When highly invasive cancer cells are cultured on an extracellular matrix substrate, they extend proteolytically active membrane protrusions, termed invadopodia, from their ventral surface into the underlying matrix. Our understanding of the molecular composition of invadopodia has rapidly advanced in the last few years, but is far from complete. To accelerate component discovery, we resorted to a proteomics approach by applying DIfference Gel Electrophoresis (DIGE) to compare invadopodia-enriched sub-cellular fractions with cytosol and cell body membrane fractions and the whole cell lysate. The fractionation procedure was validated through step-by-step monitoring of the enrichment in typical actin-related invadopodia-associated proteins. After statistical analysis, 129 protein spots were selected for peptide mass fingerprinting analysis; of these 76 were successfully identified and found to correspond to 58 proteins belonging to different functional classes including aerobic glycolysis and other metabolic pathways, protein synthesis, degradation and folding, cytoskeletal components and membrane-associated proteins. Finally, validation of a number of identified proteins was carried out by a combination of immuno-blotting on cell fractions and immunofluorescence localization at invadopodia. These results reveal newly identified components of invadopodia and open further avenues to the molecular study of invasive growth behavior of cancer cells.
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