Department of Life Sciences, Università degli Studi di Milano, Milan, Italy.
PLoS One. 2012;7(11):e51062. doi: 10.1371/journal.pone.0051062. Epub 2012 Nov 30.
We report on specific magneto-capturing followed by Multidimensional Protein Identification Technology (MudPIT) for the analysis of surface-exposed proteins of intact cells of the bacterial opportunistic pathogen Pseudomonas aeruginosa. The magneto-separation of cell envelope fragments from the soluble cytoplasmic fraction allowed the MudPIT identification of the captured and neighboring proteins. Remarkably, we identified 63 proteins captured directly by nanoparticles and 67 proteins embedded in the cell envelope fragments. For a high number of proteins, our analysis strongly indicates either surface exposure or localization in an envelope district. The localization of most identified proteins was only predicted or totally unknown. This novel approach greatly improves the sensitivity and specificity of the previous methods, such as surface shaving with proteases that was also tested on P. aeruginosa. The magneto-capture procedure is simple, safe, and rapid, and appears to be well-suited for envelope studies in highly pathogenic bacteria.
我们报告了一种特定的磁捕获方法，随后进行多维蛋白质鉴定技术（MudPIT）分析，以研究机会性病原体铜绿假单胞菌完整细胞表面暴露的蛋白质。通过将细胞包膜碎片从可溶性细胞质部分进行磁分离，使 MudPIT 能够鉴定捕获的和邻近的蛋白质。值得注意的是，我们鉴定了 63 种直接被纳米颗粒捕获的蛋白质和 67 种嵌入细胞包膜碎片中的蛋白质。对于大量的蛋白质，我们的分析强烈表明它们要么是表面暴露的，要么是定位于包膜区域。大多数鉴定出的蛋白质的定位仅被预测或完全未知。这种新方法大大提高了先前方法的灵敏度和特异性，例如用蛋白酶进行表面刮削，该方法也在铜绿假单胞菌上进行了测试。磁捕获过程简单、安全、快速，似乎非常适合高度致病性细菌的包膜研究。
