Fig. 1

Extraction pipeline to obtain multiple data layers from the same sample. A sequential extraction method followed by an untargeted mass spectrometry approach was used to obtain multiple data layers from the same samples of whole brain homogenate (50 mg) from male and female WT and 3xTg-AD mice. A first extraction (pink arrows) was performed with methanol:water (4:1, v/v), and an aliquot of the resulting supernatant was analyzed for polar metabolites. The same sample was extracted a second time with BuMe (butanol:methanol 1:1, v/v, yellow arrows), and the pool from both organic solvent extractions was evaporated, reconstituted and analyzed for complex lipids. Proteins from the remaining pellet were extracted and digested by a modified iST method (1% Na+DCA, 30mM Tris 10 mM DTT), then analyzed (green arrows). Omics data were acquired using LC-ESI-HRMS. Using MS/MS spectral matching, 119 polar metabolites, 600 unique lipid species, and 5115 proteins were annotated with high confidence. WT: wild-type; 3xTg-AD: triple transgenic Alzheimer’s disease model; LC: liquid chromatography; ESI: electrospray ionization; HRMS: high resolution mass spectrometry; MS/MS: tandem mass spectrometry