天麻蛋白质的双向电泳和肽质量指纹谱分析与鉴定

Identification of Proteins from Gastrodia elata by Two-Dimensional Electrophoresis and Peptide Mass Fingerprinting

采用双向聚丙烯酰胺凝胶电泳和质谱技术对天麻染菌球茎皮层和不染菌的新生球茎皮层进行了比较蛋白质组分析与鉴定。双向电泳后在分子量 1 2～97kD、等电点 3～ 1 0范围内 ,每块胶分离到约 90 0个蛋白质点。对新生球茎中表达量明显增加的 5个蛋白质点用基质辅助激光解吸 电离飞行时间质谱 (MALDI TOFMS)进行肽质量指纹谱的分析 ,并通过检索不同的数据库进行蛋白质鉴定与功能预测 ,初步认为第 4号蛋白点是一个与转录有关的RNA结合蛋白。同时本文在天麻蛋白质组样品制备、数据库检索策略以及蛋白质鉴定成功率等方面进行了探讨。

Proteins extracted from the cortex of fungi-infected nutritive corms and non-infected secondary corms of Gastrodia elata were separated by two dimensional polyacrylamide gel electrophoresis (2-DE) with immobilized pH gradients (IPG).Reference maps were obtained with a good reproducibility. Approximately 900 proteins per 2-DE gel were isolated with molecular weights ranging 12-97?kD and pI 3-10.The spots showed a symmetric shape indicating a good resolution in both electrophoresis dimensions.Five proteins of high levels observed in a secondary corm gel were excised and identified using peptide mass fingerprinting and matrix-assisted laser desorption ionization time-of-flight mass spectrometry.A summary of the identified proteins and their putative functions are presented.One of them,spot no.4 is identified as a RNA-binding protein involved in transcription.Technical details concerning database queries and protein identification success rates in the absence of a sequenced genome are reported and discussed.Parameters for sample preparation are critically investigated.