神经元胰岛素信号转导磷酸化蛋白质组的初步分析

PRELIMINARY ANALYSIS OF PROTEOME PHOSPHORYLATION IN NSULIN SIGNALING TRANSDUCTION IN NEURON

目的:采用同位素标记、双向电泳、放射自显影结合生物信息学技术对胰岛素(insulin,Ins)在神经元中信号转导的磷酸化蛋白质组进行分析,从蛋白质磷酸化修饰的角度深入探讨Ins在神经元中的信号转导。方法:取新生乳小鼠脑组织,进行神经元原代培养7天,用32P-NaH2PO4标记,分别加入胰岛素和培养基,分别作用0、5、20、60、120min后提取神经元蛋白。进行双向电泳,放射自显影。通过采用PDQuest2D分析软件进行图象分析,并在Swiss-Prot蛋白质数据库和PPDB磷蛋白数据库中查询,对小鼠神经元Ins信号转导磷酸化蛋白质组进行初步分析。结果:①放射双向电泳自显影图谱显示:刺激后60min、120min蛋白质磷酸化状态发生明显改变。②采用PDQuest2D分析软件对图谱差异分析及SWISSPROT蛋白质数据库和PPDB磷蛋白数据库进行查询,提示Ins在神经元的信号传递涉及到多种不同的信号途径和效应分子。结论:采用同位素标记、双向电泳、放射自显影结合生物信息技术可对Ins刺激神经元中的蛋白质可逆磷酸化修饰进行较为全面、动态的分析,寻找出相应的差异蛋白,并可进行初步的定性分析。

Objective：To investigate the way of insulin Methods： Neuron from the neonatal mice brain were cultured participating in the signal transduction in neuron. primaryly for 7 days,and labelled by 32P-NaH2PO4; then insulin and medium were added,to the stimulation group and control group respectively.Neuron proteins were extracted after 0,5,20,60,120min,and the dielectrophoresis,and radioautography were performed.Finally,the insulin stimulated phosohorylated proteome in the signal transduction of mice neuron was preliminarily omalysed by PDQuest 2D analyse software, and the SWISS PROT protein data bank and PPDB phosphoprotein data bank were inquired into.Results： （1） The analysis of icon by dielectrophoresis and radioautography showed that obvious changes of protein phosphorylation occurred after 60,120min.（2） The analysis of icon difference by PDQuest 2D analyses software,and the search results of SWISS PROT protein data bank and PPDB phosphoprotein data bank showed that a multitude of signal transduction ways and effector molecules were involved in the insulin signaling transduction in neuron.Conclusion：The Ins stimulated reversible phosphorylatiome modification of proteome in neuron maybe overall and dynamically analyed,so as to find corresponding protein difference, and its preliminary qualitative analysis may be done by isotope labeling,dielectrophoresis,and radioautography,combining with bioinformatics technology.