Department of Pathology, Northwestern University Feinberg School of Medicine, Chicago, Illinois 60611, USA.
Mol Cell Proteomics. 2010 Feb;9(2):351-61. doi: 10.1074/mcp.M900358-MCP200. Epub 2009 Nov 30.
The ability of cells to modulate interactions with each other and the substrate is essential for epithelial tissue remodeling during processes such as wound healing and tumor progression. However, despite strides made in the field of proteomics, proteins involved in adhesion have been difficult to study. Here, we report a method for the enrichment and analysis of proteins associated with the basal surface of the cell and its underlying matrix. The enrichment involves deroofing the cells with 20 mM ammonium hydroxide and the removal of cytosolic and organellar proteins by stringent water wash. Proteomic profiling was achieved by LC-FTMS, which allowed comparison of differentially expressed or shared proteins under different cell states. First, we analyzed and compared the basal cell components of mouse keratinocytes lacking the cell-cell junction molecule plakoglobin with their control counterparts. Changes in the molecules involved in motility and invasion were detected in plakoglobin-deficient cells, including decreased detection of fibronectin, integrin beta(4), and FAT tumor suppressor. Second, we assessed the differences in basal cell components between two human oral squamous cell carcinoma lines originating from different sites in the oral cavity (CAL33 and UM-SCC-1). The data show differences between the two lines in the type and abundance of proteins specific to cell adhesion, migration, and angiogenesis. Therefore, the method described here has the potential to serve as a platform to assess proteomic changes in basal cell components including extracellular and adhesion-specific proteins involved in wound healing, cancer, and chronic and acquired adhesion-related disorders.
细胞调节与彼此和基质相互作用的能力对于上皮组织在伤口愈合和肿瘤进展等过程中的重塑是至关重要的。然而，尽管在蛋白质组学领域取得了进展，但参与黏附的蛋白质仍难以研究。在这里，我们报告了一种用于富集和分析与细胞基底表面及其下基质相关的蛋白质的方法。该富集涉及用 20mM 氨水处理去细胞的屋顶，并用严格的水洗去除细胞质和细胞器蛋白。通过 LC-FTMS 进行蛋白质组学分析，可以比较不同细胞状态下差异表达或共享的蛋白质。首先，我们分析并比较了缺乏细胞间连接分子 plakoglobin 的小鼠角质形成细胞的基底细胞成分与其对照细胞。在 plakoglobin 缺陷细胞中检测到参与运动和侵袭的分子发生变化，包括纤连蛋白、整合素β4 和 FAT 肿瘤抑制因子的检测减少。其次，我们评估了源自口腔不同部位的两种人口腔鳞状细胞癌细胞系之间的基底细胞成分差异（CAL33 和 UM-SCC-1）。数据显示，这两条细胞系在与细胞黏附、迁移和血管生成相关的蛋白质的类型和丰度上存在差异。因此，这里描述的方法有可能成为评估包括参与伤口愈合、癌症以及慢性和获得性黏附相关疾病的细胞外和黏附特异性蛋白质在内的基底细胞成分的蛋白质组学变化的平台。
