Figure 1
From: Serial-omics of P53−/−, Brca1−/− Mouse Breast Tumor and Normal Mammary Gland
Workflow of the Serial-omics experiment. 10 mg of breast tumor tissue and 10 mg of normal mammary gland were harvested from the mouse and snap frozen separately in liquid nitrogen. The frozen tissue was ground to a powder over dry ice. The powder was solubilized in PBS and extracted with methyltert-butyl ether (MTBE), methanol and water. The upper non-polar liquid phase was collected, dried out and analyzed for untargeted lipidomics via DDA with a Thermo QExactive Plus high resolution Orbitrap mass spectrometer. The lower liquid phase was collected, dried out and analyzed with polarity switching for polar metabolomics via both targeted metabolomics (AB/SCIEX 5500 QTRAP hybrid triple quadrupole mass spectrometer) and untargeted metabolomics (high resolution Thermo QExactive HF Orbitrap). The precipitated protein pellet was re-suspended in sample buffer and separated via a SDS-PAGE gel, fractionated, digested with trypsin and enriched for phosphopeptides via TiO2. The digestion mixture and TiO2 enriched phosphopeptides were analyzed by DDA in pos mode with a Thermo QExactive HF.
