Department of Physiology, Division of Nutrition Physiology, Universidade Federal de São Paulo, UNIFESP, São Paulo, SP, Brazil.
Proteome Sci. 2012 Apr 20;10(1):26. doi: 10.1186/1477-5956-10-26.
The hypothalamus plays a pivotal role in numerous mechanisms highly relevant to the maintenance of body homeostasis, such as the control of food intake and energy expenditure. Impairment of these mechanisms has been associated with the metabolic disturbances involved in the pathogenesis of obesity. Since rodent species constitute important models for metabolism studies and the rat hypothalamus is poorly characterized by proteomic strategies, we performed experiments aimed at constructing a two-dimensional gel electrophoresis (2-DE) profile of rat hypothalamus proteins.
As a first step, we established the best conditions for tissue collection and protein extraction, quantification and separation. The extraction buffer composition selected for proteome characterization of rat hypothalamus was urea 7 M, thiourea 2 M, CHAPS 4%, Triton X-100 0.5%, followed by a precipitation step with chloroform/methanol. Two-dimensional (2-D) gels of hypothalamic extracts from four-month-old rats were analyzed; the protein spots were digested and identified by using tandem mass spectrometry and database query using the protein search engine MASCOT. Eighty-six hypothalamic proteins were identified, the majority of which were classified as participating in metabolic processes, consistent with the finding of a large number of proteins with catalytic activity. Genes encoding proteins identified in this study have been related to obesity development.
The present results indicate that the 2-DE technique will be useful for nutritional studies focusing on hypothalamic proteins. The data presented herein will serve as a reference database for studies testing the effects of dietary manipulations on hypothalamic proteome. We trust that these experiments will lead to important knowledge on protein targets of nutritional variables potentially able to affect the complex central nervous system control of energy homeostasis.
下丘脑在许多与维持体内平衡相关的机制中起着关键作用，例如控制食物摄入和能量消耗。这些机制的损伤与肥胖发病机制中涉及的代谢紊乱有关。由于啮齿动物物种是代谢研究的重要模型，并且大鼠下丘脑的蛋白质组学策略特征较差，因此我们进行了旨在构建大鼠下丘脑蛋白质二维凝胶电泳（2-DE）图谱的实验。
作为第一步，我们确定了组织收集、蛋白质提取、定量和分离的最佳条件。用于大鼠下丘脑蛋白质组学特征分析的提取缓冲液组成为 7M 尿素、2M 硫脲、4%CHAPS、0.5%Triton X-100，然后用氯仿/甲醇沉淀。分析了来自四个月大的大鼠下丘脑提取物的二维（2-D）凝胶；使用串联质谱和数据库查询使用蛋白质搜索引擎 MASCOT 对蛋白质斑点进行消化和鉴定。鉴定了 86 种下丘脑蛋白，其中大多数被归类为参与代谢过程，这与具有催化活性的大量蛋白质的发现一致。在这项研究中鉴定的编码蛋白质的基因与肥胖的发展有关。
本研究结果表明，2-DE 技术将有助于针对下丘脑蛋白的营养研究。本文提供的数据将作为研究饮食干预对下丘脑蛋白质组影响的参考数据库。我们相信，这些实验将为营养变量的蛋白质靶标提供重要的知识，这些靶标可能会影响复杂的中枢神经系统对能量平衡的控制。
