Francis Anthony W, Ruggiero Christy E, Koppisch Andrew T, Dong Jingao, Song Jian, Brettin Thomas, Iyer Srinivas
Bioscience Division, Los Alamos National Laboratory, P.O. 1663, Los Alamos, NM 87545, USA.
Biochim Biophys Acta. 2005 May 15;1748(2):191-200. doi: 10.1016/j.bbapap.2005.01.007.
Mass spectrometry and proteomics have found increasing use as tools for the rapid detection of pathogenic bacteria, even when they are in a mixture of non-pathogenic relatives. The success of this technique is greatly augmented by the availability of publicly accessible proteomic databases for specific pathogenic bacteria. To aid proteomic detection analyses for the causative agent of anthrax, we have constructed a comprehensive proteomic catalogue of vegetative Bacillus anthracis Sterne cells using liquid chromatography tandem-mass spectrometry. Proteins were separated by molecular weight or isoelectric point prior to tryptic digestion. Alternatively, the whole protein extract was digested and tryptic peptides were separated by cation exchange chromatography prior to Reverse Phase-LC-MS/MS. The use of three complementary, pre-analytical separation techniques resulted in the identification of 1048 unique proteins, including 694 cytosolic, 153 membrane (including 27 cell wall), and 30 secreted proteins, accounting for 19% of the total predicted proteome. Each identified protein was functionally categorized using the gene attribute database from TIGR CMR. These results provide a large proteomic catalogue of vegetative B. anthracis cells and, coupled with the recent proteomic catalogue of B. anthracis spore proteins, form a thorough summary of proteins expressed in the active and dormant stages of this organism.
质谱分析和蛋白质组学作为快速检测病原菌的工具，其应用越来越广泛，即使病原菌与非致病亲缘菌混合存在时也能进行检测。特定病原菌可公开获取的蛋白质组数据库极大地提高了这项技术的成功率。为了辅助对炭疽病病原体进行蛋白质组检测分析，我们使用液相色谱串联质谱法构建了炭疽芽孢杆菌斯terne株营养体细胞的综合蛋白质组目录。在进行胰蛋白酶消化之前，根据分子量或等电点对蛋白质进行分离。或者，将全蛋白提取物进行消化，胰蛋白酶肽段在进行反相液相色谱 - 串联质谱分析之前通过阳离子交换色谱进行分离。使用三种互补的分析前分离技术，共鉴定出1048种独特蛋白质，其中包括694种胞质蛋白、153种膜蛋白（包括27种细胞壁蛋白）和30种分泌蛋白，占预测蛋白质组总数的19%。利用TIGR CMR的基因属性数据库对每个鉴定出的蛋白质进行功能分类。这些结果提供了炭疽芽孢杆菌营养体细胞的大型蛋白质组目录，并且与最近的炭疽芽孢杆菌孢子蛋白蛋白质组目录相结合，全面总结了该生物体在活跃和休眠阶段表达的蛋白质。
