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

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。

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.