【摘要】 以陆地棉TM-1开花后6d的胚珠为对照,比较开花后6、10、14、18、22d纤维的蛋白质组双向电泳图谱,从中选取了编号为37的差异表达的蛋白质点。胶内胰蛋白酶酶切的多肽,用AB 4700蛋白质组学分析仪进行MALDI-TOF/TOF分析,获得了该点内蛋白质的高品质肽质量指纹图谱(PMF),从中挑出质量为1517.9的肽离子进行串联质谱分析,获得碰撞后断裂片段离子的串联质谱(FFP)。将获得的PMF分别用MASCOT、ProFound、Aldente和MS-Fit等常用软件对NCBInr和UniProt数据库中的绿色植物蛋白质数据库进行搜索,并用MASCOT搜索本地棉属EST数据库。将获得的FFP用MASCOT软件的离子搜索模式和序列查询模式搜索数据库结合手工质谱解析推断序列,最后确认该蛋白质是羟甲基转移酶。本文着重讨论了蛋白质组学研究中质谱分析和生物信息学软件的应用,评价常用蛋白质组学PMF检索工具及其检索结果。更多还原

【Abstract】 Using ovules of 6 days postanthesis(DPA) of the upland cotton cultivar TM-1 as the control,we compared 2-dimensional electrophoresis gel profiles of the proteins from the fibers at 6,10,14,18,and 22 DPA,and dug one of the differentially expressed spots of NO37 to digest-in-gel with trypsin,and subjected the recovered peptides to AB4700 Proteomics Analyzer for MALDI-TOF/TOF mass spectrometry(MS) analysis.We obtained a peptide mass fingerprint(PMF) of high quality,according to the peak abundance and quality of this PMF,we selected a peptide peak with mass of 1517.9 for tandem MS(MS/MS) analysis,and acquired a fragment fingerprint(FFP) in high quality.The PMF was used to search against NCBInr viridiplantae protein database with MASCOT,ProFound,and MS-Fit engines,and against UniProt viridiplantae protein database with Aldente program,respectively,this PMF was also searched against a local Gossypium EST database downloaded from NCBInr with MASCOT in-house version.Furthermore,the MS/MS data were used to search NCBInr viridiplantae protein database with MASCOT in ion search and sequence query mode respectively,followed by manual MS interpretation for de novo sequencing.The protein in No 37 spot was identified as hydroxmethyltransferase.In this paper we focus our discussion on MS analysis and the application of bioinformatic tools,we also provide our comments on the typical PMF alignment tools in proteomics and their searching results.更多还原