Detection of Differentially Expressed Basal Cell Proteins by Mass Spectrometry

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 ␤ 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. Molecular & Cellular Proteomics 9:351-361, 2010.

Plakoglobin regulates cell motility through Rho- and fibronectin-dependent Src signaling

“…The samples were trypsinized and analyzed by mass spectrometry (MS). Details of this novel method for mass spectrometry sample preparation and analysis are discussed elsewhere (Todorovic et al, 2010). Differences in protein levels were observed between integrin receptors, ECM and cytoskeletal molecules, as well as between membrane glycoproteins and matrix proteolysis regulators (Table 1).…”

“…S1; Table 1) (Todorovic et al, 2010), which may be due to the loss of FN as a primary a51 integrin ligand. Thus, because a31integrin has been recently implicated in the inhibition of keratinocyte motility on newly deposited LN-332 (Margadant et al., 2009), the decrease of 1 integrin could lead to attenuation of this inhibitory effect.…”

“…From the analysis of the expression patterns of the ECM components (Table 1; (Table 1) (Todorovic et al, 2010).…”

“…S1B). Not all ECM molecules relevant for keratinocyte adhesion and motility are differentially regulated by PG: a major basement membrane component laminin-332 was not affected (Table 1) (Todorovic et al, 2010). …”

Global Analysis Reveals the Complexity of the Human Glomerular Extracellular Matrix

“…This method was adapted from previously published methods 38 and used to reduce the complexity of protein samples for MS analysis by removing cellular components and enriching for ECM proteins. All steps were carried out at 4°C to minimize proteolysis.…”