高灵敏度和选择性，从而填补这一空白。我们选择研究更简单、定义明确的甲壳类动物口胃神经系统（STNS）及其相关的神经内分泌器官，以促进技术的开发和验证。此外，关于该模型系统中存在的神经肽的丰富信息及其明确定义的生理学为解决与神经肽能调节复杂行为（如进食）相关的基本神经科学问题提供了独特的机会。该项目的具体目标包括：(1)通过基质辅助激光解吸/电离（MALDI）傅立叶变换质谱（FTMS），通过细胞内结合（QUICC）方法，开发直接的组织原位肽谱分析和定量。从缺食和饱食动物分离的主要神经内分泌器官将分析和比较其肽含量；(2)开发体内微透析采样技术和同位素标记策略，结合纳米流LC/MS，用于循环肽对摄食反应的差异显示；(3)建立结合同位素辅助的de novo MS/MS测序和序列同源性搜索的杂交策略，以鉴定和发现新的神经肽，重点关注在摄食反应中表现出差异表达和分泌的肽；(4)观察新发现肽对胃胃神经节内胃磨神经网络和幽门神经网络的生理影响。总的来说，这些拟议的实验将发展和说明神经肽分析的改进方法和能力。该项目还将发现大量新的多肽，并为详细了解多肽能调节摄食行为的机制提供神经化学基础。从研究这样一个小系统中获得的分子见解可以转移到更大、更复杂的脊椎动物系统中，并可能导致新的喂养障碍治疗策略的发展。

DESCRIPTION (provided by applicant): Feeding behavior is critical for animal survival, and is also a fundamental aspect of energy homeostasis. The growing incidence of eating disorders and their associated health costs have led to intensive research efforts directed to understand the mechanisms and signaling pathways that control and regulate food intake and energy homeostasis. This process appears to be regulated by a highly complex neuroendocrine system involving a multitude of neuropeptides. However, given the high chemical complexity and wide distribution of neuropeptides, the precise molecular mechanisms at the cellular and network levels remain elusive. This is, in large part, due to a lack of analytical capabilities to measure and identify these low abundance endogenous signaling molecules in a complex microenvironment. Clearly, the development of highly sensitive and selective analytical tools for neuropeptide identification and quantitation is in great demand. This project aims to fill this gap by constructing and implementing a unique analytical measurement platform and developing improved mass spectrometry (MS) - based methodologies for probing peptidergic signaling in feeding with enhanced sensitivity and selectivity. We have chosen to study the simpler and well-defined crustacean stomatogastric nervous system (STNS) and its associated neuroendocrine organs, to facilitate the technology development and validation. Furthermore, the wealth of information about the neuropeptides present in this model system and its well-defined physiology provide unique opportunities to address fundamental neuroscience problems related to the neuropeptidergic modulation of complex behaviors such as feeding. The specific aims of this project include: (1) To develop direct tissue in situ peptide profiling and quantitation via in-cell combination (QUICC) methodologies by matrix-assisted laser desorption/ionization (MALDI) Fourier transform mass spectrometry (FTMS). Major neuroendocrine organs isolated from food deprived and satiated animals will be analyzed and compared for their peptide content; (2) To develop in vivo microdialysis sampling techniques and isotopic labeling strategies coupled to nanoflow LC/MS for differential display of circulating peptides in response to feeding; (3) To develop a hybrid strategy combining the use of isotope-assisted de novo MS/MS sequencing and sequence homology searching to identify and discover novel neuropeptides, with focus on the peptides showing differential expression and secretion in response to feeding; (4) To test physiological effects of the newly discovered peptides on the feeding circuits (gastric mill and pyloric neuronal networks in the stomatogastric ganglion). Collectively, these proposed experiments will develop and illustrate improved methods and capabilities for neuropeptide analysis. This project will also discover a large number of new peptides and provide the neurochemical basis toward a detailed mechanistic understanding of the peptidergic regulation of feeding behavior. The molecular insights gained from studying such a small system can be transferred to the larger, more complex vertebrate systems and could potentially lead to the development of new therapeutic strategies for feeding disorders.