白眉蝮蛇蛇毒糖基化蛋白的质谱分析

Analysis of glycoproteins of Gloydius blomhoffi Brevicaudus snake venom by mass spectrometry

关键词:  蛇毒  糖基化  Pro-Q-Emerald  电喷雾电离串联质谱

Key words:  snake venom  glycosylation  Pro-Q-Emerald dye  ESI-MS/MS

摘要:

［目的］蛋白质组学方法分析和初步鉴定旅顺产白眉蝮蛇(Gloydius blomhoffi Brevicaudus) 蛇毒中的糖蛋白。［方法］SDS-PAGE用于白眉蝮蛇粗毒蛋白质组份分离，糖蛋白凝胶荧光染料Pro-Q-Emerald染糖基化蛋白，采用胰蛋白酶酶解蛋白质，用反相C18-HPLC分离肽段混合物，用纳升电喷雾电离串联质谱(nESI-MS/MS)系统进行的质谱数据采集，蛋白质鉴定用Biowroks软件搜索NCBInr数据库完成。［结果］旅顺产白眉蝮蛇蛇毒L-氨基酸氧化酶、金属蛋白酶、谷氨酰环化酶、salmobin、纤溶酶原激活物、contortrixobin、磷脂酶A₂，神经增长因子和较小分子量的L-氨基酸氧化酶被初步鉴定为糖蛋白，其匹配的肽段数分别为8、4、7、2、3、3、4、2和5个。L-氨基酸氧化酶的纯化与表征结果验证了其糖基化，并以多种糖基化形式存在。［结论］采用蛋白质组学手段从旅顺产白眉蝮蛇蛇毒中鉴定出了8种糖蛋白，该方法简便、快速、准确。

Abstract:

［Objective］A novel proteomics approach was created to analyze and identify glycoproteins in the venom of Gloydius blomhoffi Brevicaudus snake (GBB snake).［Methods］SDS-PAGE was used to separate the protein components in snake venom complex. Pro-Q-Emerald, a fluorescent glycoprotein specific dye, was used to assess the glycoproteins. Peptides generated by the trypsin digestion of proteins were separated by RP-HPLC on a C18 column in prior to their introduction into the nESI /MS/MS system. Protein identification was carried out by Bioworks software searching against the NCBInr database.［Results］Proteins homologs of L-amino acid oxidase, metalloproteinase, glutaminyl cyclase, salmobin, plasminogen activator, halystase, phospholipase A₂, nerve growth factor and truncated/degraded L-amino acid oxidase product were identified as the glycosylated proteins in GBB snake venom. And the numbers of matched peptides for these were 8, 4, 7, 2, 3, 3, 4, 2 and 5, respectively. The results were validated by the experimental characterization of pure L-amino acid oxidase isolated from GBB snake venom.［Conclusions］Eight types of proteins in the venom of GBB snake were identified as glycoproteins. The approach established in this study merits the advantages of rapid operation, and accurate and reproducible measurement.