生物技术通报 ›› 2020, Vol. 36 ›› Issue (12): 247-255.doi: 10.13560/j.cnki.biotech.bull.1985.2020-0555

• 技术与方法 • 上一篇    下一篇

大豆种子蛋白质组样品制备与数据分析方法

牟永莹(), 王道平, 陈明, 邱丽娟, 潘映红()   

  1. 中国农业科学院作物科学研究所 农作物基因资源与基因改良重大科学工程,北京 100081
  • 收稿日期:2020-05-09 出版日期:2020-12-26 发布日期:2020-12-22
  • 作者简介:牟永莹,女,硕士研究生,研究方向:蛋白质组学;E-mail:mu_yongying@163.com
  • 基金资助:
    国家重点研发计划(2016YFD0101005);中国农业科学院科技创新工程

Sample Preparation and Data Analysis Method for Soybean Seed Proteome

MU Yong-ying(), WANG Dao-ping, CHEN Ming, QIU Li-juan, PAN Ying-hong()   

  1. Institute of Crop Science,Chinese Academy of Agricultural Sciences,Beijing 100081
  • Received:2020-05-09 Published:2020-12-26 Online:2020-12-22

摘要:

构建一种适用于大豆种子蛋白质组样品制备与液相质谱分析的技术体系,为深入研究大豆蛋白组学创造条件。以大豆品种中黄35成熟期种子为材料,分别对常用的蛋白质提取、酶切、液相分离梯度和数据库进行比较分析。结果表明,使用改进的尿素硫脲蛋白提取法配合赖氨酸C端内切酶/胰蛋白酶顺序酶切法制备的质谱分析样品,经90 min纳升级液相梯度分离和质谱分析,最后采用UniProt数据库解谱时,大豆种子蛋白鉴定数目最多。将此方法应用于大豆品种十胜长叶成熟种子样品制备和分析,3次样品制备重复试验共鉴定非冗余蛋白2 244组,其中61%蛋白质可被重复鉴定。该方法具有一定的可靠性和稳定性,适用于大豆种子蛋白质组样品制备和数据分析。

关键词: 蛋白质组, 样品制备, 蛋白酶切, 大豆种子

Abstract:

This study constructs a method for effective sample preparation and analysis of soybean seed proteomes via liquid mass spectrometry,aiming to improve the coverage of soybean proteome analysis. The mature seeds of soybean variety Zhonghuang 35 were used as materials to compare the protein extraction and enzyme digestion methods,liquid phase separation gradients and databases. The results showed that the identified proteins were the most when soybean seed proteome was extracted using modified urea thiourea buffer and digested with Lys-C/trypsin sequentially,then subjected on 90min nano liquid chromatographic separation and analyzed based on UniProt database. Finally,this method was applied to the preparation and analysis of mature seeds of soybean variety Tokachi-Nagaha,and a total of 2 244 non-redundant proteins were identified from three independent samples,in which about 61% could be repeatedly identified. Thus this method is reliable and stabile,and it is suitable for the proteome sample preparation of soybean seed and data analysis.

Key words: proteome, sample preparation, protein digestion, soybean seed