Proteomic evaluation of biological nanoparticles isolated from human kidney stones and calcified arteries.
Proteomic evaluation of biological nanoparticles isolated from human kidney stones and calcified arteries.
复制标题
从人类肾结石和钙化动脉中分离的生物纳米颗粒的蛋白质组学评估。
DOI：
10.1016/j.actbio.2010.05.004
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发表时间：
2010
影响因子：
9.7
通讯作者：
Lieske,JohnC
中科院分区：
文献类型：
--
作者：
Shiekh,FarooqA;Charlesworth,JonE;Kim,Sung-Hoon;Hunter,LarryW;Jayachandran,Muthuvel;Miller,VirginiaM;Lieske,JohnC
钙化生物纳米颗粒(NPs)是在细胞培养条件下从各种显示骨外矿化的组织样本的匀浆中发展而来的，包括肾结石和钙化的动脉瘤。在生物系统中缺乏明确识别NPs的探测器。因此，这项研究的目的是开始建立NPs的蛋白质组生物特征，以便于更明确地研究它们对疾病的贡献。从人肾结石和钙化动脉瘤中提取的生物纳米颗粒经乙二胺四乙酸过夜处理或在盐酸中短暂孵育后完全脱钙，分别通过透射电子显微镜和共聚焦显微镜观察到无钙壳和茜素红S染色。脱钙NPs含有大量蛋白质，包括一些来自牛血清的蛋白质，以及其他来自原核生物的蛋白质。后者中最突出的是EF-Tu，它似乎与表皮葡萄球菌的EF-Tu相同。抗人EF-Tu的单抗通过免疫荧光和免疫金EM识别总NP裂解物的Western blotts中的蛋白质，以及完整的NPs中的蛋白质。使用流式细胞术，抗体可以定量识别大约8%的NPs。因此，我们定义了可重复地脱钙的生物NPs的方法，并确定了其蛋白质组的关键成分。这些元素，包括EF-Tu，可以用作生物标志物，进一步定义介导生物NPs传播的过程及其对疾病的贡献。
Calcifying biological nanoparticles (NPs) develop under cell culture conditions from homogenates of diverse tissue samples displaying extraosseous mineralization, including kidney stones and calcified aneurysms. Probes to definitively identify NPs in biological systems are lacking. Therefore, the aim of this study was to begin to establish a proteomic biosignature of NPs in order to facilitate more definitive investigation of their contribution to disease. Biological NPs derived from human kidney stones and calcified aneurysms were completely decalcified by overnight treatment with ethylenediaminetetraacetic acid or brief incubation in HCl, as evidenced by lack of a calcium shell and of Alizarin Red S staining, by transmission electron microscopy and confocal microscopy, respectively. Decalcified NPs contained numerous proteins, including some from bovine serum and others of prokaryotic origin. Most prominent of the latter group was EF-Tu, which appeared to be identical to EF-Tu from Staphylococcus epidermidis. A monoclonal antibody against human EF-Tu recognized a protein in Western blots of total NP lysate, as well as in intact NPs by immunofluorescence and immunogold EM. Approximately 8% of NPs were quantitatively recognized by the antibody using flow cytometry. Therefore, we have defined methods to reproducibly decalcify biological NPs, and identified key components of their proteome. These elements, including EF-Tu, can be used as biomarkers to further define the processes that mediate propagation of biological NPs and their contribution to disease.