Reactive oxygen species (ROS) have been implicated in the pathogenesis of human breast cancer. Defining the molecular pathways linking oxidative stress to cellular dysfunction could therefore help to develop novel treatment and prevention strategies. Two dimensional fluorescence difference gel electrophoresis (2-D DIGE), which resolves multiple samples labeled with distinct fluorescent dyes on the same 2-D gel, provides a rapid and reproducible way to compare the proteome. However, one of the major drawbacks with 2-D DIGE is that frequently multiple proteins are present in each spot. Stable isotope labeled proteome standard (SILAPS), which utilizes metabolically stable isotope labeled proteome as internal standard, overcomes this problem and allows for accurate quantification of the relative abundance of each protein using mass spectrometry. In this study, we utilized both 2-D DIGE and SILAPS to explore the effect of oxidative stress on human mammary epithelial cell lines. Nonmalignant (S1) and malignant (T4-2) cells were harvested after oxidant treatment (hydrogen peroxide) and their global protein expression patterns were compared using 2-D DIGE. The differentially expressed spots were analyzed using matrix-assisted laser desorption ionization time-of-flight (MALDI/TOF/TOF) and liquid chromatography electrospry tandem mass spectrometry (LC/ESI/MS/MS). For SILAPS experiment, proteome samples containing equal amount of stable isotope labeled internal standard were resolved by 2-D gel electrophoresis. Spots of interest from the 2-D DIGE experiment were picked, trypsin digested and quantitated by LC/ESI/MS/MS. We identified differentially expressed proteins, both novel and previously associated with oxidative stress response, including 1-cys peroxiredoxin, nucleophosmin, stratifin and ras-related nuclear protein. We also observed several proteins (e.g., heat shock protein 70 and cofilin) mapping to multiple spots with disparate expression changes following oxidant treatment, suggesting that post-translational protein modification may be an important cellular response to oxidative stress. Additional experiments are underway to explore the relevance of these observations to breast cancer prevention and treatment. This work is supported by NIH RO-1 CA95586.

[Proc Amer Assoc Cancer Res, Volume 46, 2005]