Proteomic analysis of proteins expressing in regions of rat brain by a combination of SDS-PAGE with nano-liquid chromatography-quadrupole-time of flight tandem mass spectrometry
Proteomic analysis of proteins expressing in regions of rat brain by a combination of SDS-PAGE with nano-liquid chromatography-quadrupole-time of flight tandem mass spectrometry
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DOI：
10.1186/1477-5956-8-41
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发表时间：
2010-07-27
期刊：
影响因子：
2
通讯作者：
Mizukami, Yoichi
中科院分区：
文献类型：
--
作者：
Katagiri, Tomoki;Hatano, Naoya;Mizukami, Yoichi
背景：大多数由大脑控制的生物功能及其相关疾病与大脑特定区域的激活密切相关。神经蛋白质组学已应用于全脑分析，阐明了各区域蛋白质表达的一般模式。然而，每个大脑区域的综合蛋白质组仍不清楚。结果：在本研究中，我们利用Mascot Score对已鉴定蛋白质进行半定量分析，对成年大鼠大脑的六个区域：丘脑、海马、额叶皮层、顶叶皮层、枕叶皮层和杏仁核进行了比较蛋白质组学。为了有效地鉴定大脑中存在的蛋白质，在配备C18柱的纳米液相色谱仪上通过SDS-PAGE组合分离蛋白质，并通过四极杆飞行时间串联质谱法进行分析。蛋白质组数据显示，大鼠大脑中有 2,909 种肽，其中 200 多种通过半定量分析被鉴定为区域丰富的蛋白质。含有已鉴定蛋白质的区域是膜（20.0%）、细胞质（19.5%）、线粒体（17.1%）、细胞骨架（8.2%）、细胞核（4.7%）、细胞外区域（3.3%）和其他（18.0%）。在鉴定的蛋白质中，通过免疫印迹证实了胶质纤维酸性蛋白、GABA转运蛋白3、Septin 5、热休克蛋白90、突触结合蛋白、热休克蛋白70和丙酮酸激酶的表达。我们通过免疫组织化学检查了GABA转运蛋白3、神经胶质纤维酸性蛋白和热休克蛋白70在大鼠脑中的分布，发现这些蛋白定位于蛋白质组分析和免疫印迹观察到的区域周围。 IPA分析表明，与每个区域的生物功能密切相关的通路可能在大鼠大脑中被激活。结论：这些观察结果表明，成年大鼠大脑每个区域的蛋白质组学可能为阐明与大脑区域激活相关的生物作用提供一种新的方法。
Background: Most biological functions controlled by the brain and their related disorders are closely associated with activation in specific regions of the brain. Neuroproteomics has been applied to the analysis of whole brain, and the general pattern of protein expression in all regions has been elucidated. However, the comprehensive proteome of each brain region remains unclear.Results: In this study, we carried out comparative proteomics of six regions of the adult rat brain: thalamus, hippocampus, frontal cortex, parietal cortex, occipital cortex, and amygdala using semi-quantitative analysis by Mascot Score of the identified proteins. In order to identify efficiently the proteins that are present in the brain, the proteins were separated by a combination of SDS-PAGE on a C18 column-equipped nano-liquid chromatograph, and analyzed by quadrupole-time of flight-tandem-mass spectrometry. The proteomic data show 2,909 peptides in the rat brain, with more than 200 identified as region-abundant proteins by semi-quantitative analysis. The regions containing the identified proteins are membrane (20.0%), cytoplasm (19.5%), mitochondrion (17.1%), cytoskeleton (8.2%), nucleus (4.7%), extracellular region (3.3%), and other (18.0%). Of the identified proteins, the expressions of glial fibrillary acidic protein, GABA transporter 3, Septin 5, heat shock protein 90, synaptotagmin, heat shock protein 70, and pyruvate kinase were confirmed by immunoblotting. We examined the distributions in rat brain of GABA transporter 3, glial fibrillary acidic protein, and heat shock protein 70 by immunohistochemistry, and found that the proteins are localized around the regions observed by proteomic analysis and immunoblotting. IPA analysis indicates that pathways closely related to the biological functions of each region may be activated in rat brain.Conclusions: These observations indicate that proteomics in each region of adult rat brain may provide a novel way to elucidate biological actions associated with the activation of regions of the brain.