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Multivariable Difference Gel Electrophoresis and Mass Spectrometry: A Case Study on Transforming Growth Factor-β and ERBB2 Signaling *S

https://doi.org/10.1074/mcp.D600001-MCP200Get rights and content
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Multivariable DIGE/MS was used to investigate proteins altered in expression and/or post-translational modification in response to activation of transforming growth factor (TGF)-β receptors in MCF10A mammary epithelial cells overexpressing the HER2/Neu (ErbB2) oncogene. Proteome changes were monitored in response to exogenous TGF-β over time (0, 8, 24, and 40 h), 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 of protein to allow inspection of lower abundance 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-β signaling. Several proteins with a potential role in breast cancer, such as maspin and cathepsin D, were identified as novel molecules associated with TGF-β signaling.

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Published, MCP Papers in Press, October 5, 2006, DOI 10.1074/mcp.D600001-MCP200

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This work was supported in part by NCI, National Institutes of Health, RO1 Grants CA62212 (to C. L. A.), Breast Cancer Specialized Program of Research Excellence (SPORE) Grant P50 CA98131, and Vanderbilt Ingram Comprehensive Cancer Center Support Grant P30 CA68485 (to D. B. F., R. M. C., and C. L. A., members). Institutional support for proteomics was received from Vanderbilt University through the Academic Venture Capital Fund. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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The on-line version of this article (available at http://www.mcponline.org) contains supplemental material.