天津医药

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基于液质联用蛋白组学技术分析脑组织脂筏样品的属性

聂坤1,张雪竹1,2,赵岚1,2,贾玉洁1,2,韩景献1   

  1. 1. 天津中医药大学第一附属医院
    2.
  • 收稿日期:2013-01-16 修回日期:2013-04-15 出版日期:2013-09-15 发布日期:2013-09-15
  • 通讯作者: 韩景献

Properties of Lipid Rafts from Cerebral Cortex Based on HPLC MS/MS Proteomic

NIE Kun 1,ZHANG Xue zhu1, ZHAO Lan 1,3,JIA Yu jie1,3,HAN Jing xian1   

  • Received:2013-01-16 Revised:2013-04-15 Published:2013-09-15 Online:2013-09-15
  • Contact: HAN Jing xian

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摘要：

【摘要】目的 验证蔗糖密度梯度超速离心法提取脑组织脂筏的有效性。方法 用蔗糖密度梯度超速离心法提取小鼠脑组织脂筏，采用免疫印迹法、双酶胆固醇检测法结合光散射度分析样品的脂筏属性，采用液质联用蛋白组学技术和生物信息学手段，对脂筏中的蛋白质细胞定位进行分析。结果  超速离心法提取的脑组织脂筏具有典型的高散色度、高胆固醇和高表达Flotillin-1的脂筏特性；液质联用蛋白组学分析从脂筏样本中鉴定出647种蛋白质，这些蛋白质细胞定位大多是细胞膜、内质网、细胞骨架和细胞浆等常见的脂筏蛋白来源，这种脂筏样品是含有杂质的混合物，鉴定出的647种总蛋白中，有21%是细胞核、线粒体和核糖体等非脂筏来源蛋白。结论  超速离心法是提取脑组织脂筏的有效方法，但应用中要考虑杂蛋白的影响。

关键词: 膜脂质类, 计算生物学, 超速离心法, 下丘脑, 蛋白质类, 基因组, 脂筏

Abstract:

[Abstract]  Objective   To verify the validity of the sucrose density gradient ultracentrifugation method for lipid rafts
from cerebral cortex.Methods  Extract lipid rafts from cerebral cortex in mouse were extracted by the sucrose density gradient ultracentrifugation method. The properties of lipid rafts were detected by Western blotting method, double enzyme and light scattering methods. HPLC MS/MS proteomics and bioinformatics were used to locate proteins of lipid rafts in cells. Results Lipid rafts from cerebral cortex were provided with the model properties of lipid rafts such as high light scattering and cholesterol and high expression of Flotillin-1. HPLC MS/MS proteomics identified total 647proteins. Most of these proteins were from plasma membrane, endoplasmic reticulum, cytoskeleton and cytosol, however, there were 21% proteins among total647proteins were from nucleus, mitochondria and ribosomes.Conclusion  The sucrose density gradient ultracentrifugation method is a effective method to extract lipid rafts from cerebral cortex, however, the properties of mixture should be considered.

Key words: membrane lipids, computational biology, ultracentrifugation, hypothalamus, proteins, genome, lipid raft