大鼠背根神经节细胞质膜的双水相法纯化及其蛋白质组学研究(英文)

Aqueous Polymer Two-phase Partition for The Proteomic Analysis of Plasma Membrane From Rat Dorsal Root Ganglion Neurons

大鼠背根神经节(dorsal root ganglion,DRG)细胞是一种初级感觉神经元,能传导触觉、痛觉、温觉等神经冲动.为了对少量的DRG组织细胞进行质膜蛋白质组学分析,综合利用差速离心与双水相相结合的方法富集DRG质膜.然后通过SDS-PAGE、CapLC-MS/MS和生物信息学方法对其中的蛋白质进行鉴定和分析.Western blotting图谱扫描后经过Quantity One软件分析,双水相纯化后的质膜与差速离心后得到的粗质膜相比相对浓度增加了2.3倍,与匀浆液相比增加了15倍.经过大鼠IPI数据库以及相关文献检索,有729个蛋白质得到鉴定,其中547个蛋白质具有GO(gene ontology)注释信息,有159(21.8%)个蛋白质定位在质膜上.通过对大鼠DRG质膜的蛋白质组学研究,得到了大鼠DRG的质膜蛋白质的分析数据,且提供了一种适用于少量样品的蛋白质组学的分析路线.

Dorsal root ganglion （DRG） neurons are primary sensory neurons that conduct neuronal impulses related to pain,touch and temperature senses.To comprehensively identify proteins of plasma membrane （PM） from small amount of dorsal root ganglion （DRG） neurons,a proteomics strategy that utilizes aqueous polymer two-phase partition in combination with differential velocity centrifugation was adopted to enrich the PM,followed by SDS-PAGE,CapLC-MS/MS and bioinformatics analysis.Western blot analysis showed that the concentration of PM in purified plasma membrane（PPM） was 2.3 times higher than that in crude plasma membrane（CPM）,15 times higher than that in whole tissue lysate （WTL）.By searching against the rat IPI protein sequence database,a total of 729 non-redundant proteins were identified from the PM preparation,of which 547 had a gene ontology （GO） annotation indicating a cellular component,and 159 （21.8%） were unambiguously identified as PM proteins.A data set of plasma membrane proteins of DRG as well as a tool to study PM proteins were provided in a small amounts of sample.