GC-MS analysis of 13C metabolic labeling of HeLa kyoto cells expressing DadA in answer to intramitochondrial pyruvate influx by Grubraw
We analysed 13C metabolic inclusion into aminoacids, lactate, and fatty acids in HeLa cell lines stably expressing Pseudomonas aeruginosa DadA (FAD-dependent D-amino acid dehydrogenase) gene with mitochondrial targeting (named Grubraw) after the addition of 12C D-alanine comparing to no D-alanine. We used HeLa with functional DadA and mutated DadA for control. In cells expressing functionally active DadA, D-alanine generates additional influx of intra-mitochondrial pyrovate. If cells grows on labelled carbone source and unlabeled D-alanine is used, this upcoming pyruvate shifts original mass-isotopologue distribution that can be registered by GC-MS. Our cells were cultured for in a medium with labeled glucose or glutamine. To obtain current data at 90 min timescale, we changed rich growth medium with labelled glucose to pure phosphate medium containing labeled glucose as only carbon substrate. In 24h timescale, we added labelled glucose or glutamine into rich medimum and change it to fresh one 24h before the extraction. We publish original CDF data from Shimadzu TQ8040 mass-spectrometer. In “G” folders we put experiments with glucose as the only carbon source. In “GQ” folders, there are measurements for glucose+glutamine experiments. G/Q in folder name indicates the experiment with labelled glucose or glutamine correspondingly (the other source is unlabelled). “90m”/”24h” - indicate the treatment scheme (see next section). “C12”/”C13” in folder name indicates using of isotopically labeled carbon source or non-labeled one. All files are named in a similar way. “M”/“D” in file name indicate using a cell line with mutated (M) or functional (D) DadA. “+”/“-” indicates the addition of D-alanine. First number is a biological replicate, second number - technical replicate.
Steps to reproduce
90-min experiments. HeLa cells expressing normal or misfuntional Grubraw were cultured for 24 hours in RPMI medium (+10% FBS) with 1,2-C-glucose. Growth media were changed to HBSS solution containing labeled glucose. D-alanine were added (or not) into HBSS medium and cells were incubated under these conditions for 90 minutes before extraction. Cell medium were analyzed. 24-h experiments. HeLa cells expressing normal or misfuntional Grubraw were cultured for 24 hours in RPMI medium (+10% FBS) with 1,2-C-glucose and C-glutamine (G samples) or C-glucose and 5-C-glutamine (Q samples). Growth media were changed to the fresh one with the same composition and added (or not added) D-alanine 24 h before the extraction. Medium were analyzed separately from the cells. The cell medium was treated with cold methanol (4:1), refrigerated (1 hour, -22°C) and centrifuged (14000 g, 10 min). 1.5 ml of each supernatant were transferred to a new microcentrifuge tube and derivatized with MSTFA as described in (Long, Antoniewicz, 2019). D-Norvaline was added before the procedure as an internal standard for concentration measurements. From cellular fractions we precipitated proteins with acetone. Cells were centrifuged (1000 g, 5 min), prechilled water-acetone (1:4) mixture was added and pipetted, the resulting suspension was frozen (2 h, -20°C). Proteins were then precipitated (14000 g, 25 min), washed with dry acetone, precipitated again, and dried in air. The protein sediment then was used to obtain proteinogenic aminoacids by hydrolysis in 6N HCl (24 h, +100°C). Then samples were dried under vacuum (25°C) and derivatized with MSTFA as described in (Long, Antoniewicz, 2019). Lipids were extracted by standard Folch’s procedure. Then lipids were dried from chloroform under constant stream of nitrogen and then processed according to (Long, Antoniewicz, 2019). Lipids were then resuspended with methanol : sulfuric acid mixture for further hydrolysis and simultaneous production of methyl ether of fatty acids (2h, 100°C). The mixture was colded to RT and then vortexed with MS-grade hexane. Non-polar fraction was extracted for further GC-MS analysis. The GC–MS system was a Shimadzu TQ8040 GC–MS, with an Agilent CP-Sil 8 CB (50m × 0.25 mm i.d. × 0.25 μm) column. The carrier gas was UHP-grade helium at 0.88 mL/min. GC–MS analysis of the prepared samples was performed as described in (Long, Antoniewicz, 2019). 0.5 μL was injected in the split mode at a ratio of 1:50 with two or three replicates. For control of the GC–MS system, data collection and initial preprocessing, the Shimadzu GCMS Postrun Analysis software was used. Mass isotopomer distributions (MIDs) for lactate were extracted from raw spectra using xcms library (see comcon1/fluxNotebooks GitHub repository).