【摘要】 目的 利用邻近标记技术建立检测心肌细胞分泌蛋白组的新方法。方法 通过分子克隆技术构建质粒,用于表达内质网(ER)定位的Turbo ID蛋白。在生物素处理的HEK293T细胞中,通过免疫荧光成像和免疫印迹实验(Western blot)对Turbo ID蛋白的表达、亚细胞定位和邻近标记功能进行验证。在上述质粒基础上包装腺病毒,侵染原代新生大鼠心室肌细胞(NRVM),加入生物素处理后,使用链霉亲和素磁珠对NRVM培养上清中NRVM细胞分泌的生物素化蛋白进行富集,进行蛋白质质谱分析,检测分泌蛋白组。结果 在HEK293T细胞和NRVM中,免疫荧光成像结果显示,TurboID蛋白能够定位在细胞核周围对应的ER区域。在添加生物素的作用下,Western blot结果显示,TurboID能将ER蛋白生物素化。用链霉亲和素磁珠对NRVM培养上清中的生物素化蛋白进行富集后,通过蛋白质质谱分析,结果发现心肌细胞的ER、细胞膜和细胞外的蛋白质被显著富集,包括经典的心肌细胞分泌蛋白质心房利尿钠肽。对不含ER-TurboID的NRVM培养上清进行蛋白质组分析,主要获得白蛋白等培养基中的成分,难以检测到心肌细胞的分泌蛋白质。结论 ER-TurboID技术可有效富集和检测心肌细胞通过ER分泌的蛋白质组,并排除培养基中非细胞来源蛋白的干扰。更多还原

【Abstract】 Objective To establish a new method for detecting the secretory proteome of cardiomyocytes by using proximity-labeling-based analysis.Methods We constructed a plasmid using molecularcloning technology to express endoplasmic reticulum(ER)-localized TurboID protein.Biotin was added to the culture medium of HEK293T cells and the expression,subcellular localization,and proximity labeling function of TurboID protein were verified by immunofluorescence imaging and Western blot.After that,plasmid was packaged into adenovirus and transfected primary neonatal rat ventricular cardiomyocytes(NRVM) with it.Then streptavidin-coated magnetic beads were used to enrich the biotinylated proteins secreted by NRVMs from the medium and analyzed these proteins by mass spectrometry.Results Immunofluorescence imaging showed that TurboID protein could be appropriately localized in the ER region surrounding the nucleus in HEK293T cells and NRVMs.And the results of Western blot showed that TurboID could biotinylate proteins that pass through ER.We used streptavidin-coated magnetic beads to enrich the biotinylated proteins from the NRVM culture supernatant and analyzed them by mass spectrometry.The results indicated that the ER,cell membrane,and extracellular proteins of cardiomyocytes were significantly enriched by the magnetic beads,including the classical cardiomyocytes secretory protein ANP(atrial natriuretic peptide).Proteome analysis of the NRVM culture supernatant without ER-TurboID only presented protein supplements of the culture medium making it difficult to detect secretory proteins of cardiomyocytes.Conclusions The ER-TurboID techniques can eliminate the interference of non-cell-derived proteins and effectively detect the proteins secreted by cardiomyocytes that pass through the ER.更多还原