Chemicals
Stable isotope-labeled substrates were purchased at the following company: u-13C-glucose and 13C1-glucose are from Campro Scientific (Berlin, Germany). Extraction chemicals methanol and chloroform were products of Merck (Whitehouse Station, NJ, USA). All other chemicals were bought in highest quality at Sigma-Aldrich (St. Louis, MO, USA) unless otherwise noted.
Cell culture
Dr. Ulrike Ziebold (MDC Berlin-Buch, T98G, HeLa, HCT-116) and Dr. Markus Landthaler (MDC Berlin-Buch, HEK293) kindly provided the cell lines. The cell lines were cultivated in glucose-free Dulbecco's modified Eagle's medium (DMEM; Invitrogen, Renfrew, Scotland) supplemented with 2.5 g/l glucose, 10% fetal bovine serum (Invitrogen), and 1% penicillin/streptomycin (Invitrogen) and cultivated at 37°C in 5% CO2. The cells were passaged with appropriate split ratios every 3 days. Viable cell numbers were determined by trypan blue staining (0.04%, Invitrogen) and automated counting (BioRad, Hercules, CA, USA).
pSIRM experiment
Cell culture/cell seeding
The number of cells for plating was determined to avoid contact inhibitory effects during the experiment for each cell line and nutrient condition separately. After seeding, the cells were cultured for 2 or 3 days. During that time, cell culture media was replaced 24 and 4 h prior to harvest.
Cell labeling/harvest
The adherent growth behavior of the used cell lines allowed the labeling with 13C substrates directly on the cell culture dish. Therefore, the cell culture medium was replaced with pre-warmed full label medium containing all carbon sources and supplements like standard cell culture for a defined time range. One carbon source was substituted with its carbon-13 variant according to the setup of the experiment. Hereafter, the cells were quickly flushed with label buffer (140 mM NaCl, 5 mM HEPES (Roth, Karlsruhe, Germany) with pH 7.4, major carbon sources according full label media) to remove extracellular metabolites. Immediately, the cells were quenched with 5 ml -20°C cold 50% methanol (containing cinnamic acid (2 μg/ml)). The cells were scratched from the culture dish in the solvent, transferred into a 15-ml falcon, and stored on ice or at -25°C until proceeding with metabolite extraction. In the pSIRM experiments with an application of 13C substrates for less than 5 min, the cells were incubated in label buffer directly.
Metabolite extraction
Methanol-chloroform-water extraction provides an effective extraction and subsequent separation of lipid and polar intermediates. One milliliter chloroform was added to 5 ml of methanolic cell extracts, shaken for 30 min at 4°C, and centrifuged at maximum speed for 15 min for phase separation. Both phases were collected separately and dried under vacuum. The extracts were stored at -25°C.
GC-MS analysis
Derivatization was carried out as described with modifications[19]. The dried cell extracts were dissolved in 20 μl of methoxyamine hydrochloride solution (Sigma, 40 mg/ml in pyridine (Roth)) and incubated for 90 min at 30°C with constant shaking followed by the addition of 80 μl of N-methyl-N-[trimethylsilyl]trifluoroacetamide (MSTFA; Machery-Nagel, Dueren, Germany) and incubation at 37°C for 45 min. The extracts were centrifuged for 10 min at 10,000 × g, and aliquots of 30 μl were transferred into glass vials (Th. Geyer, Berlin, Germany) for gas chromatography-mass spectrometry (GC-MS) measurement.
Retention index standard
Nine alkanes (n-decane, n-dodecane, n-pentadecane, n-octadecane, n-nonadecane, n-docosane, n-octacosane, n-dotriacontane, and n-hexatriacontane) were dissolved in hexane, combined at a final concentration of 2 mg/ml and stored at 4°C. Retention index standard was added to the solvent (MSTFA) at a final concentration of 2% (v/v) during derivatization.
Quantification standard
The quantification mixture was composed of 63 compounds (stock concentration 1 mg/ml, 20% MeOH). A dilution series from 1:1, 1:2, 1:5, 1:10, 1:20, 1:50, 1:100, and 1:200 was prepared, portioned, dried under vacuum, and stored at -20°C. One set of quantification standard was treated in parallel with cell extracts during derivatization and measured in technical replicates within an experiment.
GC-MS measurement
Metabolite analysis was performed on a gas chromatography coupled to time of flight mass spectrometer (Pegasus III- TOF-MS-System, LECO Corp., St. Joseph, MI, USA), complemented with an auto-sampler (MultiPurpose Sampler 2 XL, Gerstel, Mülheim an der Ruhr, Germany). The samples and quantification standards were injected in split mode (split 1:5, injection volume 1 μl) in a temperature-controlled injector (CAS4, Gerstel) with a baffled glass liner (Gerstel). The following temperature program was applied during sample injection: initial temperature of 80°C for 30 s followed by a ramp with 12°C/min to 120°C and a second ramp with 7°C/min to 300°C and final hold for 2 min. Gas chromatographic separation was performed on an Agilent 6890 N (Agilent, Santa Clara, CA, USA), equipped with a VF-5 ms column of 30-m length, 250-μm inner diameter, and 0.25-μm film thickness (Varian, Palo Alto, CA, USA). Helium was used as carrier gas with a flow rate of 1.2 ml/min. Gas chromatography was performed with the following temperature gradient: 2-min heating at 70°C, first temperature gradient with 5°C/min up to 120°C and hold for 30 s; subsequently, a second temperature increase of 7°C/min up to 350°C with a hold time of 2 min. The spectra were recorded in a mass range of 60 to 600 U with 20 spectra/s at a detector voltage of 1750 V.
Data analysis
The vendor software ChromaTOF Version 4.42 (LECO) was used for metabolite evaluation with the following parameters: baseline offset of 1, peak width of 4 s, signal/noise of 20, and peak smoothing of 11 data points. Retention indices were calculated based on retention index standards. The Golm Metabolome Database (GMD) provided mass spectra and retention information for peak identification[20]. The quantification routine of ChromaTOF was used for external calibration based on the measured quantification standards. Exported .txt files included the following: name, quantification mass retention index, first dimension retention time, second dimension retention time, area, concentration, match, reverse, quantitative signal/noise, type, concentration units, and the peak true spectrum in absolute values. For further data analysis, the tool MetMax was developed in cooperation with the MPIMP in Potsdam-Golm (http://gmd.mpimp-golm.mpg.de/apps/metmax)[21]. MetMax provided the extraction of peak areas and quantities (retention analysis mode) and intensities of pre-defined mass ranges (isotope concentrator mode) from the exported .txt files. The in-house-developed pSIRM-wizard enabled the determination of 13C-label incorporation based on the exported data following the descriptions and equations stated in the paper. The R package is available on request.
Experimental procedures
Reproducibility
T98G cells were seeded (6.5 × 105 cells/10-cm cell culture dish) in 10 ml DMEM (2.5 g/l glucose, 4 mM glutamine, 10% FBS, and 1% pen/strep) and cultivated for 3 days. Media changes were performed 24 and 4 h prior the harvest to avoid nutrient deprivation.
Stable isotope labeling with 13C glucose was applied at three independent dishes for 3 min. Cell harvest and extraction was carried out as described above. The extracts of all dishes were pooled, portioned, and measured six times to evaluate technical reproducibility. The biological variance was determined by the measurement of five individually handled dishes of T98G cells measured in four technical replicates. In addition, two plates of T98G cells were harvested with 12C glucose for the acquisition of reference spectra.
Quantification addition
Seven dishes of T98G cells were harvested as described above. The cells were extracted a second time with 20% (v/v) MeOH containing an internal standard (cinnamic acid, 2 μg/ml), and pooled. Quantification mixtures and quantification mixtures with spiked in cell extracts as well as cell extracts were measured, and recovery was calculated from these data.
Verification of calculation strategy by the measurement of known ratios of 13C1-Glc and 12C-Glc
One milligram per milliliter stock solutions of glucose and 13C1-glucose (Campro Scientific) were prepared in 20% MeOH and mixed in known ratios of 0%, 2%, 5%, 10%, 25%, 50%, 75%, 90%, 95%, 98%, and 100% of 13C1-glucose; three independent batches were prepared. Twenty microliters were dried under vacuum, derivatized, and measured as described above. Measurement and data extraction were performed as described and uncorrected, targeted, and position-independent strategies applied.
Metabolic profiling of cell lines
T98G, HEK293, HeLa, and HCT-116 were grown for analysis of the metabolic profile under identical nutrient conditions (DMEM, 10% FBS, 2.5 g/l glucose, 4 mM glutamine). Seeding densities were evaluated in advance to avoid contact inhibition. Labeling with 13C-glucose and cell harvest were performed as described. Three dishes for each cell line were pooled, processed as described above, and measured in technical replicates.
Glycolytic inhibition
T98G cells were seeded and harvested as described for the reproducibility experiment. Inhibitors were added in the following concentrations separately for 12 min: 2 mM BrPyr and 2, 4, and 10 mM 2DG. On a separate dish, 2 mM mannitol was applied as osmotic control. Subsequently, media were replaced with 5ml pre-warmed labeling buffer containing 2.5 g/l u-13C-glucose, 2 mM glutamine, and inhibitors for 3 min. Cell harvest, extraction, and metabolite measurement were performed as described above.