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目的提取小鼠肝细胞线粒体磷酸化蛋白并利用双向凝胶电泳技术对其进行分离。方法提取小鼠肝细胞线粒体蛋白并利用金属磷酸盐亲和层析树脂富集磷酸化蛋白后,进行一维等电聚焦分离和二维聚丙烯酰胺凝胶电泳分离,质谱鉴定感兴趣的蛋白点。结果成功提取了小鼠肝细胞线粒体磷酸化蛋白,并通过双向凝胶电泳分离技术成功建立了小鼠肝细胞线粒体磷酸化蛋白质组图谱。结论磷酸化蛋白纯化技术与二维凝胶电泳分离技术的结合是研究亚细胞器磷酸化蛋白质组的有效方法,为进一步全面鉴定和研究线粒体磷酸化蛋白的功能打下了基础。
Objective To extract mitochondrial phosphorylated proteins from mouse hepatocytes and separate them by two-dimensional gel electrophoresis. Methods Mitochondrial proteins were extracted from mouse liver cells and the phosphoproteins were enriched with metal phosphate affinity chromatography resin. The proteins were isolated by one-dimensional isoelectric focusing and separated by two-dimensional polyacrylamide gel electrophoresis. Mass spectrometry identified the protein spots of interest . Results Mitochondrial phosphorylated protein of mouse hepatocytes was successfully extracted and the mitochondrial phosphoproteome map of mouse hepatocytes was successfully established by two-dimensional gel electrophoresis. Conclusion The combination of phosphoprotein purification and two-dimensional gel electrophoresis separation is an effective method to study the phosphorylation of subcellular organelles. It laid the foundation for the further comprehensive identification and study of the function of mitochondrial phosphoproteins.