【作者】 曹锐；

【导师】 王贤纯； 梁宋平；

【作者基本信息】 湖南师范大学 ， 生物化学与分子生物学， 2004， 硕士

【摘要】 采用多维色谱-串联质谱联用技术分析鉴定小鼠肝脏质膜蛋白质。以强阳离子交换柱(5mm×320μm)为第一相，反相柱(150mm×75μm)为第二相，在两相之间通过六通阀连接一预柱(5mm×300μm)用来脱盐和浓缩肽段。用密度梯度超速离心方法获得的小鼠肝脏细胞质膜样品经酶解并适当酸化后通过离子交换柱吸附，然后分别用10个不同的盐浓度进行洗脱。洗脱物经预柱脱盐和浓缩后进入毛细管反相柱进行反相分离，分离后的肽段直接进入质谱仪离子源进行一级和二级质谱的分析。质谱仪采得的数据经计算机处理后用Mascot软件进行库搜寻鉴定。通过这种方法，从小鼠肝组织质膜蛋白提取物中一共鉴定出126种蛋白质，其中43种为膜蛋白或与膜相关的蛋白质，包括用2DE方法所未曾鉴定的具有多跨膜区的部分膜蛋白质。本研究为建立质膜蛋白质组学研究的多维色谱-质谱联用技术提供了有价值的基础性研究资料。 我们还首次对大鼠海马组织质膜蛋白质组进行了研究。通过密度梯度超速离心的方法获得质膜蛋白质后，采取一维聚丙烯酰胺凝胶电泳(1DE)和二维聚丙烯酰胺凝胶电泳(2DE)结合串联质谱的方法对其进行了分离和鉴定，并对两种不同方法鉴定膜蛋白质的效果进行了比较。发现1DE对膜蛋白质鉴定的效果要比2DE好。在本研究中，通过1DE结合串联质谱的方法一共鉴定出51种基因表达的不同蛋白质，其中有19种位于质膜上。而通过2DE结合质谱的方法只鉴定出19种蛋白质，只有1种蛋白质明确位于质膜上。更多还原

【Abstract】 Multidimensional liquid chromatography coupled with tandem massspectrometry was used to identify mouse liver plasma membrane(PM) proteins. A strong cation-exchange(SCX) column worked as the first phase and a reversed-phase capillary column (150 mm 75 m)as the second phase. A pre-column was incorporated between the two phases of chromatography .The mouse liver plasma membrane was obtained by the density-gradient centrifugation and the PM proteins were extracted by detergents-assisted solution. After an optimized tryptic digestion of the PM proteins , the digested peptide mixture was loaded onto the SCX column (5 mm 320 m) . 10 different concentrations of ammonium acetate solution were injected to elute the ion-exchanged peptides respectively .Following desalinisation the eluted peptides were further purified by reversed-phase capillary column(150 mm 75 m) and online analysed by the coupled tandem mass spectrometry. The acquired MS and MS/MS spectra , after being processed with analytical software Proteinlynx ,were utilized to search the Swiss-Prot database with Mascot search engine for protein identification .By this method , a total of 126 proteins were identified ,of which 43 were known membrane proteins .14 of the membrane proteins were integral proteins or proteins with multiple transmembrane domains (TMDs), which were unidentified by using two-dimensional polyacrylamide gel electrophoresis followed bymass spectrometry (2DE-MS). This work provides fundamental information of the mouse liver plasma membrane proteome .We have also made a preliminary study on the proteome of adult rat hippocampus plasma membrane. The hippocampal plasma membrane isolated from the brains of adult rat with sucrose density-gradient centrifugation. The plasma membrane protein were extracted and subjected to one-dimensional (IDE) and two-dimensional gel electrophoresis, followed by in-gel tryptic digestion and analysis through liquid chromatography coupled with tandem mass spectrometry. We Compared the results of the two methods for proteomic analysis of adult rat hippocampus plasma membrane. In the present study, 51 proteins were identified, of which 19 proteins were known plasma membrane proteins, by IDE in combination with liquid chromatography coupled with tandem mass spectrometry. But only one of 19 identified proteins was plasma membrane protein by 2-DE-LC-MS/MS. It appears that 1DE-LC-MS/MS is more suitable for separating and analyzing plasma membrane proteins than 2DE/LC-MS/MS.更多还原