小鼠肝细胞线粒体磷酸化蛋白质组的初步研究

Pilot study on mitochondrial phosphoproteome in mouse liver

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 phosphoproteins from liver cells of mice and develop an approach for fractionating phosphoprotein mixtures by two-dimensional electrophoresis, so as to further study the biological function of mitochondrial phosphoproteins. Methods First, the mitochondrial protein was extracted from mouse liver and the purity was measured by transmissive electron micrography. The mitochondrial phosphoproteins were then enriched through the phosphate metal affinity chromatography resin （PMAC） and detected by Western blotting to observe its enrichment effect. Subsequently, the phosphoproteins were separated by one-dimensional iso-electric focusing and two-dimensional SDS electrophoresis.Finally, these protein spots were identified by matrix-assisted laser desorption ionization mass spectrometry （MALDI-TOF/TOF）. Mitochondria of liver cells from ten mice were dissociated by differential centrifugation combinated with density-gradient eentrifugation. The mitoehondrial proteins were extracted with lysis buffer and mitochondrial phosphoproteome were extracted by PMAC. Results Transmissive electron micrograph of mitoehondria showed that the pure mitochondria were obtained. The results of Western blot showed that the enrichment of mitochondrial phosphoproteome was efficient. The patterns of mitochondrial phosphoprotcome were established by two-dimensional electrophoresls, and mass spectrometry showed that the identified spots were really phosphorylated and localized in mitochondria, which confirmed the efficacy of the methods established in present study. Conclusions Combination of phosphoproteins enrichment and two-dimensional electrophoresis is an effective approach for phosphoproteomic stud ies, which provides a foundation for further studies on the functions of rnitochondrial phosphoproteome and differential phosphoprotcome of discease.Analysis for proteomic post-translational modifications （PTMs） may contribute to the understanding of disease etiology and deliver many new targets for research against diseases.