构建小鼠肝细胞磷酸化蛋白质组分析鉴定的方法模型

Model of identifying phosphoproteome in mice hepatocytes

背景：磷酸化蛋白质组的分析鉴定是目前蛋白质组学技术研究的主要目标之一，然而以质谱为核心的鉴定技术尚不完善。目的：建立一种简便易行、适于在一般实验室普及的分析鉴定磷酸化蛋白质组的方法并应用于小鼠肝细胞磷酸化蛋白质组的模型。设计、时间及地点：观察对比实验，于2003-07／2004-06在泸州医学院生物化学教研室及医学分子生物学实验室完成。材料：封闭群乳昆明小鼠100只，年龄1-3d，体质量3-6g，雌雄各半；^32P-NaH2PO4由中国北京原子能研究所提供。方法：将长势相当的体外培养的乳小鼠肝细胞随机分成两组。一组作同位素^32P标记后液氮终止反应，裂解细胞，收集蛋白并定量，双向电泳分离蛋白，干胶及放射自显影，建立小鼠肝细胞磷酸化蛋白质组的放射自显影图谱，初步确定6种靶标磷酸化蛋白EPs15等；另一组直接裂解细胞，收集蛋白并定量，双向电泳分离蛋白后半干式电转移至PVDF膜，根据靶标蛋白EPs15等的理论等电点和分子量剪下6张小膜。主要观察指标：采用持久性化学发光检测技术确证初步鉴定的6种靶标蛋白EPs15等。结果：①小鼠肝细胞磷酸化蛋白质组的放射自显影图谱显示约100个蛋白质斑点，经PDQuest 2D软件结合蛋白质数据库初步鉴定出已知的6种磷酸化蛋白EPs15等。②6种靶标蛋白的免疫印迹-化学发光图谱上均只出现了1个特异蛋白质斑点，与放射自显影图谱上的斑点基本匹配。结论：实验建立的同位素标记结合免疫印迹-化学发光的方法分析鉴定磷酸化蛋白质组简单、实用、灵敏、高效。

BACKGROUND： Nowadays one of the main aims of proteome technology study is to identify phosphoproteome, but there are some shortages in mass spectrometry, which is the core of identification techniques. OBJECTIVE： To provide a new convenient method for the analysis and identification of phosphoproteome, and apply in phosphoproteome model of mice hepatocytes. DESIGN, TIME AND SETTING： An observation controlled experiment was done at the Department of Biochemistry/Laboratory of Molecular Biology in Luzhou Medical College from July 2003 to June 2004. MATERIALS： There were 100 closed colony Kunming mice, aged 1-3 days and weighing 3-6 g, were adopted irrespective of genders. ^32P-NaH2PO4 was purchased from Beijing Atomic Energy Research Institute. METHODS： The mice hepatocytes cultured in vitro were divided into two groups at random. One group of cultured hepatocytes were labeled with ^32P orthophosphate, then were put in liquid nitrogen to terminate reactions. After cell lysis, phosphoproteins were collected and quantified. Subsequently, proteins were separated by two-dimensional polyacrylamide gel electrophoresis （2-DE）, and the dried gel was autoradiographed. Finally, autoradiography map of phosphoproteome model in mice hepatocytes was established, and six target phosphoproteins were identified preliminarily as EPs15. The other group of cultured hepatocytes were lysed directly, then collected protein contents were estimated. After 2-DE, proteins were transferred to PVDF membrane, from which six small membranes were cut down according to molecular weight and isoelectric point. MAIN OUTCOME MEASURES： Chemiluminescence technology was utilized to detect six target phosphoproteins, which were identified preliminarily. RESULTS： Autoradiography map of the phosphoproteome of mice hepatocytes revealed about 100 phosphoproteins, among which six were identified preliminarily as EPs15 via using PDQuest 2D and protein databases. There was only one protein blot on each chemiluminescence map of six target proteins, which was almost identical with the blot on chemiluminescence-Western blotting map of the same protein. CONCLUSION： The co-application of isotope labeling and chemiluminescence-Western blotting has proven to be simple, applicable, sensitive and specific in analysis of phosphoproteome.