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

Published: 10 November 2023| Version 1 | DOI: 10.17632/ws58h3crzh.1


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.


Steps to reproduce

HeLa cells expressing normal or misfuntional Grubraw were cultured for 24 hours in RPMI medium (+10% FBS) with labeled carbon sources: 1,2-[13]C-glucose or 5-[13]C-glutamine. Growth media were changed to HBSS solution containing labeled carbon substrate with only labeled glucose (G), or a mixture of glucose and glutamine with only one labeled component (GQ* or G*Q). D-alanine were added (or not) into HBSS medium and cells were incubated under these conditions for 90 minutes before extraction. Medium were analyzed separately from the cells. Samples were derivatized with phenylhydrazine (PH) using a procedure described in details in our paper (missing citation). The derivatizing reagent (DR) was a freshly prepared 2 mM solution of PH base in acetonitrile-methanol-water mixture (2:2:1 v/v). For intracellular samples the cultural medium was removed and cells were incubated with 1 ml of DR (1h, −20°C). For extracellular samples an aliquot of cultural medium was mixed with DR (1:9) and incubated (1h, −20°C). Then the liquid phases were centrifuged and dried under vacuum (25°C) and dissolved in solvent A (see below). The column in the HPLC system was a Thermo C18 Hypersil Gold 100×2.1mm, 1.9 μm. Solvent A consisted of 5% acetonitrile, 0.3% triethylammonium formate (TEA) in water, and solvent B consisted of 0.3% TEA in acetonitrile. The following gradient was used: 0-3 min: isocratic 5%B, 23 min: 45%B, 24-29 min: isocratic 90% B. The mass-spectrometer (Thermo Q-Exactive HF, HESI-II ion source) was operated in negative ion mode. To register MS1 for PH derivatives we used single ion monitoring mode with 60 000 resolution at three m/z ranges: 176.066-180.066 (Pyr); 220.100-224.100 (OA); 234.100-238.100 (α-KG). MS2 spectra of these substances were registered in separate runs of parallel reaction monitoring mode for precursor ions: 177.066, 178.070, 179.073, 221.056, 222.060, 223.063, 235.072, 236.075, 237.0786, 238.0795 m/z at 30,000 resolution and HCD energy 10. MIDs were extracted from MS1 runs and corrected to natural isotope occurrence utilizing MS2 data. Additionally, we extracted the probabilities of tracer inclusion into the first position in a molecule from MS2 data. Details on spectra processing, the equation for MID correction, and statistical inference are provided in the Supplementary Methods of our main study (missed citation).


Institut bioorganiceskoj himii RAN, Rossijskij nacional'nyj issledovatel'skij medicinskij universitet imeni N I Pirogova, Federal'noe mediko-biologicheskoe agenstvo


Metabolism, Cancer Cell Line


Russian Science Foundation