Multifaceted Mass Spectrometric Investigation of Neuropeptides in Callinectes sapidus during Hypoxia

 描述（由申请人提供）：缺氧或低氧（O2）水平对许多生物体（包括水生无脊椎动物和哺乳动物）构成巨大的生理挑战。没有适当的O2水平，细胞无法呼吸，这意味着葡萄糖不能被利用，这可能对细胞有害。氧气的缺乏经常出现在几种医疗条件下（如哮喘），尽管我们缺乏对缺氧引起的主要生化变化的分子理解。神经肽被认为是这些应激诱导的反应的主要类别的调节剂，然而，完整的补充神经肽和它们的表达变化在应激过程中没有得到很好的表征。这主要是由于哺乳动物神经系统的复杂性以及目前缺乏探测这种复杂系统的工具。无脊椎动物（如甲壳类动物）具有简单的神经系统和良好的生理特征，是理解神经肽在应激反应中作用的极好模型。甲壳类动物也含有几种神经肽同源物，以更高的目的动物，这表明这项研究可以很容易地转化为哺乳动物模型系统。随着高分辨率和质量精确仪器的发展，质谱技术已成为研究神经肽的首选技术。此外，质谱成像（MSI）的发展已经允许在没有先验知识的情况下对生物组织中的分子种类进行高通量分析，从而在一个实验中获得数百种分析物的空间信息。由于其高灵敏度和高选择性，质谱将用于定量研究缺氧应激反应引起的神经肽组的变化。因此，我提出以下目标：1）利用ESI-和MALDI-MS研究缺氧环境引起的甲壳动物神经系统中神经肽的体内表达变化，2）利用MALDI-MSI分析缺氧暴露引起的神经肽分布变化，和3）开发一种通过MALDI-MSI用定制的多重质量差同位素标签测定神经肽绝对量的方法（iDiLeu）。总的来说，这些目标将开发MS作为一种使能分析工具，以促进理解甲壳动物神经系统内缺氧调节的分子机制，这将使我们对哺乳动物神经系统如何因缺氧条件而变化有新的见解。

 DESCRIPTION (provided by applicant): Hypoxia, or low oxygen (O2) levels, poses a great physiological challenge for many organisms, including aquatic invertebrates and mammals. Without proper O2 levels, cells are unable to respire, meaning glucose cannot be utilized, which can be detrimental to the cell. Lack of O2 is often seen in several medical conditions (e.g. asthma), although we lack a molecular understanding on the major biochemical changes due to hypoxia. Neuropeptides are thought to be a major class of regulators for these stress-induced responses; however, the full complement of neuropeptides and their expression changes during stress are not well characterized. This is mainly due to the complexity of the mammalian nervous system and current lack of tools to probe such intricate systems. Invertebrates (e.g. crustaceans), with their simple nervous system and well-characterized physiology, are an excellent model for understanding the roles of neuropeptides in response to stress. Crustaceans also contain several neuropeptide homologs to higher order animals, suggesting that this research can be easily translated to a mammalian model system. With the development of high resolution and mass accurate instrumentation, mass spectrometry (MS) has become the preferred technique to study neuropeptides. Also, the development of mass spectrometric imaging (MSI) has allowed for high-throughput analysis of molecular species in a biological tissue with no prior knowledge, thus obtaining the spatial information of hundreds of analytes in one experiment. With its high sensitivity and selectivity, MS will be used to quantitatively study the changes in the neuropeptidome due to the hypoxic stress response. Hence, I propose the following goals: 1) to investigate the in vivo expression changes of neuropeptides in the crustacean nervous system caused by a hypoxic environment using ESI- and MALDI-MS, 2) to analyze the neuropeptide distribution changes due to hypoxia exposure via MALDI-MSI, and 3) to develop a method to determine absolute quantities of neuropeptides via MALDI-MSI with custom multiplexed mass difference isotopic tags (iDiLeu). Collectively, these aims will develop MS as an enabling analytical tool to facilitate the understanding of the molecular mechanisms underlying the regulation of hypoxia inside the crustacean nervous system, which will give us novel insight into how the mammalian nervous system changes due to hypoxic conditions.