Novel application for pseudopodia proteomics using excimer laser ablation and two-dimensional difference gel electrophoresis
Novel application for pseudopodia proteomics using excimer laser ablation and two-dimensional difference gel electrophoresis
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DOI：
10.1038/labinvest.2012.98
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发表时间：
2012-09-01
影响因子：
5
通讯作者：
Kondo, Tadashi
中科院分区：
文献类型：
--
作者：
Ito, Akihiko;Mimae, Takahiro;Kondo, Tadashi
我们开发了一种新的应用程序来进行伪足蛋白质组学。伪足是一种富含肌动蛋白的腹侧突起，在细胞迁移中起着重要作用。伪足蛋白的鉴定有助于进一步了解肿瘤细胞的恶性表型和新的治疗策略。在我们的应用中，肿瘤细胞被放置在纤维连接蛋白涂层的多孔膜上形成伪足。根据细胞的运动能力，细胞在毛孔内形成伪足突起。一种用于眼科屈光手术的准分子激光，水平消融细胞膜表面的细胞以去除细胞体。显微镜观察和蛋白质表达研究表明，激光处理对伪足没有明显的损伤。将全细胞和伪足部分中的蛋白质分别溶解，用高灵敏的荧光染料标记，然后用双向差示凝胶电泳法分离。在观察到的2508个蛋白质斑点中，211个在整个细胞和伪足部分之间具有不同的强度(差异超过4倍，P-值
We developed a novel application to conduct pseudopodia proteomics. Pseudopodia are ventral actin-rich protrusions and play functional roles in cell migrations. Identification of pseudopodia proteins leads to a further understanding of malignant phenotypes of tumor cells and novel therapeutic strategies. In our application, tumor cells were placed on a fibronectin-coated porous membrane to form pseudopodia. According to the motile potentials of the cells, the cells formed pseudopodial nnicroprocesses in the pores. An excimer laser, which was used for ophthalmic refractive surgeries, horizontally ablated cells at the membrane surface to remove the cell body. The microscopic observations and the protein expression studies suggested that the laser treatment caused no apparent damages to pseudopodia. Proteins in whole cells and pseudopodia fractions were individually solubilized, labeled with a highly sensitive fluorescent dye, and separated using two-dimensional difference gel electrophoresis. Among 2508 protein spots observed, 211 had different intensity between whole cells and pseudopodia fractions (more than fourfold differences and P-value of