The details of the MS experiments have been described in Alves et al.
Mass spectrometry: MS analysis was performed on the QTRAP 6500+ System equipped with SelexION Technology and an IonDrive™ Turbo V Ion Source. The gradient conditions for both are described in Table 1. The TCA cycle intermediates were analyzed using a Hypercarb 100 x 2.1 mm column (Thermofisher). 1 In short, glycolytic intermediates were analyzed using an Amino Acid 50 x 2.1 mm, 3 µm column (Imtakt). Samples were resuspended in water and 2 µL were injected on column.Ĭhromatography: The M.I.M.O.S.A chromatography has been described in Alves et al. Cells were frozen in V-bottom plates (Bioexpress, Cat #T-3025-8B) and lyophilized overnight to dryness. Cells were subsequently washed with ice-cold PBS, and cellular metabolism was quenched with ice-cold 20% methanol in 0.1% formic acid supplemented with 10 µM d 4-taurine (Cambridge Isotopes), 3 mM sodium fluoride (Sigma), 1 mM phenylalanine (Sigma), and 100 µ M EDTA. Cells were incubated in glucose labeling media for 4 h.
Cells were washed with PBS and resuspended in 13C-glucose labeled media that consisted of 5.5 mM -D-glucose (CLM-2001 Cambridge Isotopes). Cells were harvested and incubated with fresh cold media without glucose or glutamine (MP Biomedicals) that had been supplemented with sodium bicarbonate (3.7 g/L), 10 mM HEPES, 0.2% essentially FFA free BSA (Sigma), 0.05 mM sodium pyruvate, 4 mM glutamine, and 0.45 mM L-lactate for 1 h. 4 Briefly, cells were unstimulated or stimulated with ImmunoCult. Sample preparation: A protocol detailing the isolation, culture, and activation of primary human CD4+ T-cells is described in Hiemer et al. This automated workflow has removed the barriers and improved the rigor for fluxomics research. MIMOSA has been fully incorporated into the Polly software (Elucidata) with their LC-MS/MS labeled workflow. For separation of the glycolytic and TCA cycle intermediates, the method employs two 5-min LC gradients in conjunction with differential mobility separation. 1 The MIMOSA technique utilizes a targeted MRM-based method to understand each isotopologue as well as the position of the labeled carbon atoms. (Mass Isotopomer Multi-Ordinate Spectral Analysis), which is focused on energy metabolism. The Kibbey lab has developed a targeted fluxomics technique, designated M.I.M.O.S.A. While fluxomics is a powerful technique in understanding the dynamic changes in metabolism, these experiments require an expert level of understanding of the pathways and are mathematically rigorous. For a metabolite with an increased enrichment of label, a flux rate can distinguish if this is due to the pathway speeding up and forming more product, or accumulation due to subsequent steps in the pathway slowing down.
A flux rate reveals details about how a biological system changes between 2 or more cohorts.
Unlike isotope labeling experiments, which mainly focus on the incorporation of labeled atoms into certain metabolites, the output of a flux experiment is a rate. The experimental design can include a single time point once the system reaches a steady-state or a series of time points for a kinetic understanding of how the metabolites are changing over time. Commonly, 13C or 15N labeled precursor molecules are fed into a biological system and allowed to be metabolized for a given amount before the experiment is quenched. Fluxomics is a relatively recent application of mass spectrometry-based techniques that employs the use of stable labeled isotopes to trace how specific metabolites and molecules are metabolized through biological pathways.