Monitoring of changes in the membrane proteome during stationary phase adaptation of Bacillus subtilis using in vivo labeling techniques
Monitoring of changes in the membrane proteome during stationary phase adaptation of Bacillus subtilis using in vivo labeling techniques
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DOI：
10.1002/pmic.200701081
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发表时间：
2008-05-01
期刊：
影响因子：
3.4
通讯作者：
Voelker, Uwe
中科院分区：
文献类型：
--
作者：
Dreisbach, Annette;Otto, Andreas;Voelker, Uwe
枯草芽孢杆菌已被开发为生理蛋白质组学的模型系统。然而，到目前为止，这些研究主要局限于细胞质，细胞外和细胞壁附着蛋白。虽然对于细胞生理学来说肯定是重要的，但是由于传统的基于2-DE的工作流程的不可及性和可靠定量的局限性，膜蛋白级分还没有被以可比的深度研究。在这项研究中，我们现在比较了稳定同位素标记氨基酸（SILAC）和N-14/N-15标记的潜力，用于生理驱动的蛋白质组学研究中细菌膜组分的分析。使用B的适应。枯草杆菌的氨基酸（赖氨酸）和葡萄糖饥饿作为证明的原则方案，我们表明，这两种方法提供了类似的有价值的数据，细菌膜蛋白的定量。即使用稳定的氨基酸标记允许更直接的数据分析，N-14/N-15-标记通常具有一些优点，例如标记氨基酸，从而增加用于定量的肽的数量。SILAC和N-14/N-15标记均与2-DE、2-D LC-MS/MS和GeLC-MS/MS兼容，因此将允许全面同时询问细胞质和富集的膜蛋白质组。
Bacillus subtilis has been developed as a model system for physiological proteomics. However, thus far these studies have mainly been limited to cytoplasmic, extracellular, and cell-wall attached proteins. Although being certainly important for cell physiology, the membrane protein fraction has not been studied in comparable depth due to inaccessibility by traditional 2-DE-based workflows and limitations in reliable quantification. in this study, we now compare the potential of stable isotope labeling with amino acids (SILAC) and N-14/N-15-labeling for the analysis of bacterial membrane fractions in physiology-driven proteomic studies. Using adaptation of B. subtilis to amino acid (lysine) and glucose starvation as proof of principle scenarios, we show that both approaches provide similarly valuable data for the quantification of bacterial membrane proteins. Even if labeling with stable amino acids allows a more straightforward analysis of data, the N-14/N-15-labeling has some advantages in general such as labeling of an amino acids and thereby increasing the number of peptides for quantification. Both, SILAC as well as N-14/N-15-labeling are compatible with 2-DE, 2-D LC-MS/MS, and GeLC-MS/MS and thus will allow comprehensive simultaneous interrogation of cytoplasmic and enriched membrane proteomes.