Solubilization of cellular membrane proteins from Streptococcus mutans for two-dimensional gel electrophoresis
Solubilization of cellular membrane proteins from Streptococcus mutans for two-dimensional gel electrophoresis
复制标题
DOI：
10.1002/elps.200410349
复制
发表时间：
2005-03-01
期刊：
影响因子：
2.9
通讯作者：
Brady, LJ
中科院分区：
文献类型：
--
作者：
Zuobi-Hasona, K;Crowley, PJ;Brady, LJ
膜蛋白很少在二维电泳 (2-DE) 蛋白质组图中被识别。这是由于丰度低、溶解度差和固有的疏水性导致第一维期间的自聚集。在这项研究中，使用三种不同的方法溶解来自革兰氏阳性细菌变形链球菌的膜蛋白，并通过 2-DE 进行评估。在第一种方法中，使用十二烷基硫酸钠（SDS）进行提取，然后用离液缓冲液溶解并用甲醇/氯仿沉淀。第二种方法基于使用 Triton X-114 的温度依赖性相分配，然后使用 Bio-Rad 的 ReadyPrep 2-D 净化试剂盒进行纯化。第三种方法涉及使用有机溶剂三氟乙醇 (TFE) 和氯仿进行萃取，产生三个独立的相。上层水相富含 TFE，具有最高的总蛋白质产量和最佳的 2-DE 分辨率。通过纳电喷雾四极杆飞行时间 (OTOF)-串联质谱 (MS/MS) 鉴定蛋白质点，揭示了已知的膜和表面相关蛋白质。这是第一份描述革兰氏阳性细菌膜蛋白成功溶解和二维电泳的报告。
Membrane proteins are rarely identified in two-dimensional electrophoretic (2-DE) proteomics maps. This is due to low abundancy, poor solubility, and inherent hydrophobicity leading to self-aggregation during the first dimension. In this study, membrane proteins from the Gram-positive bacterium Streptococcus mutans were solubilized using three different methods and evaluated by 2-DE. In the first method, the extraction was performed using sodium dodecyl sulfate (SDS) followed by solubilization with a chaotropic buffer and precipitation with methanol/chloroform. The second method was based on temperature-dependent phase partitioning using Triton X-114 followed by purification using the ReadyPrep 2-D clean-up kit from Bio-Rad. The third method involved extraction using the organic solvents trifluoroethanol (TFE) and chloroform, which produced three separate phases. The upper aqueous phase, enriched with TFE, gave the highest overall protein yield and best 2-DE resolution. Protein spot identification by nanoelectrospray quadrupole time of flight (OTOF)-tandem mass spectrometry (MS/MS) revealed known membrane and surface-associated proteins. This is the first report describing the successful solubilization and 2-D electrophoresis of membrane proteins from a Gram-positive bacterium.