Targeted metabolomics in control or PERKKO AML cells incubated with vehicle or GSK621
Description
Related to Figure 3. Results are provided as amounts of metabolites in pmol/106 cells. ATP: adenosine triphosphate; ADP: adenosine diphorphate; AMP: adenosine monophosphate; G6P: glucose-6-phosphate; FBP: fructose 1,6 biphosphate; 23BPG: 2,3 biphosphoglyceric acid; PEP: phosphoenolpyruvic acid; 6PG: 6-phosphogluconate; P5P: ribose_ribulose_xylulose 5-phosphate; SED7P: Sedoheptulose 7-phosphate; AKG: alpha ketoglutarate; GMP: guanosine monophosphate; GDP: guanosine diphosphate; CMP: cytosine monophosphate; CDP: cytosine diphosphate; CTP: cytosine triphosphate; UMP: uridine monpphosphate; UDP: uridine biphosphate; UTP: uridine triphosphate; UDP gLC: UDP-glucose; UDP AcGlnc: UDP-N-acetylglucosamine; G3P: Glycerol 3-phosphate; MAN 6P: mannose 6-phosphate.
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Sampling of central metabolites by fast filtration We harvested 1-3x106 cells by vacuum filtration (nylon, 0.45 μm pore size, 47 mm, Sartorius, Göttingen, Germany) and washed with the same volume of NaCl (0.9%, 4°C) solution during 20s or less. Metabolism was quenched by placing the cell-containing filter in a centrifugation tube filled with 3 ml of a mix of 80% methanol, 20% water and 100 μl Isotope-Dilution Mass Spectrometry (IDMS) at -80°C during 15 min.. Samples were then centrifuged (2000 g, -10°C, 5min), and cell pellet were incubated with 1 ml of the quenching solution before another centrifugation (2000g, -10°C, 5min). Pellets were lyophilized, dissolved in 200μl water, centrifuged (2,000 g, 4°C, 5min) and then stored at -80°C. Mass spectrometry Ion chromatography mass spectrometry (IC/MS) analysis of intracellular central metabolites was performed by high performance anion exchange chromatography (Dionex ICS 2000 system, Sunnyvale, USA) coupled to a LTQ Orbitrap Velos mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) equipped with a heated ESI probe. All samples were analyzed FTMS mode at a resolution of 60,000 (at m/z 400) with the following source parameters: capillary temperature was 350°C, source heater temperature was 350 °C, sheath gas flow rate was 50, auxiliary gas flow rate was 5, S-Lens RF level was 60%, and source voltage was 2,7 kV. The injection volume was 15 μl. Samples were injected on a Dionex IonPac AS11 column (250 x 2mm) equipped with a Dionex AG11 guard column (50 x 2mm). Mobile phase was composed of a KOH gradient which varied as follows: 0min 0.5; 1min 0.5; 9.5min 4.1; 14.6min 4.1; 24min 9.65; 36min 60; 36.1min 90; 43min 90; 43.1min 0.5; 45min 0.5. Data processing We used the TraceFinder software (Thermo Fisher Scientific) for processing of metabolomic data. Mass spectrometry quantifications are performed on 3x106 cells, and concentrations are compared to a range of concentration for each metabolite determined using 13C-chemical labeled internal standards through the IDMS method (Bennett et al., 2008). The application of the IDMS approach performed with the calibration curve of a known amount of a fully 13C-labeled E. coli extract reduces errors caused by variations occurring during analysis and sample processing. Then the ratio between the unlabeled metabolites of interest and corresponding 13C labelled metabolites are measured and used to determine the exact concentrations of the metabolites of interest. Data are expressed in pM of metabolite per 106 cells. Experiments were done in experimental triplicate, and repeated three time independently. For each replicate, adenylate energy charge (AEC) was calculated as ([ATP]+0,5*[ADP])/ ([ATP]+[ADP]+[AMP]) and used as a quality control of the metabolomic sampling process, and samples with AEC ≤ 0.75 were excluded.