LC-MS/MS measurements of ketoacids isotopologues distribution in HeLa cells after 90 min of intramitochondrial pyruvate influx by Grubraw

We analysed 13C metabolic inclusion into ketoacids 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 cell lines with functional DadA and mutated DadA for control. In cells expressing functionally active DadA, D-alanine generates additional influx of intra-mitochondrial pyruvate. 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 LC-MS. Our cells were cultured for in a medium with labeled glucose or glutamine. To obtain current data, we changed rich growth media to pure phosphate medium containing labeled carbon substrate (with only labeled glucose, or a mixture of glucose and glutamine with only one labeled component), added D-alanine, and incubated cells under these conditions for 90 minutes before extraction. Using this scheme, we focused on acute effects of Grubraw activation. We publish original RAW data from Orbitrap mass-spectrometer of intra- and extracellular ketoacid analysis. In “G” folders we put experiments with glucose as the only carbon source. In “GQ” folders, there are measurements for glucose+glutamine experiments. All files are named in a similar way. C12/C13 in the beginning indicates using of isotopically labeled carbon source or non-labeled one. “M”/“D” at the second position indicate using a cell line with mutated (M) or functional (D) DadA. “Plus”/“minus” indicates the addition of D-alanine. G/Q in the end of files inside “GQ” folder indicate the experiment with labelled glucose or glutamine correspondingly (the other source is unlabelled). First number is a biological replicate, second number - technical replicate.