Steely H Thomas, Dillow Glen W, Bian Liangqian, Grundstad Jason, Braun Terry A, Casavant Thomas L, McCartney Mitchell D, Clark Abbot F
Alcon Research, Ltd., Fort Worth, TX 76134, USA.
Mol Vis. 2006 Apr 18;12:372-83.
Characterization of the human trabecular meshwork (TM) proteome is hindered by the small mass of intact tissue and the slow growth of cultured cell strains. We have previously characterized a transformed TM cell strain (GTM3) that demonstrates many of the same protein expression and cell signaling systems of nontransformed cell strains. The aim of this study was to initiate a proteomic survey of GTM3 cells as the initial step toward characterization of the complete human TM proteome.
GTM3 cells were cultured to confluence, harvested and solubilized in urea/Nonidet. The protein extract (600 mug) was focused in immobilized isoelectric focusing (IEF) strips, separated by 10% SDS PAGE, and visualized with colloidal Coomassie Blue. Spots of interest were excised, destained, and the contained proteins subjected to in-gel reduction, derivatization, and tryptic digestion. Tryptic peptides were extracted and analyzed by electrospray LC/MS/MS. Protein identification was made using the TurboSequest search algorithm and a recent version of the nonredundant human protein database downloaded from the National Center for Biotechnology Information (NCBI).
Eighty-seven (87) primary proteins and 93 variants of these proteins were identified. A website was created (TM proteome) that combines data such as graphic spot location within the gel, peptide sequence, apparent and calculated pI, apparent and calculated mass, percentage of coverage, and protein informatic website links.
Proteomic analysis of a transformed human TM cell line has been initiated combining preparative two-dimensional PAGE separation, LC/MS/MS analysis of major proteins, and bioinformatic cataloging of the data. Further investigation of data from the transformed cell strain will be used in a comparative fashion for spot identification of analytical proteomic gels of human TM tissue and cultured normal cells. These initial data will form the base from which the characterization of protein expression in the normal and glaucomatous TM can be accomplished.
完整组织量少以及培养细胞株生长缓慢阻碍了对人小梁网（TM）蛋白质组的表征。我们之前已对一种转化的TM细胞株（GTM3）进行了表征，该细胞株表现出许多与未转化细胞株相同的蛋白质表达和细胞信号系统。本研究的目的是启动对GTM3细胞的蛋白质组学调查，作为表征完整人TM蛋白质组的第一步。
将GTM3细胞培养至汇合状态，收获并在尿素/去氧胆酸钠中溶解。蛋白质提取物（600微克）在固定化等电聚焦（IEF）条带中聚焦，通过10% SDS-PAGE分离，并用考马斯亮蓝胶体染色可视化。感兴趣的斑点被切除、脱色，其中所含蛋白质进行胶内还原、衍生化和胰蛋白酶消化。提取胰蛋白酶肽段并通过电喷雾液相色谱/串联质谱（LC/MS/MS）分析。使用TurboSequest搜索算法和从美国国立生物技术信息中心（NCBI）下载的最新版非冗余人类蛋白质数据库进行蛋白质鉴定。
鉴定出87种主要蛋白质及其93种变体。创建了一个网站（TM蛋白质组），该网站整合了凝胶内图形斑点位置、肽序列、表观和计算得到的pI、表观和计算得到的质量、覆盖率百分比以及蛋白质信息网站链接等数据。
已启动对一种转化的人TM细胞系的蛋白质组学分析，结合了制备性二维PAGE分离、主要蛋白质的LC/MS/MS分析以及数据的生物信息学编目。来自转化细胞株的数据将以比较的方式用于人TM组织和培养的正常细胞的分析蛋白质组凝胶的斑点鉴定。这些初始数据将构成完成正常和青光眼性TM中蛋白质表达表征的基础。
