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.