Abstract
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.
Highlights
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
In epithelial tissues, reduced expression of the cellcell adhesion molecule E-cadherin correlates with epithelial to mesenchymal transition, tissue invasion, and metastasis and is a prognostic biomarker of poor clinical outcome in many cell types [7,8,9]
This method removes organelles and cytoskeletal and cytosolic proteins while leaving behind secreted, transmembrane, and peripheral membrane proteins associated with the ventral aspect of the cell, which remain adsorbed to the glass coverslip (Fig. 1A). These proteins can be digested with a protease to generate peptides that can be identified after separation by LC-FTMS
Summary
Cell Culture—Keratinocyte cultures established from PG knock-out (PGϪ/Ϫ, PGϪ/Ϫ B2, and PGϪ/Ϫ B3) or heterozygous control (PGϩ/Ϫ) mouse skin [25] were cultured in defined keratinocyte serum-free medium (Invitrogen) supplemented with 10 ng/ml epidermal growth factor, and 10Ϫ10 M cholera toxin. Cells were rinsed with PBS, fixed in anhydrous methanol for 2 min at Ϫ20 °C, stained with 6D8 (1:100) antibody, and visualized with Alexa Fluor 488-conjugated secondary goat anti-mouse antibody (1:400). Cells were detached with cell detachment solution (sterile, calcium-free PBS containing 2.5 mM EDTA and 0.1% glucose), resuspended in their growth medium, and collected by centrifugation at 1500 rpm for 10 min at 4 °C. Because cell culture media contained serum, proteins of serum origin were identified using control samples (surfaces without cells). This allowed serum proteins to be filtered out of the protein lists generated for the deroofed cells, leaving only monolayer-isolated proteins for comparison by spectral counting. ProteinCenterTM (Proxeon, Cambridge, MA) was used to report gene ontologies of proteins detected
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