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Originally published In Press as doi:10.1074/mcp.T100007-MCP200 on November 19, 2001.
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Molecular & Cellular Proteomics 1:91-98, 2002.
© 2002 by The American Society for Biochemistry and Molecular Biology, Inc.

Technology

Evaluation of Two-dimensional Differential Gel Electrophoresis for Proteomic Expression Analysis of a Model Breast Cancer Cell System*

Severine Gharbi, Piers Gaffney, Alice Yang, Marketa J. Zvelebil, Rainer Cramer, Michael D. Waterfield and John F. Timms{ddagger}

From the Ludwig Institute for Cancer Research, 91 Riding House Street, London W1W 7BS, United Kingdom

The technique of fluorescent two-dimensional (2D) difference gel electrophoresis for differential protein expression analysis has been evaluated using a model breast cancer cell system of ErbB-2 overexpression. Labeling of paired cell lysate samples with N-hydroxy succinimidyl ester-derivatives of fluorescent Cy3 and Cy5 dyes for separation on the same 2D gel enabled quantitative, sensitive, and reproducible differential expression analysis of the cell lines. SyproRuby staining was shown to be a highly sensitive and 2D difference gel electrophoresis-compatible method for post-electrophoretic visualization of proteins, which could then be picked and identified by matrix-assisted laser-desorption ionization mass spectroscopy. Indeed, from these experiments, we have identified multiple proteins that are likely to be involved in ErbB-2-mediated transformation. A triple dye labeling methodology was used to identify proteins differentially expressed in the cell system over a time course of growth factor stimulation. A Cy2-labeled pool of samples was used as a standard with all Cy3- and Cy5-labeled sample pairs to facilitate cross-gel quantitative analysis. DeCyder (Amersham Biosciences, Inc.) software was used to distinguish clear statistical differences in protein expression over time and between the cell lines.

{ddagger} To whom correspondence should be addressed. Tel.: 44-207-8784126; Fax: 44-207-8784040; E-mail: jtimms{at}ludwig.ucl.uk.


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