二维电泳-质谱分析斯氏按蚊中约氏疟原虫卵囊黑化相关的差异表达蛋白

目的　用 2 DE和PMF分离和鉴定约氏疟原虫卵囊黑化相关的斯氏按蚊中肠和血淋巴差异表达蛋白。方法　用固相pH梯度二维电泳分离中肠和血淋巴差异表达蛋白 ,考马斯亮蓝染色 ,ImageMasterVDS CL凝胶扫描和PDQuest2DElite软件分析 ,差异蛋白质点用基质辅助激光解析电离飞行时间质谱测定其胶内酶解后的肽质指纹图谱 ,用PeptIdent软件查询SWISS PROT数据库。结果　获得了分辨率和重复性均较好的 2 DE考马斯亮蓝图谱 ,吸硝喹糖水组蚊血淋巴和中肠蛋白点分别有 10 1个和 10 4个 ,感染组分别有 115个和 162个 ,匹配点分别为 5 1个和 44个。斯氏按蚊血淋巴和中肠蛋白质组中蛋白点分别主要分布于酸性端和碱性端 ,血淋巴和中肠差异蛋白也分别主要分布于酸性端和碱性端。随机取2 5个差异蛋白点进行胶内原位酶解 肽质指纹图谱分析得到了 16个蛋白质肽质指纹图 ,查询数据库初步鉴定了 8个蛋白质 ,分别为与抗疟免疫、黑化、结构、糖代谢、细胞通讯等相关蛋白。结论　建立了一套重复性和分辨率较好的斯氏按蚊蛋白质分离和鉴定的二维凝胶电泳 肽质谱指纹分析法 ,约氏疟原虫卵囊黑化前后 ,斯氏按蚊血淋巴和中肠伴随大量差异表达蛋白 ,部分蛋白可能与黑化有关。

Analysis of differentially expressed proteins from Anopheles stephensi related with Plasmodium yoelii oocyst melanization by two-dimensional electrophoresis and MALDI-TOF-mass spectrometry

Objective To separate and identify the differentially expressed proteins in the midguts and hemolymph from Anopheles stephensi adult females by two dimensional polyacrylamide gel electrophoresis and peptide mass fingerprinting (PMF). Methods A series of methods, including immobilized pH gradient two dimensional polyacrylamide gel electrophoresis, Coomassie brilliant blue staining, scanning by the Image Master VDS CL apparatus, PDQuest 2D Elite analysis software, peptide mass fingerprinting based on matrix assisted laser desorption/ionization time of flying mass spectrometry (MALDI TOF MS) by in gel enzymologic extract, and SWISS PROT database searching, were used to separate and identify the differentially expressed proteins in the midguts and hemolymph. Results The 2 DE pattern with higher resolution and reproducibility was obtained. After Coomassie brilliant blue staining, the 2 DE image analysis by PDQuest 2 DE software detected 101 spots in the hemolymph and 104 spots in the midgut from the nitroquine acetate feeding group, respectively, and 115 spots in the hemolymph and 162 spots in those from infected blood feeding group, respectively. Fifty one matched spots in the hemolymph and 44 matched spots in the midgut were shared between the two groups. Differentially expressed proteins spots in the hemolymph were mainly located at isoelectric points of the basic end, but the differential spots in the midgut at the acidic end. Twenty five spots were incised from Coomassie brilliant blue staining gel randomly and digested in gel by TPCKtrypsin. Sixteen peptide mass fingerprint maps were obtained by MALDI TOF MS. The typical peptide masses were searched in the SWISS PROT database by PeptIdent software. Eight proteins or proteinases, related with anti malarial immunity, melanization, structure, carbohydrate metabolism, and cell communication, were identified in the database. Conclusion We have established an analytic method of protein separation by 2 DE and identification by PMF with the advantages of higher resolution and better reproducibility. Our results show that markedly differentially expressed proteins can be observed in the midgut and the hemolymph before and after melanization of Plasmodium yoelii oocysts. Partial differential protein spots might be related with melanization.