AbstractThis study demonstrates a liquid-liquid extraction for the sequential tandem mass spectrometry (LC-MS/MS) analysis of non-polar lipids, polar metabolites, proteins and phosphorylation sites from a single piece of tissue. Extraction of 10 mg BRCA−/−, p53−/− breast tumor tissue or normal mammary gland tissue with methyl-tert-butyl ether (MTBE) results in three phases: an upper non-polar phase containing 1,382 lipids, a lower polar phase with 805 metabolites and a precipitated protein pellet with 4,792 proteins with 1,072 phosphorylation sites. Comparative analysis revealed an activated AKT-mTOR pathway in tumors. Tumors also showed a reduction of phosphorylation sites involved in transcription and RNA splicing and decreased abundance of enzymes in lipid synthesis. Analysis of polar metabolites revealed a reduction in glycolysis, pentose phosphate pathway, polyamines and nucleotides, but an increase in TCA and urea cycle intermediates. Analysis of lipids revealed a shift from high triglycerides in mammary gland to high phospholipid levels in tumors. The data were integrated into a model showing breast tumors exhibit features on the proteomic, lipidomic and metabolomic level that are distinct from normal breast tissue. Our integrative technique lends itself to samples such as tumor biopsies, dried blood spots and fluids including urine and CSF to develop biomarkers of disease.