Two-dimensional map of the proteome of Haemophilus influenzae
Two-dimensional map of the proteome of Haemophilus influenzae
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DOI：
10.1002/(sici)1522-2683(20000101)21:2
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发表时间：
2000-01-01
期刊：
影响因子：
2.9
通讯作者：
Fountoulakis, M
中科院分区：
文献类型：
--
作者：
Langen, H;Takács, B;Fountoulakis, M
我们已经构建了一个流感嗜血杆菌蛋白质组的二维数据库，这是一种具有医学意义的细菌，其完整基因组由大约1742个开放阅读框组成，已经被测序。利用不同pH区的固定化pH梯度条、不同浓度的丙烯酰胺凝胶和不同拖尾离子的缓冲液，对该微生物的可溶性蛋白质组分进行了双向电泳分析。为了可视化低拷贝数的基因产物，我们采用了一系列的蛋白质提取和样品应用方法以及几个层析步骤，包括肝素层析、层析聚焦和疏水作用层析。我们还使用固定化pH梯度条或两种洗涤剂系统分析了细胞包膜结合的蛋白质组分，第一种是阳离子洗涤剂，第二种是阴离子洗涤剂。用基质辅助激光解吸电离质谱仪和氨基酸组成分析鉴定了不同蛋白质(502)。这是目前最大的二维蛋白质组数据库之一。
We have constructed a two-dimensional database of the proteome of Haemophilus influenzae, a bacterium of medical interest of which the complete genome, comprising about 1742 open reading frames, has been sequenced. The soluble protein fraction of the microorganism was analyzed by two-dimensional electrophoresis, using immobilized pH gradient strips of various pH regions, gels with different acrylamide concentrations and buffers with different trailing ions. In order to visualize low-copy-number gene products, we employed a series of protein extraction and sample application approaches and several chromatographic steps, including heparin chromatography, chromatofocusing and hydrophobic interaction chromatography. We have also analyzed the cell envelope-bound protein fraction using either immobilized pH gradient strips or a two-detergent system with a cationic detergent in the first and an anionic detergent in the second-dimensional separation. Different proteins (502) were identified by matrix-assisted laser desorption/ionization mass spectrometry and amino acid composition analysis. This is at present one of the largest two-dimensional proteome databases.