建立BioID-质谱联用技术筛选细胞内蛋白质相互作用

BioID combined with mass spectrometry in screening intracellular protein-protein interaction

目的通过BioID-质谱联用技术建立一种新的蛋白质相互作用的筛选系统,并采用免疫共沉淀-蛋白质印迹法验证与Yes相关蛋白1(YAP1)相互作用的蛋白质。方法构建YAP1-BirA*融合表达质粒,将质粒转染进293T细胞后,在细胞培养基中加入50μmol/L生物素,继续培养24 h后提取细胞总蛋白质。利用YAP1特异抗体,采用蛋白质印迹法检测YAP1-BirA*融合蛋白的表达。利用HRP偶联的亲和素检测细胞内蛋白质被生物素标记的水平。通过亲和素磁珠对细胞内生物素化的蛋白质进行亲和纯化,多次洗涤去除亲和素磁珠上非特异性结合的蛋白质后对亲和素磁珠纯化的蛋白质进行溶解。取40μg总蛋白行SDS-PAGE,然后采用蛋白质银染试剂盒对凝胶中的蛋白质进行染色,确定亲和素磁珠对生物素化蛋白的纯化效果。用非标记定量质谱法鉴定生物素化的蛋白质。针对鉴定得到的蛋白质,采用蛋白质印迹法对生物素化的SMAD家族成员3(SMAD3)进行验证。通过YAP1的免疫共沉淀-蛋白质印迹法确定SMAD3能否与YAP1相互作用。结果成功构建YAP1-BirA*融合表达质粒,该质粒在293T细胞中能够高效表达融合蛋白。在生物素存在的情况下,YAP1-BirA*融合蛋白能够利用生物素标记细胞内与YAP1-BirA*融合蛋白邻近的蛋白质。在细胞裂解后,这些在细胞内被生物素标记的蛋白质能够被偶联亲和素的磁珠纯化,经过多次洗涤,能够富集生物素标记的蛋白质。质谱鉴定显示,能够获得细胞内与YAP1在空间上相互靠近的蛋白质。免疫共沉淀-蛋白质印迹法结果证明SMAD3蛋白能与YAP1相互作用。结论BioID-质谱联用方法是一种能用于蛋白质相互作用筛选的简单、高效技术,与免疫共沉淀-蛋白质印迹法联合使用可以成为一种新的蛋白质相互作用研究体系。

Objective To establish a new screening system for protein-protein interaction using BioID combined with mass spectrometry,and to verify the interacting protein of Yes-associated protein 1(YAP1)through co-immunoprecipitation and Western blotting.Methods The YAP1-BirA*fusion expression plasmid was constructed.After the plasmid was transfected into 293T cells,50μmol/L biotin was added to the cell culture medium,and the total proteins of the cells were extracted after 24 h of culture.The expression of YAP1-BirA*fusion protein was detected by Western blotting with YAP1 specific antibody.Horseradish peroxidase(HRP)-avidin was used to detect the level of proteins labeled by biotin in cells.The biotin-labeled proteins in cells were purified through affinity magnetic beads,and the proteins purified by affinity magnetic beads were dissolved after washing the nonspecific binding proteins on affinity magnetic beads for many times.A total of 40μg total proteins were subjected to sodium dodecyl sulphate-polyacrylamide gel electrophoresis(SDS-PAGE),and then the proteins in the gel were stained by protein silver staining kit to determine the purification effect of affinity magnetic beads on biotinylated protein.Biotinylated proteins were identified by label-free mass spectrometry.For the identified proteins,the biotinylation of SMAD family member 3(SMAD3)was verified by Western blotting.The interaction between SMAD3 and YAP1 was determined by co-immunoprecipitation and Western blotting.Results The YAP1-BirA*fusion expression plasmid was successfully constructed,which could efficiently expressed the fusion protein in 293T cells.In the presence of biotin,YAP1-BirA*fusion protein could biotin-label the proximity proteins close to YAP1-BirA*fusion protein in cells.After cell lysis,these biotin-labeled proteins in cells could be purified by magnetic beads coupled with avidin.After repeated washing,biotin-labeled proteins could be enriched.Mass spectrometry showed that the proteins close to YAP1 in space could be obtained.The interaction between SMAD3 and YAP1 was confirmed by co-immunoprecipitation and Western blotting.Conclusion BioID combined with mass spectrometry is a simple and efficient technology for protein-protein interaction screening.Combined with co-immunoprecipitation and Western blotting,it may become a new protein-protein interaction research system.