We have used a unique metastasis model system in combination with quantitative, comparative proteomics to identify novel markers associated with the ability of cancer cells to colonize and form metastasis, and subsequently analyzed the clinical relevance of these proteins using breast cancer biopsies from patients with known clinical outcome within a 10-year follow-up. The model system consists of two isogenic human breast cancer cell lines that are equally tumorigenic in mice, but one gives rise to metastasis while the other disseminates single cells that remain dormant in distant organs. Using stable isotopic labeling by amino acids in cell culture and subcellular fractionation, the nuclear, cytosol and mitochondria proteomes were analyzed by LC-MS/MS, identifying seven proteins that exhibited altered expression between the cell lines, including L-lactate dehydrogenase A, NADH-cytochrome b5 reductase 3 (CYB5R3), niemann-pick c1 protein, and nucleolar RNA helicase 2.