家蚕5龄第3天血液蛋白的双向电泳及质谱分析

基于为家蚕血液比较蛋白质组学的研究提供基础信息的目的,利用双向电泳技术对家蚕5龄幼虫的血液蛋白进行分析,并采用基质辅助质量飞行时间质谱(MALD I-TOF MS)对其中一些表达量较高的蛋白进行了肽质量指纹图谱分析鉴定。120μg血液蛋白经过双向凝胶电泳及考马斯亮蓝染色后可以检测出106个蛋白点,60μg血液蛋白经双向电泳及银染后可以检测出126个蛋白点,这些蛋白点主要集中在分子质量15~80 kD区域,等电点4~7。MALD I-TOF MS鉴定的52个蛋白点中都有较强的肽质量指纹信号峰,其中成功鉴定了46个蛋白点。在这些蛋白中,不仅包含了SP贮存蛋白和30 K低分子量脂蛋白等家蚕血液蛋白的主要成分,还包含了大量与代谢相关的蛋白酶类、蛋白酶抑制剂、离子转运蛋白、免疫相关因子、分子伴侣等不同种类的蛋白。

Study on Hemolymph Proteins of Silkworm (Bombyx mori) Using Two-dimensional Electrophoresis and Mass Spectrometry

To provide assistance for comparative proteomics of silkworm hemolymph,two-dimensional polyacrylamide gel electrophoresis,in this job,was used to investigate hemolymph of silkworm,Bombyx mori.The matrix-assisted laser desorption ionization time of flight mass spectrometry were applied for identifying the major spots of the 2D map.The result showed that about 106 spots were obtained by Coomassie brilliant blue staining from 120 μg hemolymph protein,while 126 spots were obtained from 60 μg hemolymph protein by silver staining.Most of them were distributed in the area from 15 kD to 80 kD with pI 4~7.A total of 52 spots were excised to identify by MALDI-TOF MS after digested with trypsin.The result showed that 46 proteins were identified sucessfully.They included not only the SP2,30K protein,but also plenty of proteins involved in metabolism enzyme,protease inhibitor,ion transportation,immunity,and molecular chaperone etc.