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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。
The protein was extracted and recaptured by medium phenol-ammonium acetate method, acetone-trichloroacetic acid method and urea-thiourea method using the mature seeds of Zhonghuang 13 soybean as raw materials. Nano LC- MS / MS) were used to analyze and compare the initial extraction of urea and thiourea by phenol-methanol-ammonium acetate method after primary extraction with urea-thiourea method and the primary extraction of urea-thiourea by one-dimensional electrophoresis. Effectively improve the protein extraction rate and identification of the sample preparation method. The results showed that the initial yield of urea-thiourea method combined with phenol-methanol-ammonium acetate method was the highest, reaching 29.18% and 4.08%, respectively. The urea-thiourea method and urea- A total of 2 246 proteomes were identified in the replicate samples, and the difference between the initial and duplicate samples was very clear. For each sample prepared independently, 1 167 proteomes were identified on average, and 1 167 proteomes were identified by one-dimensional electrophoresis After pre-separation, an average of 1868 proteomes were identified. Studies have shown that the use of appropriate protein sample preparation and pre-separation methods can effectively improve the soybean seed protein extraction rate and identification rate.