Mass Spectrometric Studies of Neuropeptides in Feeding

描述(申请人提供)：摄食行为是动物生存的关键，也是能量平衡的一个基本方面。饮食失调及其相关的健康成本的增加导致了密集的研究努力，旨在了解控制和调节食物摄入量和能量平衡的机制和信号通路。这一过程似乎受到高度复杂的神经内分泌系统的调控，涉及多种神经肽。然而，鉴于神经肽高度的化学复杂性和广泛的分布，在细胞和网络水平上的确切分子机制仍然难以捉摸。这在很大程度上是由于缺乏分析能力，无法在复杂的微环境中测量和识别这些低丰度的内源信号分子。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是非常必要的。该项目旨在通过构建和实施独特的分析测量平台，并开发基于改进的质谱学(MS)方法来探测饲喂过程中的肽能信号，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和定义明确的甲壳类口胃神经系统(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，这个模型系统中存在的关于神经肽的丰富信息及其明确的生理学为解决与摄食等复杂行为的神经肽能调节有关的基础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)傅立叶变换质谱仪(FTMS)建立直接组织原位多肽图谱和细胞内结合(QUIC)方法。将分析和比较从食物缺乏和饱食动物分离的主要神经内分泌器官的多肽含量；(2)发展体内微渗析采样技术和与纳米流LC/MS相结合的同位素标记策略，以区分显示摄食反应的循环肽；(3)发展结合同位素辅助从头测序和序列同源性搜索的混合策略，以发现和发现新的神经肽，重点放在那些在摄食反应中表现出差异表达和分泌的多肽；(4)测试新发现的多肽对摄食回路(胃磨和胃神经节中的幽门神经网络)的生理影响。总而言之，这些拟议的实验将开发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，并为详细了解多肽对摄食行为的调节提供神经化学基础。从研究这样一个小系统中获得的分子洞察力可以转移到更大、更复杂的脊椎动物系统，并可能导致开发新的摄食障碍治疗策略。

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.