State Key Laboratory for Molecular Virology and Genetic Engineering, Institute of Pathogen Biology, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, PR China.
BMC Genomics. 2011 Jan 18;12:40. doi: 10.1186/1471-2164-12-40.
Tuberculosis is an infectious bacterial disease in humans caused primarily by Mycobacterium tuberculosis, and infects one-third of the world's total population. Mycobacterium bovis bacillus Calmette-Guérin (BCG) vaccine has been widely used to prevent tuberculosis worldwide since 1921. Membrane proteins play important roles in various cellular processes, and the protein-protein interactions involved in these processes may provide further information about molecular organization and cellular pathways. However, membrane proteins are notoriously under-represented by traditional two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) and little is known about mycobacterial membrane and membrane-associated protein complexes. Here we investigated M. bovis BCG by an alternative proteomic strategy coupling blue native PAGE to liquid chromatography tandem mass spectrometry (LC-MS/MS) to characterize potential protein-protein interactions in membrane fractions.
Using this approach, we analyzed native molecular composition of protein complexes in BCG membrane fractions. As a result, 40 proteins (including 12 integral membrane proteins), which were organized in 9 different gel bands, were unambiguous identified. The proteins identified have been experimentally confirmed using 2-D SDS PAGE. We identified MmpL8 and four neighboring proteins that were involved in lipid transport complexes, and all subunits of ATP synthase complex in their monomeric states. Two phenolpthiocerol synthases and three arabinosyltransferases belonging to individual operons were obtained in different gel bands. Furthermore, two giant multifunctional enzymes, Pks7 and Pks8, and four mycobacterial Hsp family members were determined. Additionally, seven ribosomal proteins involved in polyribosome complex and two subunits of the succinate dehydrogenase complex were also found. Notablely, some proteins with high hydrophobicity or multiple transmembrane helixes were identified well in our work.
In this study, we utilized LC-MS/MS in combination with blue native PAGE to characterize modular components of multiprotein complexes in BCG membrane fractions. The results demonstrated that the proteomic strategy was a reliable and reproducible tool for analysis of BCG multiprotein complexes. The identification in our study may provide some evidence for further study of BCG protein interaction.
结核病是一种由结核分枝杆菌引起的人类传染性细菌性疾病，感染了全球三分之一的人口。自 1921 年以来，卡介苗（BCG）疫苗已在全球范围内广泛用于预防结核病。膜蛋白在各种细胞过程中发挥着重要作用，而这些过程中涉及的蛋白质-蛋白质相互作用可能提供关于分子组织和细胞途径的进一步信息。然而，传统的二维聚丙烯酰胺凝胶电泳（2-D PAGE）对膜蛋白的代表性严重不足，关于分枝杆菌膜和膜相关蛋白复合物的信息知之甚少。在这里，我们通过一种结合蓝色 native PAGE 和液相色谱串联质谱（LC-MS/MS）的替代蛋白质组学策略来研究牛分枝杆菌 BCG，以表征膜部分中的潜在蛋白质-蛋白质相互作用。
使用这种方法，我们分析了 BCG 膜部分中蛋白质复合物的天然分子组成。结果，在 9 个不同的凝胶条带中鉴定出 40 种蛋白质（包括 12 种整合膜蛋白）。使用 2-D SDS PAGE 对鉴定出的蛋白质进行了实验验证。我们鉴定出了参与脂质转运复合物的 MmpL8 和四个相邻蛋白，以及 ATP 合酶复合物的所有亚基均处于单体状态。在不同的凝胶条带中获得了属于单个操纵子的两个酚磷酸转移酶和三个阿拉伯糖基转移酶。此外，确定了两种巨大的多功能酶，Pks7 和 Pks8，以及四种分枝杆菌 Hsp 家族成员。此外，还发现了参与多核糖体复合物的 7 种核糖体蛋白和琥珀酸脱氢酶复合物的两个亚基。值得注意的是，在我们的工作中，一些疏水性或具有多个跨膜螺旋的蛋白质也得到了很好的鉴定。
在这项研究中，我们利用 LC-MS/MS 结合蓝色 native PAGE 来表征 BCG 膜部分中多蛋白复合物的模块化组件。结果表明，蛋白质组学策略是分析 BCG 多蛋白复合物的可靠且可重复的工具。我们的研究结果可能为进一步研究 BCG 蛋白相互作用提供一些证据。
