Quantification of metabolite levels in TIF and plasma

In order to quantitate metabolites in TIF and plasma samples, we first constructed a library of 149 chemical standards of plasma polar metabolites (see Supplementary file 1 for suppliers for each chemical standard). These compounds were selected to encompass a number of metabolic processes and have previously been included in efforts to profile plasma polar metabolites by LC/MS (Cantor et al., 2017; Evans et al., 2009; Lawton et al., 2008; Mazzone et al., 2016). We pooled these metabolites into seven separate chemical standard pools (Supplementary file 1). To do this, each metabolite in a given pool was weighed and then mixed (6 cycles of 1 min. mixing at 25 Hz followed by 3 min. resting) using a Mixer Mill MM301 (Retsch, Düsseldorf, Germany), and mixed metabolite powder stocks were stored at −20°C prior to resuspension and analysis. Stock solutions of the mixed standards pools containing ~5 mM,~1 mM,~300 µM,~100 µM,~30 µM,~10 µM,~3 µM and ~1 µM of each metabolite were made in HPLC grade water and were stored at −80°C (see Supplementary file 1 for the concentration of each metabolite in the external standard pools). We refer to these stock solutions as ‘external standard pools’ throughout. External standard pools were used to confirm the retention time and m/z for each analyte and provide standards to quantitate concentrations of stable isotope labeled internal standards used in downstream analysis, as well as to quantitate metabolite concentrations in TIF and plasma samples directly where internal standards were not available (see below for details).

We had three classes of samples: plasma, TIF and the external standard pool dilutions (prepared in water). We extracted polar metabolites (Figure 1—figure supplement 2) from each sample type using the same extraction mix and protocol: 5 µL of sample (plasma, TIF or external standard pool dilution) was mixed with 45 uL of acetonitrile:methanol:formic acid (75:25:0.1) extraction mix including the following isotopically labeled internal standards: ¹³C labeled yeast extract (Cambridge Isotope Laboratory, Andover, MA, ISO1), ¹³C₃ lactate (Sigma Aldrich, Darmstadt, Germany, 485926), ¹³C₃ glycerol (Cambridge Isotope Laboratory, Andover, MA, CLM-1510), ¹³C₆ ¹⁵N₂ cystine (Cambridge Isotope Laboratory, Andover, MA, CNLM-4244), ²H₉ choline (Cambridge Isotope Laboratory, Andover, MA, DLM-549), ¹³C₄ 3-hydroxybutyrate (Cambridge Isotope Laboratory, Andover, MA, CLM-3853), ¹³C₆ glucose (Cambridge Isotope Laboratory, Andover, MA, CLM-1396), ¹³C₂ ¹⁵N taurine (Cambridge Isotope Laboratory, Andover, MA, CNLM-10253), ²H₃ creatinine (Cambridge Isotope Laboratory, Andover, MA, DLM-3653), 8-¹³C adenine (Cambridge Isotope Laboratory, Andover, MA, CLM-1654), ¹³C₅ hypoxanthine (Cambridge Isotope Laboratory, Andover, MA, CLM-8042), 8-¹³C guanine (Cambridge Isotope Laboratory, Andover, MA, CLM-1019), ¹³C₃ serine (Cambridge Isotope Laboratory, Andover, MA, CLM-1574) and ¹³C₂ glycine (Cambridge Isotope Laboratory, Andover, MA, CLM-1017). All solvents used in the extraction mix were HPLC grade. Samples were then vortexed for 10 min. at 4°C and insoluble material was sedimented by centrifugation at 15 kg for 10 min. at 4°C. 20 µL of the soluble polar metabolite extract was taken for LC/MS analysis.

LC/MS analysis was performed on the sample extracts using a QExactive orbitrap mass spectrometer using an Ion Max source and heated electrospray ionization (HESI) probe coupled to a Dionex Ultimate 3000 UPLC system (Thermo Fisher Scientific, Waltham, MA). External mass calibration was performed every 7 days, and internal mass calibration (lock masses) was not used. 2 μL of each sample was injected onto a ZIC-pHILIC 2.1 × 150 mm analytical column equipped with a 2.1 × 20 mm guard column (both 5 μm particle size, EMD Millipore). The autosampler and column oven were held at 4°C and 25°C, respectively. Buffer A was 20 mM ammonium carbonate, 0.1% ammonium hydroxide; buffer B was acetonitrile. The chromatographic gradient was run at a flow rate of 0.150 mL/min as follows: 0–20 min: linear gradient from 80% to 20% B; 20–20.5 min: linear gradient from 20% to 80% B; 20.5–28 min: hold at 80% B. The mass spectrometer was operated in full scan, polarity-switching mode with the spray voltage set to 3.0 kV, the heated capillary held at 275°C, and the HESI probe held at 350°C. The sheath gas flow rate was set to 40 units, the auxiliary gas flow was set to 15 units, and the sweep gas flow was set to one unit. The MS data acquisition was performed in a range of 70–1000 m/z, with the resolution set to 70,000, the AGC target at 1e6, and the maximum injection time at 20 msec.

After LC/MS analysis, metabolite identification was performed with XCalibur 2.2 software (Thermo Fisher Scientific, Waltham, MA) using a 5ppm mass accuracy and a 0.5 min. retention time window. For metabolite identification, external standard pools were used for assignment of metabolites to peaks at given m/z and retention time, and to determine the limit of detection for each metabolite, which ranged from 100 nM to 3 µM (see Supplementary file 1 for the m/z, retention time and limit of detection for each metabolite analyzed).

After metabolite identification, quantification was performed by two separate methods for either quantification by stable isotope dilution or external standard calibration (Figure 1—figure supplement 3). For quantification by stable isotope dilution, where internal standards were available, we first compared the peak areas of the stable isotope labeled internal standards with the external standard pools diluted at known concentrations. This allowed for quantification of the concentration of labeled internal standards in the extraction mix. Subsequently, we compared the peak area of a given unlabeled metabolite in the TIF and plasma samples with the peak area of the now quantified internal standard to determine the concentration of that metabolite in the TIF or plasma sample. 70 metabolites were quantitated using this internal standard method (see Supplementary file 1 for the metabolites quantitated with internal standards).

For metabolites without internal standards, quantification by external calibration was performed as described below (Figure 1—figure supplement 3). First, the peak area of each externally calibrated analyte was normalized to the peak area of a labeled amino acid internal standard that eluted at roughly the same retention time to account for differences in recovery between samples (see Supplementary file 1 for the labeled amino acid paired to each metabolite analyzed without an internal standard). This normalization was performed in both biological samples and external standard pool dilutions. From the normalized peak areas of metabolites in the external standard pool dilutions, we generated a standard curve describing the relationship between metabolite concentration and normalized peak area. The standard curves were linear with fits typically at or above r² = 0.95. Metabolites which did not meet these criteria were excluded from further analysis. These equations were then used to convert normalized peak areas of analytes in the TIF or plasma samples into analyte concentration in the samples. 74 metabolites were quantitated using this method. The relationship between metabolite concentration and normalized peak area is matrix dependent, and the external standards are prepared in water, which is a different matrix than either TIF or plasma. Therefore, we consider metabolite measurements using this external standard method semi-quantitative.