| Breast cancer is the most common gynecological cancer in developed nations and represents a significant public health concern. In an attempt to better understand the underlying biology of breast cancer, several studies were carried out to characterize the MCF10 model of human breast cancer. In these studies, intact proteins from whole cell lysates were separated first by isoelectric point (pI) and then by reverse phase high performance liquid chromatography (RP-HPLC). Effluent was directed to an electrospray ionization time-of-flight mass spectrometer (ESI-TOF-MS) for intact protein molecular weight determination, or collected as fractions for proteolytic digest and peptide mass fingerprint (PMF) identification by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). These data were then recombined to form a proteomic profile of the cell lines which could then be compared to determine differential protein expression.; The first study compared one normal (MCF10A) and two malignant cell lines (MCF10CA1a and MCF10CA1d) using two pI fractions from the Rotofor(TM) isoelectric focusing chamber. Forty proteins from these fractions were identified and quantitated, with 27 of them designated as differentially expressed. These differentially expressed proteins include various cytokeratins (CK7, 8, 18, 19), heat shock proteins (HSP73 and GRP78), and Annexin II.; As an outgrowth of this initial study, an automated data analysis package, Protein Trawler(TM) (PT), was tested and evaluated for automated processing of ESI-TOF-MS datasets. PT significantly speeds data interpretation, reduces user-induced errors, and improves the scope protein mass mapping. Applications of Protein Trawler to MCF10 cell lines are presented and potential biomarkers from PT-processed data are discussed.; In a second study, a more in-depth profile of acidic protein expression was obtained for one transformed (MCF10AT1) and the two malignant cell lines (MCF10CA1a and MCF10CA1d). Utilizing chromatofocusing (CF) as the pI separation dimension and automated data analysis for mass map generation, seven CF fractions between pI 4.6 & 6.0 were analyzed for differential protein expression. Nearly 250 common proteins were observed between the cell lines, of which 135 were identified and 69 were found to be up/down-regulated. The broader impact of these changes on cell morphology, metabolism, and stress adaptation is addressed. |
