大豆种子蛋白质组样品制备与数据分析方法

doi:10.13560/j.cnki.biotech.bull.1985.2020-0555

摘要/Abstract

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

牟永莹, 王道平, 陈明, 邱丽娟, 潘映红. 大豆种子蛋白质组样品制备与数据分析方法[J]. 生物技术通报, 2020, 36(12): 247-255.

MU Yong-ying, WANG Dao-ping, CHEN Ming, QIU Li-juan, PAN Ying-hong. Sample Preparation and Data Analysis Method for Soybean Seed Proteome[J]. Biotechnology Bulletin, 2020, 36(12): 247-255.

图/表 7

图1 大豆种子蛋白质组分析技术流程图

图2 UT法和mUT法提取大豆种子蛋白的质谱鉴定结果 A：UT法和mUT法制备的大豆种子蛋白、肽段、谱图匹配及二级谱图数目的均值,*：P<0.05;B：UT和mUT法3次独立重复试验制备的大豆种子蛋白的鉴定数比较

图3 TD、Lys-C/TD、SDC-TD和HT-TD 4种酶切方法的质谱鉴定结果 A：蛋白和肽段鉴定数目,a、b、c：5%显著水平差异;B：蛋白序列覆盖度,NS：无显著差异;C：漏切位点比例

图4 三种液相梯度分离蛋白的总离子流图

表1 不同液相梯度时间蛋白分离效果比较

液相梯度	蛋白	肽段	二级谱图数	肽段-谱图匹配数
30 min	709±11 b	2 890±53 c	13 015±129 c	4 747±124 c
60 min	1 364 b±4 a	5 620±22 b	36 751±173 b	10 966±30 b
120 min	1 396±47 a	5 771±60 a	53 129±301 a	25 256±694 a

表2 不同数据库搜库结果比较

数据库名称	数据库总蛋白条目数	鉴定蛋白数
数据库名称	数据库总蛋白条目数	有功能注释	无功能注释
Phytozome：Gmax_275_Wm82.a2.v1	88 647	1 411	560
UniProt：Glycine max_cv.Williams 82	88 687	1 237	753
NCBI：Glycine_max_v2.1	88 647	1 501	457

图5 十胜长叶质谱定性定量分析结果 A：定性鉴定肽段和蛋白数;B：肽段和蛋白定性鉴定重复率;C：3次生物重复鉴样品肽段和蛋白定量Spearman相关系数;D：定量鉴定蛋白质信号强度分布

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