分级提取蛋白技术在蛋白质组学中的应用

Application of Sequential Extraction Protein Technology on Proteomics

以小鼠急性肝脏氧化损伤为模型,通过采用不同浓度的丙酮对小鼠肝脏蛋白进行分级提取和沉淀,用SDS-聚丙烯酰胺连续密度梯度凝胶电泳(SDS-PAGE)试验寻找差异表达蛋白质。通过试验发现,模型组中水溶性总蛋白、非水溶性总蛋白和膜蛋白总体表达量较空白组均有明显降低,进而对水溶性总蛋白进行的分级提取试验中发现有2个蛋白条带具有显著的差异性。进一步以基质辅助激光解吸电离飞行时间质谱(MALDI-TOF-MS)检测差异蛋白的肽质量指纹,在蛋白质数据库中检索鉴定差异蛋白的种类,经鉴定两种差异蛋白分别为一种未命名蛋白和Cu/Zn超氧化物歧化酶(Cu/Zn superoxide dismutase),为进一步研究其损伤机理奠定基础。通过分级提取蛋白质样品的试验方法,降低了蛋白组学当中蛋白质分离的成本和技术难度,在保证蛋白纯度的同时提高了电泳的分辨率,电泳图中的条带清晰、分明,便于后期进行质谱分析,同时也为蛋白质组学的应用提供了一种新思路和新方法。

This paper is modeled by acute oxidative injury in liver of mice.The protein is sequentially extracted by acetone of different concentrations,the differences on expression being is found out by SDS-PAGE grads electrophoresis experiment,which show the expression of water-soluble protein（WSP）,water-insoluble protein and membrane protein in model group（the mice are injected with alloxan）decreased heavily,much lower than control group.Then during the sequential extraction from WSP,highly significant differences have been discovered in two bands.Furthermore,by means of MALDI-TOF-MS to test the peptide mass fingerprinting of two different proteins,searching in the data base,one is believed to be Cu/Zn superoxide dismutase,other to be untitled,which lay a foundation to study injury reasons.Sequential extraction is a good method to reduce the cost of protein separation and level of skills,while maintaining protein purity,to raise the electrophoretic resolution.The band in the electrophoretogram is very clear and distinct and easy to be identified using Mass Spectrometry.Meanwhile,it is an innovation to proteomics application in the future.