A proteomic characterization of one premalignant (MCF10AT1) and two malignant (MCF10CA1a and MCF10 CA1d) human breast cancer cell lines has been performed using a combination of two-dimensional liquid separations and mass spectrometry. Chromatofocusing (CF) and NPS-RP-HPLC are combined with ESI-TOF-MS to resolve and detect intact proteins. Simultaneously, fractions are collected and digested for protein identification using MALDI-MS peptide mass fingerprinting. Following protein identification a small database was compiled for use in comparison between IDs and measured masses taking into account variables such as pI, hydrophobicity and potential modifications. Out of 239 mass bands detected between pH 4.6 and 6.0, 133 have been definitively associated with identified proteins and 67 show consistent up/down regulation in two malignant breast cancer cell lines relative to the precursor premalignant cell line. Of these, 8 are also altered in the premalignant MCF10AT1 cell line by treatment with estradiol. Differentially expressed proteins indicate significant changes to the cytoskeleton, cellular metabolism, and adaptation to environmental stressors in malignant cell lines.

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