Cells were handled in identical manor as described above for bulk RNAseq experiment, with the exception of plating in 6-well dishes at approximately 4.36 × 105 cells per well. Four hours following the media change (stim or control), polar metabolites were extracted for GCMS analysis. Immediately following the last incubatory period of each experiment, plates were removed from the incubator and placed on ice, the media was removed, cells were washed twice with ice cold 0.9% NaCl (VWR BDH Analytical #BDH9286-2.5KG), then 1mL of ice cold 50% MeOH [HPLC-grade (Sigma-Aldrich #A456-4); containing 20μM L-norleucine (Sigma-Aldrich #N6877-1G)] was added to quench cellular metabolic activity followed by 10 min incubation at −80 °C to ensure cell lysis. After removing from the freezer, plates were placed on ice, wells scraped with a cell scraper and the entire contents collected into a tube, vortexed briefly and placed on ice until all samples were collected. Tubes were then placed on a Disruptor Genie Cell Disruptor Homogenizer (Scientific Industries) for 5 min at 3,000 rpm, followed by centrifugation at 24,000 × g for 10 min at 4 °C. The aqueous fraction was isolated to a new tube for further processing, the resulting pellet was briefly dried at 10−3 mbar CentriVap vacuum concentrator (LabConco) to evaporate any remaining methanol. Following drying, the protein content of the pellet was determined using a BCA assay (ThermoFisher #23225) to normalize metabolite concentrations to total protein amount. The aqueous fraction containing polar metabolites was dried at 10−3 mbar followed by derivatization. The dried polar metabolite pellet was derivatized by a two-step methoxyamine protocol first by addition of 50 μL methoxyamine HCl (Sigma-Aldrich #226904-5G) in pyridine (20 mg/mL; Sigma-Aldrich #TS25730) followed by 90 min dry heat incubation at 30 °C. Samples were then centrifuged at 20,000 × g for 10 minutes after which 50μL of each sample was transferred to an amber V-shaped glass chromatography vial (Agilent #5184-3554) containing 80μL N-methyl-trimethylsilyl-trifluoroacetamide (MSTFA; ThermoFisher #TS48915) and gently vortexed followed by 30 min dry heat incubation at 37°C. The samples were allowed to cool to room temperature, and then analyzed via gas chromatography (GC) mass spectrometry (MS).
Samples were analyzed on an Agilent 8890 GC / 5977B MS (Agilent Technologies, Santa Clara, CA, USA). A GC temperature gradient of 130°C was held for 4 min, rising at 6°C/min to 243°C, rising at 60°C/min to 280°C and held for 2 min. Electron ionization energy was set to 70eV. Scan mode for m/z: 50–550 was used for steady-state metabolomics and scan mode for m/z: 50–800 was used for stable-isotope resolved metabolomics. Spectra were translated to relative abundance using the Automated Mass Spectral Deconvolution and Identification System (AMDIS) v2.73 software with retention time and fragmentation pattern matched to FiehnLib library1 64 (link) with a confidence score of > 80.
Samples were analyzed on an Agilent 8890 GC / 5977B MS (Agilent Technologies, Santa Clara, CA, USA). A GC temperature gradient of 130°C was held for 4 min, rising at 6°C/min to 243°C, rising at 60°C/min to 280°C and held for 2 min. Electron ionization energy was set to 70eV. Scan mode for m/z: 50–550 was used for steady-state metabolomics and scan mode for m/z: 50–800 was used for stable-isotope resolved metabolomics. Spectra were translated to relative abundance using the Automated Mass Spectral Deconvolution and Identification System (AMDIS) v2.73 software with retention time and fragmentation pattern matched to FiehnLib library1 64 (link) with a confidence score of > 80.
