Difference Gel Electrophoresis (DIGE)

Volume 854 of the series Methods in Molecular Biology pp 269-286


Proteomic Profiling of the Epithelial-Mesenchymal Transition Using 2D DIGE

  • Rommel A. MathiasAffiliated withLudwig Institute for Cancer Research
  • , Hong JiAffiliated withLudwig Institute for Cancer Research
  • , Richard J. SimpsonAffiliated withLudwig Institute for Cancer Research Email author 

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Metastasis remains the primary cause of cancer patient death. Although the precise molecular mechanisms at play remain largely unknown, tumor progression is currently hypothesized to follow a series of sequential steps known as the metastatic cascade. An important component, thought to be involved early in this cascade, is the process known as epithelial-mesenchymal transition (EMT), whereby epithelial cells undergo morphogenetic alterations and acquire properties typical of mesenchymal cells. EMT confers a metastatic advantage to the cancer cells through the loss of cell-cell adhesion, enhanced proteolytic activity, and increased cell migration and invasiveness. This chapter describes the experimental workflow for the secretome analysis of MDCK cells undergoing oncogenic Ras, and Ras/TGF-β-mediated EMT. To enable this comparison, serum-free cell culture conditions were optimized, and a secretome purification methodology established. Secretome samples were then subjected to DIGE analysis to reveal and quantify proteins that are differentially expressed during EMT. The proteomic strategy detailed within successfully identified several EMT modulators and broadens our understanding of the extracellular facets of the EMT process.

Key words

Epithelial-mesenchymal transition EMT MDCK DIGE Ras Secretome Quantitative proteomics