Metabolite Extraction and Mass Spectrometry

A detailed description of the metabolite extraction and mass spectrometry analysis is described in^{47 (link)}. In brief, samples were thawed on ice and 100 µL was mixed with ice-cold methanol spiked with a cocktail of internal standards. After vortexing and 30 min incubation in − 20 °C followed by centrifugation in 12 min at 4 °C, the samples were dried down and reconstituted in 100 µL 5% MeOH, 0.1% formic acid and 94.9% deionized MilliQ water upon analysis. 10 µL was injected in a randomized order constrained to the factor age into a Thermo Ultimate 3000 HPLC equipped with a Thermo Accucore aQ RP C18 column (100 × 2.1 mm, 2.6 µm particle size) and coupled to a Thermo Q-Exactive Orbitrap. A global pool of all samples was injected repeatedly, followed by a blank injection for quality control and filtering purposes. Finally, a twofold serial dilution series ranging from 0.5 to 32.0 µL QC was injected.