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

  • Rommel A. Mathias
  • Hong Ji
  • Richard J. SimpsonEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 854)


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 



Two-dimensional gel electrophoresis


Ammonium persulfate


Biological variation analysis


Conditioned medium


Differential in-gel analysis


Two-dimensional fluorescence difference gel electrophoresis


Dulbecco’s Modified Eagle Medium






Extended data analysis


Epithelial-mesenchymal transition


Fetal calf serum


Liquid chromatography


Madin Darby canine kidney


Mass spectrometry


Room temperature



This work was supported, in part, by the National Health & Medical Research Council of Australia (program grant #487922 (R.J.S)), and funds from the Operational Infrastructure Support Program provided by the Victorian Government of Australia.


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Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  • Rommel A. Mathias
    • 1
  • Hong Ji
    • 1
  • Richard J. Simpson
    • 1
    Email author
  1. 1.Ludwig Institute for Cancer ResearchParkvilleAustralia

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