人红细胞20S蛋白酶体的蛋白质组学表征及不同来源20S蛋白酶体异质性的研究  

Proteomic Characterization of Human Erythrocyte 20S Proteasome and Analysis of Species-dependent 20S Proteasome Heterogeneity

作　　者：陈国强[1] 刘辉[1] 张海婧[1] 邓艳春[1] 李智立[1] 

机构地区：[1]中国医学科学院基础医学研究所北京协和医学院基础学院,北京100005

出　　处：《分析化学》2009年第12期1711-1716,共6页Chinese Journal of Analytical Chemistry

基　　金：国家自然科学基金(No.20675088);863计划(No.2006AA02Z154);高等学校博士学科点专项科研基金(No.20070023023)资助项目

摘　　要：通过整合差速离心和非变性聚丙烯酰胺凝胶电泳(native-PAGE)技术,建立了能有效分离20S蛋白酶体(20S core particle,CP)的方法。与传统纯化方法比较,此方法具有经济、快速的特点,并且能够对不同组织细胞来源的CP进行分离。利用本方法对人红细胞来源的CP亚基进行了2-DE分离和MALDI-TOF/TOFMS鉴定。结果显示,可鉴定出33个具有不同相对分子量和等电点的蛋白点,此数量远远多于CP亚基的14种。此外,利用非变性/变性十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(native/SDS-PAGE)技术,比较了来源于酵母、小鼠肝脏、人红细胞、人胰腺癌细胞系SW1990和PANC-1的CP及其亚基在电泳行为方面的差异,进行了蛋白酶体异质性初探。Abstract A method was developed for purification of 20S proteasome （20S core particle, CP） by combining differential centrifugations with nondenaturing polyacrylamide gel electrophoresis （native-PAGE） , irrespective of species origins of CPs. CP purified from human erythrocytes was subjected to proteomic analysis by two-dimensional gel electrophoresis （2-DE） and matrix-assisted laser desorption/ionization mass spectrometry （ MALDI-MS）, revealing 33 spots of subunit isoforms with different molecular weights and isoelectric points, more than 14 constituent subunits. Furthermore, other four CPs were purified from yeast, mouse liver, two pancreatic cancer cell lines SW1990 and PANC-1 using this method mentioned above, and subjected to protea- some heterogeneity analysis by native/SDS-PAGE （native/sodium dodecyl sulphate polyacrylamide gel electro-phoresis）, together with CP from erythrocytes. The method described acts as a rapid and effective tool for CP isolations, and the results obtained may be served as a footstone for the investigations of species-dependent proteasome heterogeneity.

关 键 词：蛋白酶体 异质性 非变性聚丙烯酰胺凝胶电泳 差速离心 细胞系 

分 类 号：Q55[生物学—生物化学] TQ464.8[化学工程—制药化工]