[1]林杨杰,赵明,杨生超,等.大豆种子蛋白质组样品制备方法研究[J].大豆科学,2016,35(05):810-817.[doi:10.11861/j.issn.1000-9841.2016.05.0810]
　LIN Yang-jie,ZHAO Ming,YANG Sheng-chao,et al.A Study on Sample Preparations for Proteomics of Soybean Seeds[J].Soybean Science,2016,35(05):810-817.[doi:10.11861/j.issn.1000-9841.2016.05.0810]
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大豆种子蛋白质组样品制备方法研究

《大豆科学》[ISSN:1000-9841/CN:23-1227/S] 卷: 第35卷 期数: 2016年05期 页码: 810-817 栏目: 出版日期: 2016-09-20
Title:
A Study on Sample Preparations for Proteomics of Soybean Seeds
作者:
林杨杰1; 2; 3; 赵明1; 杨生超1; 潘映红2; 3
（1.云南农业大学 农学与生物技术学院，云南 昆明 650201； 2.中国农业科学院 国家农作物基因资源与基因改良重大科学工程，北京 100081； 3.中国农业科学院 作物科学研究所，北京 100081）
Author(s):
LIN Yang-jie1; 2; 3 ;  ZHAO Ming1;  YANG Sheng-chao1;  PAN Ying-hong2; 3
（1.Institute of agriculture and biotechnology, Yunnan Agricultural University, Kunming 650201, China; 2.The National Key Facility for Crop Gene Resources and Genetic Improvement, Chinese Academy of Agricultural Sciences, Beijing 100081, China; 3.Crop Science Institute of Chinese Academy of Agricultural Sciences, Beijing 100081, China）
关键词:
大豆种子; 蛋白质组学; 样品制备; 纳升级液相色谱串联质谱
Keywords:
Soybean seed;  Proteomics;  Sample preparation;  Nano LC-MS/MS
DOI:
10.11861/j.issn.1000-9841.2016.05.0810
文献标志码:
A
摘要:
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（NanoLC-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。
Abstract:
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.

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备注/Memo

基金项目：中国农业科学院科技创新工程（作物分子标记技术及其应用科研创新团队）。第一作者简介：林杨杰(1992-)，男，硕士，主要从事蛋白质组学研究。E-mail: Lin_YangJie@163.com。通讯作者：潘映红(1962-)，男，研究员，主要从事蛋白质组学研究。E-mail: panyinghong@caas.cn。

更新日期/Last Update: 2016-09-25