Proteomic analysis of the Escherichia coli outer membrane
Proteomic analysis of the Escherichia coli outer membrane
复制标题
DOI：
10.1046/j.1432-1327.2000.01296.x
复制
发表时间：
2000-05-01
期刊：
影响因子：
--
通讯作者：
Gooley, AA
中科院分区：
文献类型：
--
作者：
Molloy, MP;Herbert, BR;Gooley, AA
革兰氏阴性菌的外膜蛋白（OMPs）是连接细胞与环境的关键分子。传统的生物化学和遗传学方法已经产生了大量与omp功能有关的知识。尽管如此，随着大肠杆菌基因组测序项目的完成，有机会进一步扩大我们对这种革兰氏阴性细菌中omp的组织、表达和功能的理解。在本报告中，我们描述了一种蛋白质组学方法，它为omp的平行分析提供了一个平台。我们提出了一种快速分离细菌OMPs的方法，采用碳酸盐培养、纯化和二维电泳蛋白质阵列，然后用质谱法鉴定蛋白质。应用该方法检测在最小培养基中生长的大肠杆菌K-12细胞，我们鉴定了26个（80%）预测的完整omp中的21个，这些omp在SWISS-PROT release 37中有注释，预计在pH 4-7和分子质量10-80 kDa的范围内分离。还鉴定出5种外膜脂蛋白，仅观察到非膜蛋白的轻微污染。重要的是，这项研究很容易证明，通常在二维凝胶图中缺失的整体omp可以通过二维电泳分离。鉴定出的两个omp （YbiL, YeaF）以前只从它们的orf中知道，它们的鉴定证实了同源基因的转录和翻译。此外，我们发现与大肠杆菌铁受体FhuE和FhuA一样，YbiL的表达因铁限制而显著增加，这表明该蛋白可能在铁运输中起作用。在另一个演示中，我们展示了平行蛋白分析在记录受培养温度影响的大肠杆菌OMP表达变化方面的价值。
Outer membrane proteins (OMPs) of Gram-negative bacteria are key molecules that interface the cell with the environment. Traditional biochemical and genetic approaches have yielded a wealth of knowledge relating to the function of OMPs. Nonetheless, with the completion of the Escherichia coli genome sequencing project there is the opportunity to further expand our understanding of the organization, expression and function of the OMPs in this Gram-negative bacterium. In this report we describe a proteomic approach which provides a platform for parallel analysis of OMPs. We propose a rapid method for isolation of bacterial OMPs using carbonate incubation, purification and protein array by two-dimensional electrophoresis, followed by protein identification using mass spectrometry. Applying this method to examine E. coli K-12 cells grown in minimal media we identified 21 out of 26 (80%) of the predicted integral OMPs that are annotated in SWISS-PROT release 37 and predicted to separate within the range of pH 4-7 and molecular mass 10-80 kDa. Five outer membrane lipoproteins were also identified and only minor contamination by nonmembrane proteins was observed. Importantly, this research readily demonstrates that integral OMPs, commonly missing from 2D gel maps, are amenable to separation by two-dimensional electrophoresis. Two of the identified OMPs (YbiL, YeaF) were previously known only from their ORFs, and their identification confirms the cognate genes are transcribed and translated. Furthermore, we show that like the E. coli iron receptors FhuE and FhuA, the expression of YbiL is markedly increased by iron limitation, suggesting a putative role for this protein in iron transport. In an additional demonstration we show the value of parallel protein analysis to document changes in E. coli OMP expression as influenced by culture temperature.