| 马驰,刘淑清,宗军卫,袁章利,孙明忠,吴玉群,李建立,孙立新,郑庆印.2009.双向电泳结合质谱初步分析蛇岛蝮蛇毒蛋白质组.动物学杂志,44(4):70-77. |
| 双向电泳结合质谱初步分析蛇岛蝮蛇毒蛋白质组 |
| Proteomic Analysis of Gloydius shedaoensis shedaoensis Venom by Two-dimensional Electrophoresis Combined to High Performance Liquid Chromatography-electro-spray Ionization Tandem Mass Spectrometry:a Preliminary Study |
| 投稿时间：2009-01-16 修订日期：2009-04-23 |
| DOI： |
| 中文关键词: 蛇岛蝮 蛇毒 双向电泳 高效液相色谱 电喷雾质谱 |
| 英文关键词:Gloydius shedaoensis shedaoensis Snake venom Two-dimensional electrophoresis High performance liquid chromatography Electro-spray ionization tandem mass spectrometry |
| 基金项目:大连医科大学大学生科研活动资助项目;辽宁省自然科学基金项目(No.2008S077);大连科技厅资助项目(No.2008J22JH014);辽宁省百千万人才工程资助项目(No.2008921069) |
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| 中文摘要: |
| 采用荧光染料(Cy5)标记中国辽宁蛇岛蝮(Gloydius shedaoensis shedaoensis,GSS)蛇毒(snake venom,SV,GSS-SV)蛋白质,获得了该蛇毒的双向十二烷基硫酸钠聚丙烯酰胺凝胶电泳(2D SDS-PAGE)图谱,经DeCyder软件分析,分辨出1000多个蛋白点,分子量范围在10～150ku间,等电点在4～7的蛋白质点占78.8%。凝胶后染色(post-staining)采用蛋白荧光染料Deep Purple,选取的5个蛋白点经胶内酶解,产生的肽段经高效液相色谱-电喷雾串联质谱(high performance liquid chromatography/HPLC-electro-spray ionization tandem mass spectrometry/ESI-MS/MS,HPLC-ESI-MS/MS)进行序列测定,质谱数据经Sequest Bioworks软件分析,为蛇毒L-氨基酸氧化酶、金属蛋白酶、类凝血酶、纤溶酶原激活物和磷脂酶A2的同源蛋白。本研究采用的荧光标记2DSDS-PAGE结合HPLC-ESI-MS/MS的技术适于高通量研究蛇毒蛋白组成。 |
| 英文摘要: |
| The protein components contained in the snake venom of Gloydius shedaoensis shedaoensis（GSS-SV） were labeled with the fluorescent dye of Cy5.Then,the two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis（2D SDS-PAGE） profile of GSS-SV proteome was obtained.Gel image analysis by DeCyder software resolved over 1 000 protein spots with the apparent molecular weights ranging between 10 and 150 ku on the gel.The protein spots with the pI ranging between 4 and 7 covered about 78.8% of the overall protein spots detected by the 2D SDS-PAGE.Post-staining of the gel was performed by a high sensitive fluorescent dye,Deep Purple.Five protein spots were excised from the gel and digested by trypsin in-gel.The tryptic peptides were separated by high performance liquid chromatography（HPLC） and subsequently sequenced by electro-spray ionization tandem mass spectrometry（ESI-MS/MS）.The mass spectrometry data were searched against the NCBInr database through the software of Sequest Bioworks for protein identifications.The above mentioned five gel spots were identified as the snake venom L-amino acid oxidase,metalloproteinase,thrombin-like enzyme（salmobin）,plasminogen activator and phospholipase A2,respectively.The results prove that the proteomic techniques based on two-dimensional electrophoresis,protein fluorescent labeling technique and HPLC-ESI-MS/MS can be utilized in the analysis of protein components in the snake venom complex at high throughput level. |
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