Abstract
This 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.
Highlights
It is important for a complete view on diseases such as cancer to investigate changes on multiple levels of biological activity as each one gives a unique but partial profile
We analyzed breast tumor and normal mammary tissue from the same mouse in triplicate in order to develop a strategy that can be applied to tumor biopsied samples or dried blood spots using a comprehensive one-step methyl tert-butyl ether (MTBE) based liquid-liquid extraction protocol for serial-omics[19,20]. It creates a methyl-tert-butyl ether (MTBE), methanol/water and solid phase separation where the non-polar lipids are soluble in the upper phase, the aqueous polar metabolites are soluble in the middle phase and proteins, long DNA/RNA and cell debris are found as a precipitated insoluble pellet at the bottom of the tube
We started with 10 mg of mouse breast tumor tissue and 10 mg of mouse mammary gland tissue from the same mouse (K14-Cre BRCA1f/fp53f/f female)[23] and propagated in syngeneic littermate as described[24,25,26]
Summary
It is important for a complete view on diseases such as cancer to investigate changes on multiple levels of biological activity as each one gives a unique but partial profile. We show a comprehensive serial study from MTBE extractions from mouse breast tumor tissue and mammary gland tissue samples where we identified and quantified more than 4,792 proteins, 1,072 unique phospho-Ser/Thr/Tyr containing peptides, 805 polar metabolites and 1,382 non-polar lipids from 10 mg of starting material using tandem mass spectrometry (LC-MS/MS). We utilized a combination of high resolution and triple quadrupole mass spectrometry for both untargeted and targeted analyses This method offers a chance to evaluate metabolites, lipids, phosphopeptides and proteins from a single liquid-liquid extraction without technical variation in handling. It provides the possibility to illustrate the biological pathways in a tumor that drive its growth This strategy could form the basic concept for using mass spectrometry based –omics technologies to identify potential new therapeutic targets and helping to identify the most effective treatment. We present a method that allows the detection of the non-genetic molecular biology of tumor samples on the proteomic, lipidomic and metabolomic level in a single step
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