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A more recent version of this article appeared on January 1, 2007.
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Submitted on March 31, 2006
Revised on September 25, 2006
Accepted on October 5, 2006

Multi-variable Difference Gel Electrophoresis and Mass Spectrometry: A Case Study on TGF-beta and ErbB2 signaling

David B. Friedman, Shezhen E. Wang, Corbin W. Whitwell, Richard M. Caprioli, and Carlos L. Arteaga

Biochemistry, Mass Spectrometry Research Center, Vanderbilt University Medical Center, Nashville, TN 37232

Corresponding Author: david.friedman{at}vanderbilt.edu

Multi-variable difference gel electrophoresis coupled with mass spectrometry (DIGE/MS) was used to investigate proteins altered in expression and/or post-translational modification in response to activation of TGF-beta receptors in MCF10A mammary epithelial cells overexpressing the HER2/Neu (ErbB2) oncogene. Proteome changes were monitored in response to exogenous TGF-beta over time (0, 8, 24 and 40 hours), and proteins were resolved using medium range (pH 4-7) and narrow range (pH 5.3-6.5) isoelectric focusing combined with up to 2 mg protein to allow inspection of lower-abundant proteins. Triplicate samples were prepared independently and analyzed together across multiple DIGE gels using a pooled-sample internal standard to quantify expression changes with statistical confidence. Unsupervised Principle Component Analysis and hierarchical clustering of the individual DIGE proteome expression maps provided independent confirmation of distinct expression patterns from the individual experiments and demonstrated high reproducibility between replicate samples. Fifty-nine proteins (including some isoforms) that exhibited significant kinetic expression changes were identified using mass spectrometry and database interrogation, and were mapped to existing biological networks involved in TGF-beta signaling. Several proteins with a potential role in breast cancer, such as maspin and cathepsin D, were identified as novel molecules associated with TGF-beta signaling.


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