利用邻近生物素标记技术结合质谱分析鉴定核仁相关蛋白

Identification of nucleolus-related proteins using proximate biotin labeling technique combined with mass spectrometry

目的:在小鼠胚胎干细胞(mouse embryonic stem cell,mESC)中,基于抗坏血酸过氧化物酶(engineered ascorbate peroxidase,APEX2)的邻近标记技术和质谱鉴定核仁相关蛋白。方法:构建稳定过表达APEX2-FBL细胞系,通过APEX2邻近标记技术获得生物素标记蛋白,用链酶亲和素包裹的磁珠对生物素标记的蛋白进行富集,随后运用液相色谱-串联质谱(liquid chromatography-tandem mass spectrometry,LC-MS/MS)鉴定,并对数据进行生物信息学分析,筛选差异蛋白。结果:成功构建稳定高表达APEX2-FBL细胞系;Western blot结果显示,通过APEX2邻近标记技术获得大量生物素标记蛋白;质谱鉴定出837个差异蛋白,且生物学过程多与核仁相关。结论:利用邻近生物素标记技术结合质谱分析是一种可行的鉴定核仁相关蛋白的方法。

Objective:In mouse embryonic stem cells(mESC),proximity labeling technology based on engineered ascorbate peroxidase(APEX2)and mass spectrometry were used to identify nucleolar related proteins.Methods:An APEX2-FBL overexpression stable cell line was constructed,biotin-labeled protein was obtained by APEX2 proximity labeling technology,the biotin-labeled protein was enriche with magnetic beads coated with streptavidin,and then liquid chromatography-tandem mass spectrometry(LC-MS/MS),and bioinformatics analysis was used to screen differential proteins.Results:Highly expressed APEX2-FBL stable cell lines were suuessfully constructed;Western blot results showed that a large number of biotin-labeled proteins were obtained by APEX2 proximity labeling technology;837 differential proteins were identified by mass spectrometry,and the biological processes were mostly related to nucleoli.Conclusion:The use of adjacent biotin labeling technology combined with mass spectrometry is a feasible method to identify nucleolar related proteins.