巴西橡胶树叶片蛋白样品的制备及质谱初步分析

采用TCA-丙酮沉淀法并稍做改进提取巴西橡胶树叶片总蛋白，蛋白溶解后，用试剂盒对蛋白上清液进行纯化和浓度测定。十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)和双向凝胶电泳(2-DE)的结果均表明，所制得的蛋白样品质量较高，并且纯化过的蛋白样品2-DE(双向电泳)图谱效果更好(7 cm，考染)。根据预实验结果，选用17 cm IPG预制干胶条对纯化的蛋白样品进行2-DE实验，采用快速银染法进行染色，随机挖取银染胶上6个蛋白点进行MALDI-TOF MS分析和数据库检索，鉴定了3个蛋白，它们是：1-氨基环丙烷-1羧酸合酶2，核酮糖-1，5-二磷酸羧化酶/加氧酶大亚基和F11O4.2。本实验为进一步对巴西橡胶树叶片中具有重要生物学功能蛋白的筛选和鉴定打下了方法学基础。

Preparation and Mass Spectrometry Primary Analysis of Protein Samples of Rubber Tree Leaves

The method for improved TCA-acetone precipitation was used to extract proteins from rubber tree (Hevea brasiliensis) leaves, and the protein supernatant samples were purified and quantified by kits (Bio-Rad). The result of SDS-PAGE and two-dimensional gel electrophoresis (2-DE) both showed that the protein samples prepared were good in quality and that the purified protein samples had a better 2-DE map when compared with the nonpurified one (7 cm, Coomassie brilliant blue stained). Purified protein samples were separated by 2-DE with 17 cm IPG strips, and the protein spots were detected by fast silver staining. Six protein spots were randomly excised from the silver stained gel for primary analysis by MALDI-TOF MS. Four proteins were identified, and they were 1-aminocyclopropane-1-carboxylate synthase 2, ribulose-1, 5-bisphosphate carboxylase/oxygenase large subunit, F11O4.2 and Rubisco small subunit. This experiment has provided a methodological foundation for screening and identification of biologically important functional proteins in rubber tree leaves.