Steady state-polar metabolomics and lipidomics

Soleus muscles of C57BL/6J mice were treated with AAV6:lacZ-shRNA or AAV6:Yap-shRNA vectors and excised after 4 weeks for homogenisation in pre-chilled solutions on dry ice. The extraction of metabolites was initiated by the addition of 80:20 Methanol:Water containing 2 μM each of ¹³C-AMP, ¹³C-UMP, ¹³C Sorbitol, and ¹³C Valine to the already snap-frozen samples, as internal standards. Muscles were homogenised using a micro homogeniser connected to a pre-chilled pellet pestle. Samples were then vortexed for 30 s before being sonicated for 5 min in an ice bath. Samples were incubated for 5 min at 4 °C before being fractionated into two groups of 250 μL each for steady-state-polar metabolomics and lipidomics analyses. The 250 μL volume for steady state-polar metabolomics was thermomixed for 10 min at 4 °C, then centrifuged at 18928 × g for 10 min at 4 °C before injecting 7 μL into the Agilent hydrophilic interaction (HILIC) LC and high-resolution mass spectrometry (QTOF-6545). Metabolite peak calling and quality check was performed using QTOF MassHunter Quant software (Agilent). The remaining 250 μL of extract was used for lipidomic analysis following rigorous vortex agitation to resuspend the pellet. A 100 μL aliquot of this resuspended solution was used for subsequent lipid extraction. Lipids were extracted using 2:1 Methanol:Chloroform containing 2 μM each of internal standards of PC 19:0/19:0, PG 17:0/17:0, PE-d31, and TG-d5. Samples were then incubated in a Thermomix at 20 °C for 15 min, before centrifugation at 25200 × g at 4 °C for 10 min. The supernatant was evaporated in a rotational vacuum concentrator with nitrogen to complete dryness. Finally, the samples were reconstituted in 9:1 Butanol:Methanol before 2 μL injection into the Lipid Liquid Chromatography Mass Spectrometer (QQQ-6590, Agilent). Metabolite peak calling and quality check was performed using QQQ MassHunter Quant software (Agilent). Samples were normalised according to the soluble protein concentration as determined by DC Protein assay kit (Bio-Rad). Heatmaps were generated using MetaboAnalyst 4.0⁷³.