Quantitative Peptidomics of Food Deprivation

描述（由申请人提供）：肥胖是一个主要的健康问题，使人们容易患糖尿病、癌症和心脏病。尽管付出了相当大的努力，但调节体重的精确分子机制尚未得到很好的表征。神经肽被认为在调节摄食中起着至关重要的作用，但由于它们的小尺寸和低丰度，这些分子经常被蛋白质组学技术遗漏。我们最近开发了一种从肥胖小鼠大脑中分离神经肽加工中间体的方法，并使用差异同位素标记来提供来自两种不同动物（或动物池）的肽的相对定量。利用这种定量神经肽组学方法，我们在小鼠下丘脑中检测到大量的多肽，其中20-30%的多肽在2天的食物剥夺中大幅上调或下调。在Aim 1中，我们将使用串联质谱法鉴定这些肽。同时，在目标2中，我们将开发额外的同位素标签，允许定量更多的肽，并从每个实验中提供更多的信息。目标3将使用目标2中开发的新标签（如果成功）或目前使用的现有标签来检查食物剥夺和重新喂养的其他范例。预计这些研究将导致鉴定出由于食物剥夺和/或禁食后重新进食而改变的新型神经肽。在进一步的研究中，重要的是测试这些肽是否在野生型小鼠中受到类似的调节，以及微注射这些肽是否会影响摄食和/或体重；这些额外的研究超出了本R21申请的范围，但将在后续的R01申请中继续进行。R21的初步研究将对食物剥夺所改变的神经肽进行公正的调查，从而可能参与调节食物摄入和/或体重。

DESCRIPTION (provided by applicant): Obesity is a major health problem that predisposes people to diabetes, cancer, and heart disease. Despite considerable effort, the precise molecular mechanisms that regulate body weight are not well characterized. Neuropeptides are thought to play a critical role in regulating feeding, but these molecules are often missed by proteomics techniques due to their small size and low abundance. We have recently developed a method to isolate neuropeptide-processing intermediates from fat/fat mouse brain, and have used differential isotopic labeling to provide relative quantitation of peptides from two different animals (or pools of animals). Using this quantitative neuropeptidomics method, we have detected a large number of peptides in mouse hypothalamus, of which 20-30% are substantially up- or down-regulated by 2 days of food deprivation. In Aim 1, we will identify these peptides using tandem mass spectrometry. At the same time, in Aim 2 we will develop additional isotopic labels that allow for the quantitation of a larger number of peptides, and which provide more information from each experiment. Aim 3 will use either the new labels developed in Aim 2 (if successful) or the existing labels currently in use to examine additional paradigms of food deprivation and re-feeding. It is anticipated that these studies will lead to the identification of novel neuropeptides that are altered by food deprivation and/or re-feeding after fasting. In further studies, it would be important to test if these peptides are similarly regulated in wild type mice, and whether microinjection of the peptides influence feeding and/or body weight; these additional studies are beyond the scope of this R21 application, but would be continued in a subsequent R01 application. The studies in this initial R21 will provide an unbiased survey of neuropeptides that are altered by food deprivation, and which may therefore be involved in regulating food intake and/or body weight.