Biology Reference
In-Depth Information
Chapter 19
Proteomic Profi ling of the Epithelial-Mesenchymal
Transition Using 2D DIGE
Rommel A. Mathias , Hong Ji , and Richard J. Simpson
Abstract
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 sequen-
tial 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 workfl ow for the secre-
tome 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 purifi cation 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 identifi ed
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
Abbreviations
2DE
Two-dimensional gel electrophoresis
APS Ammonium persulfate
BVA Biological variation analysis
CM Conditioned medium
DIA Differential in-gel analysis
DIGE Two-dimensional fl uorescence difference gel electrophoresis
DMEM Dulbecco's Modifi ed Eagle Medium
DMF
Dimethylformamide
DTT
Dithiothreitol
EDA
Extended data analysis
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