家蚕丝腺蛋白质组学研究方法的建立

通过高精度的双向电泳技术对家蚕中部丝腺组织的蛋白质进行分离,采用基质辅助激光解析电离飞行时间质谱(matrix-assistedlaserdesorption/ionizationtimeofflightmassspectrometry,MALDI-TOF-MS)对其中一些表达量较高的蛋白点进行鉴定,并利用GPMAW(GeneralProtein/MassAnalysisforWindows)软件结合家蚕基因组预测的蛋白质数据库构建本地的肽质量指纹图谱数据库,对所得到的肽质量指纹图谱进行分析。研究发现,经过双向凝胶电泳及其图象分析技术,硝酸银染色和考马斯亮蓝染色分别能分离出500个以上和100个以上的蛋白点。这些蛋白质点主要集中在分子量15~90kD区域,等电点pH3·5~7之间。MALDI-TOF-MS鉴定的25个考染蛋白点中有60%以上的PMF(PeptideMassFingerprint)的信号峰较强。在数据库检索过程中,利用家蚕肽质量指纹数据库所得检索结果与在Mascot的检索结果相比,前者不仅能够准确鉴定出一些已有研究报道的蛋白,从而验证检索方法的可行性,而且还能够对一些已经被家蚕基因组数据库所预测但未曾报道的新蛋白质进行鉴定,从而建立了一整套适合于家蚕蛋白质组研究的方法,并为其它绢丝昆虫蛋白质组研究提供了重要参考。

An Establishmented Methods of Researching Silk Gland of Silkworm(Bombyx mori L.) with Proteomics

We separated proteins of the middle silk gland through high resolution two-dimensional polyacrylamide gel electrophesis, and identified the high expressional proteins using MALDI-TOF-MS and analyzed the PMF in protein database by GPMAW( General Protein/Mass Analysis for Windows) . The protein database was forecasted through silkworm genome database. More than 500 spots were obtained from each gel by silver stain and more than 100 protein spots were obtained from gel by Coomassie brilliant blue stain. Most of them were distributed in the area from 15kD to 90kD with pH 3.5-7. Among the 25 Coomassie brilliant blue stained spots identified by MALDI-TOF-MS, more than 60% have relatively strong signal. Comparing with the result of using Mascot, the method using PMF database of silkworm not only can identify some known proteins, but also can identify some unknown proteins that have been forecasted in silkworm genome database. Accordingly, we founded a complete set of method that fit for researching proteome of silkworm.