Metabolomics: anion-exchange chromatography mass spectrometry for the analysis of anionic metabolites

Extracted metabolites were resuspended in 200 μl of Optima UPLC/MS grade water (Thermo Fisher Scientific). After 15 min incubation on a thermomixer at 4°C and a 5 min centrifugation at 16,000g at 4°C, 100 μl of the cleared supernatant was transferred to polypropylene autosampler vials (Chromatography Accessories Trott). The samples were analyzed using a Dionex ion chromatography system (Integrion, Thermo Fisher Scientific) as described previously (61). In brief, 5 μl of the polar metabolite extract were injected in full-loop mode using an overfill factor of 1, onto a Dionex IonPac AS11-HC column (2 mm × 250 mm, 4 μm particle size, Thermo Fisher Scientific) equipped with a Dionex IonPac AG11-HC guard column (2 mm × 50 mm, 4 μm, Thermo Fisher Scientific). The column temperature was held at 30°C, whereas the autosampler was set to 6°C. A potassium hydroxide gradient was generated using a potassium hydroxide cartridge (Eluent Generator, Thermo Fisher Scientific), which was supplied with deionized water. The metabolite separation was carried out at a flow rate of 380 μl/min, applying the following gradient conditions: 0–3 min, 10 mM KOH; 3–12 min, 10–50 mM KOH; 12–19 min, 50–100 mM KOH, 19–21 min, 100 mM KOH, 21–22 min, 100-10 mM KOH. The column was re-equilibrated at 10 mM for 8 min.

For the analysis of metabolites and their isotopologues, the eluting compounds were detected in negative ion mode using full scan measurements in the mass range m/z 50–750 on a Q-Exactive HF high-resolution MS (Thermo Fisher Scientific). The heated electrospray ionization (ESI) source settings of the mass spectrometer were: spray voltage 3.2 kV, the capillary temperature was set to 275°C, sheath gas flow 50 AU, aux gas flow 14 AU at a temperature of 380°C, and a sweep gas flow of 3 AU. The S-lens was set to a value of 45.

The semi-targeted LC-MS data analysis was performed using the TraceFinder software (Version 4.1; Thermo Fisher Scientific). The identity of each compound was validated by authentic reference compounds, which were measured at the beginning and the end of the sequence.

For data analysis, the area of the deprotonated [M-H+]− monoisotopic mass peak and the corresponding isotopologues of each compound were extracted and integrated using a mass accuracy of <5 ppm and a retention time (RT) tolerance of <0.05 min as compared with the independently measured reference compounds. Areas of the cellular pool sizes were normalized to the internal standards added to the extraction buffer, followed by total ion count (TIC) normalization.