大豆种子蛋白质组样品制备方法研究

以中黄13大豆成熟种子为材料,采用苯酚甲醇醋酸铵法、三氯乙酸丙酮法和尿素硫脲法进行了蛋白质初提与复提,并利用纳升级液相色谱串联质谱技术(Nano LC-MS/MS)分析比较了尿素硫脲法初提和尿素硫脲法初提后苯酚甲醇醋酸铵法复提的样品,以及一维电泳预分离的尿素硫脲法初提样品,旨在构建能有效提高蛋白质提取率和鉴定率的样品制备方法。结果表明:尿素硫脲法初提结合苯酚甲醇醋酸铵法复提的蛋白得率最高,分别达到29.18%与4.08%;尿素硫脲法初提与尿素硫脲法初提后苯酚甲醇醋酸铵法复提样品中共鉴定到2 246个蛋白质组,初提和复提样品之间差异十分明显;每次独立制备的尿素硫脲法初提样品平均可鉴定到1 167个蛋白质组,经一维电泳预分离后平均可鉴定到1 868个蛋白质组。研究表明,采用适当的蛋白质样品制备和预分离方法,能有效提高大豆种子蛋白质的提取率和鉴定率。

A Study on Sample Preparations for Proteomics of Soybean Seeds

By using pheno meth anolammonium acetate,TCA acetone or urea thiourea methods,seed proteins of soybean Zhonghuang 13 were extracted at first and second step.In order to explore the efficient methods of preparing sample for proteomics of soybean seeds.The protein extraction obtained by urea thiourea method at first step and the protein extraction obtained by pheno meth anolammonium acetate method at second step,and the first step extracted protein,which was separated further by one-dimensional electrophoresis,were analyzed with nano-LC-MS/MS respectively.The results showed that the protein yield of first and second extractions obtained by urea thiourea method and following by pheno meth anolammonium acetate method reached 29.18％ and 4.08％ respectively,and was the highest among extractions obtained by other combined methods.A total of 2 246 protein groups was identified from three replicate samples of first and second extraction,and there were significant differences between them.Average 1 167 protein groups were identified from the sample extracted by urea thiourea,and average 1 868 protein groups from the urea thiourea extracted sample which were separated further by one-dimensional electrophoresis.The results indicated that the effective sample preparation and pre-separation could enhance the yields and identification of the protein from soybean seeds.