Biological Sciences Division, Pacific Northwest National Laboratory, Richland, Washington, USA.
J Proteome Res. 2010 Sep 3;9(9):4454-63. doi: 10.1021/pr100215h.
A simple and effective subcellular proteomic method for fractionation based on osmotic lysis, differential centrifugation, and Sarkosyl solubilization was applied to the Gram-negative bacterium Shewanella oneidensis to gain insight into its subcellular architecture. Global differences in bacterial cytoplasm, inner membrane, periplasm, and outer membrane protein fractions were observed by SDS PAGE and heme staining, and tryptic peptides were analyzed using high-resolution liquid chromatography-tandem mass spectrometry. Proteins predicted to be localized to each subcellular fraction were enriched approximately 2-fold (on average) in each fraction compared to crude cell lysates. In addition, the Sarkosyl solubilization method facilitated separation of the inner and outer membranes, making the procedure amenable for effective probing of the subcellular proteome of Gram-negative bacteria via liquid chromatography-tandem mass spectrometry. With 40% of the observable proteome represented, this study provides extensive information on both subcellular architecture and relative abundance of proteins in S. oneidensis and provides a foundation for future work on subcellular organization and protein-membrane interactions in other Gram-negative bacteria.
一种基于渗透压裂解、差速离心和 Sarkosyl 溶解的简单有效的亚细胞蛋白质组学分离方法被应用于革兰氏阴性菌希瓦氏菌，以深入了解其亚细胞结构。通过 SDS-PAGE 和血红素染色观察到细菌细胞质、内膜、周质和外膜蛋白组分的全局差异，并使用高分辨率液相色谱-串联质谱分析肽段。与粗细胞裂解物相比，预测定位于每个亚细胞区室的蛋白质在每个区室中富集了约 2 倍（平均水平）。此外，Sarkosyl 溶解方法促进了内膜和外膜的分离，使得通过液相色谱-串联质谱有效地探测革兰氏阴性菌的亚细胞蛋白质组成为可能。在可观察到的蛋白质组的 40%中，本研究为希瓦氏菌的亚细胞结构和蛋白质相对丰度提供了广泛的信息，并为其他革兰氏阴性细菌的亚细胞组织和蛋白质-膜相互作用的未来工作奠定了基础。
