基于DIA LC-MS的烟草根尖蛋白质样品检测分析  

Detection and analysis of protein samples of tobacco root tip tissue based on DIA LC-MS

作　　者：莫泽君 陈小翔[3] 蒲媛媛 段丽丽[1,2] 田宗林 滕建辉 罗雯 阙远慧 刘仁祥 MO Ze-jun;CHEN Xiao-xiang;PU Yuan-yuan;DUAN Li-li;TIAN Zong-lin;TENG Jian-hui;LUO Wen;QUE Yuan-hui;LIU Ren-xiang(College of Agricultural,Guizhou University,Guiyang 550025,China;Key Laboratory of Tobacco Quality in Guizhou Province,Guiyang 550025,China;Zhejiang Zhongyan Industry Co.,Ltd.,Hangzhou 310000,China;College of Tobacco,Guizhou University,Guiyang 550025,China)

机构地区：[1]贵州大学农学院,贵阳550025 [2]贵州省烟草品质研究重点实验室,贵阳550025 [3]浙江中烟工业有限责任公司,杭州310000 [4]贵州大学烟草学院,贵阳550025

出　　处：《南方农业学报》2021年第11期2962-2968,共7页Journal of Southern Agriculture

基　　金：国家自然科学基金项目(32060510);贵州省科技厅项目(〔2016〕5663);贵州省科技厅项目(〔2019〕1405)。

摘　　要：【目的】分析数据非依赖性采集高分辨率色谱—质谱技术(DIA LC-MS)对根尖差异蛋白质样品检测的适用性,为深入研究烟草蛋白质组学提供技术支撑。【方法】以6个烟草品种(系)移栽后74 d的根尖组织为试验材料,采用DIA LC-MS法对裂解的蛋白质肽段进行扫描,从而分析该方法对根样组织的适用性。【结果】采用TCA/丙酮沉淀+SDT裂解法提取根尖总蛋白质,能得到横向条带平行性好,电泳条带清晰的蛋白质样品,样品等级评价均达到A级。构建的DDA数据库包含28042条肽段,共鉴定出7084个蛋白质。DIA数据的色谱峰平均数据点为7,平均的峰容量达260,主要肽段的保留时间(iRT)均被检测出,且整体较稳定,在Q值为0.01标准下的一致性评分(Cscore)为0.948,变异系数(CV)中值为11.5%(小于20.0%),相关系数大于0.9。DIA全息扫描模式下从6个烟草品种(系)的根尖组织中鉴定出的蛋白质数目为4149~5069个。【结论】DIA LC-MS分析鉴定的蛋白质范围广、数量大、可信度高,具有一定的可靠性和稳定性,各项质控指标均能达到试验分析要求,说明DIA LC-MS适用于烟草根尖蛋白质组样品的检测分析。【Objective】The applicability of data-independent acquisition high-resolution chromatography-mass spec-trometry(DIA LC-MS)for the detection of differential protein samples in root tips was analyzed to provide technical sup-port for in-depth study of tobacco proteomics.【Method】The root tip tissue of 6 tobacco varieties(lines)was used as the test material at 74 d after transplantation,and the cleaved protein peptides were scanned by DIA LC-MS method to ana-lyze the applicability of this method to root-like tissue.【Result】The total protein of root apex was extracted by TCA/ace-tone precipitation+SDT lysis method,and protein samples with good parallelism of horizontal bands and clear electro-phoresis bands were obtained,and the sample grade evaluation all reached grade A.The constructed DDA database con-tained 28042 peptides,and a total of 7084 proteins were identified.The average chromatogram peak of the data was 7,the average peak capacity was 260,the main retention time(iRT)was detected,and the retention time was generally stable,the consistency score(Cscore)was 0.948 under the Q value of 0.01 standard,the median coefficient of variation(CV)was 11.5%(less than 20.0%),and the correlation coefficient was greater than 0.9.The total number of proteins identified from the root tip tissue of six tobacco varieties(lines)under DIA holographic scanning mode ranged from 4149 to 5069.【Conclusion】The proteins identified by DIA LC-MS analysis have a wide range,a large number,high relia-bility,and certain reliability and stability.All quality control indicators can meet the requirements of experimental analy-sis,indicating that DIA LC-MS is suitable for detection and analysis of proteomic samples from tobacco root tips.

关 键 词：烟草 DIA LC-MS 根尖 蛋白质 检测 适应性 

分 类 号：S572.035.3[农业科学—烟草工业]