应用IP-2D nano-HPLC-MALDI-TOF-TOF鉴定蛋白质泛素化修饰  

Identification of Protein Ubiquitination by IP-2D nano-HPLC-MALDI-TOF-TOF

作　　者：毛建华[1,2,3] 张群业[2] 黄秋花[2] 陈竺[2] 陈俭[1] 陈赛娟[1,2] 

机构地区：[1]中国科学院动物研究所,北京100101 [2]上海交通大学医学院附属瑞金医院上海血液学研究所,医学基因组学国家重点实验室,上海200025 [3]中国科学院研究生院,北京100049

出　　处：《生物化学与生物物理进展》2008年第7期839-847,共9页Progress In Biochemistry and Biophysics

基　　金：国家自然科学基金资助项目(30600262,30772744,30572143,30623010);上海市教育委员会重点学科资助项目(Y0201)~~

摘　　要：近年来,在蛋白质研究中,特别是在蛋白质翻译后修饰(PTM)的研究中,生物质谱技术的应用越来越广泛,与纳升级HPLC的联合应用,使这一技术手段更加有效.针对泛素化在细胞功能调控中发挥关键作用的PTM的特点,将免疫沉淀、2Dnano-HPLC和基质辅助激光解吸/电离串联飞行时间质谱(IP-2D nano-HPLC-MALDI-TOF-TOF)有机整合,建立了天然状态下蛋白质泛素化位点的鉴定方法,并应用这一方法确定出K562细胞内具有酪氨酸激酶活性的蛋白c-ABL的泛素化位点.为定性鉴定生理和病理状态下内源性蛋白的泛素化修饰提供了借鉴.Mass spectrometry has been extensively used in the identification of protein and its posttranslational modification （PTM）. Mass spectrometry coupled with nano high performance liquid chromatography （HPLC） could improve its resolution and efficiency. Ubiquitination, one kind of the important protein posttranslational modifications （PTM）, plays a pivotal role in dynamic balance and functional regulation of protein. A new strategy taking the advantage of immunoprecipitation, 2D nano HPLC and matrix-assisted laser desorption/ionization-time of flight mass spectrometry was established to identify the ubiquitination of endogenous protein in mammalian cells. By using this strategy, the ubiquitinated sites of c-ABL in K562 leukemia cells was successfully identified. Thus, it may provide a valuable tool to characterizing the ubiquitination of endogenous proteins under physiologic and pathologic conditions.

关 键 词：泛素化 蛋白质翻译后修饰 IP-2D nano-HPLC-MALDI-TOF-TOF 

分 类 号：Q7[生物学—分子生物学] R3[医药卫生—基础医学]