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

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.