Metabolism meets signaling to fine-tune cellgrowth naturecellbio
AMP ; and 0.2 μl deuterated citric acid was added to each tube. The samples were vortexed for 10 s immediately after the addition of the extraction buffer and then incubated for 30 min on an orbital shaker at 4 °C. The proteins were pelleted by centrifugation and the supernatants were transferred to a clean 2-ml tube.
The lipid pellets were resuspended in 200 µl ultra-performance liquid chromatography -grade acetonitrile:isopropanol 70:30 . The samples were vortexed for 10 s, incubated for 10 min in a thermomixer at 4 °C and then centrifuged for 5 min at 10,000and 4 °C. The cleared supernatants were transferred to 2-ml glass vials with 200 µl glass inserts . All samples were placed in an Acquity iClass UPLC sample manager at 6 °C.
All samples were analysed in a randomized run order. Targeted data analysis was performed using the quan module of the TraceFinder 4.1 software in combination with a sample-specific in-house-generated compound database. For measurements of intracellular TAG abundance , the sum of all values corresponding to the peak areas for the different individual TAGs was calculated for each sample.An OPP incorporation assay was used to test for de novo protein synthesis.
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