基于质谱和生物信息学分析的小菜蛾蛋白质鉴定

本研究以非模式昆虫小菜蛾Plutella xylostella为材料, 对比2, 3, 4龄幼虫的蛋白质组双向电泳图谱, 得到24个蛋白质差异点, 从中选取了编号为1111的差异表达蛋白质点进行质谱鉴定和生物信息学分析. 采用胶内酶解的多肽进行MALDI-TOF/TOF分析, 获得该点的肽质量指纹图谱（PMF）及串联质谱（MS/MS）图谱。将获得的PMF分别用MASCOT和ProFound等常用软件在NCBInr的Metazoa蛋白质数据库进行搜索, 匹配结果不理想. 进一步用PMF+MS/MS谱图搜索NCBInr的Metazoa蛋白质数据库, 以及小菜蛾EST数据库。 在NCBInr库中匹配结果为拟暗果蝇Drosophila pseudoobscura中的一种假定蛋白GA18218-PA, 而用EST库搜索的结果为家蚕Bombyx mori的ATP合酶的亚基。为验证搜索结果, 将该蛋白质点进行磺基异硫氰酸苯酯（SPITC）化学衍生后de novo测序, 最后确认该点可能为ATP合酶的一个亚基。最后着重讨论了蛋白质的质谱鉴定与生物信息学分析的联合使用, 希望据此选择出最适合于非模式昆虫蛋白质组学鉴定的方法。

Protein identification for the diamondback moth,Plutella xylostella,based on mass spectrometry and bioinformatics analysis

Protein expression in different larval instars of the diamondback moth (DBM), Plutella xylostella, was characterized by two dimensional gel electrophoresis (2-DE). Twenty-four differentially expressed protein spots were discriminated. The spot 1111 was selected for peptide mass fingerprinting (PMF) and peptide sequence using matrix-assisted laser desorption ionization of time-of-flight tandem mass spectrometry (MALDI-TOF/TOF MS). The PMF was used to search for the matched protein based on NCBInr Metazoa database with MASCOT and ProFound softwares, but the match was unsatisfied. The PMF +MS/MS spectrum was further used to search for the matched protein, and the protein was identified as GA18218-PA of Drosophila pseudoobscura based on the NCBInr Metazoa database while it was identified as the subunit d of H~+ transporting ATP synthase of Bombyx mori based on the EST database of DBM population. To validate the result of matching, the spot was used for chemical N-terminal derivatization of the tryptic peptides with 4-sulfophenyl isothiocyanate (SPITC) prior to MALDI-TOF/TOF MS, and the protein was identified as a subunit of ATP synthase again. Finally, potential application of MS analysis and bioinformatics as tools to select the optimal method in proteomic research on non-model insects was discussed in some detail.