Protein expression becomes altered in breast epithelium during malignant transformation. Knowledge of these perturbations should provide insight into the molecular basis of breast cancer, as well as reveal possible new therapeutic targets. To this end, we have performed an extensive comparative proteomic survey of global protein expression patterns in proliferating MCF-7 breast cancer cells and normal human mammary epithelial cells using gel-free shotgun tandem mass spectrometry. Pathophysiological alterations associated with the malignant breast cancer phenotype were detected, including differences in the apparent levels of key regulators of the cell cycle, signal transduction, apoptosis, transcriptional regulation, and cell metabolism.