Metabolic analysis

A 600 MHz Avance III spectrometer (BrukerBioSpin, Rheinstetten, Germany) was used to acquire one-dimensional (1D) 1H and two-dimensional (2D) 1H–13C heteronuclear single-quantum correlation spectra as published before⁶¹. Absolute concentrations from 1D and 2D nuclear magnetic resonance (NMR) data were calculated employing individual peak calibration factors and MetaboQuant⁵⁸. Amino acids, tryptophan metabolites, and intermediates of the methionine and polyamine metabolism were determined by mass spectrometry (MS) as previously described^60,62,63. For each metabolite, a stable isotope labelled internal standard was used.

Organic acids of the TCA cycle were analysed by high performance liquid chromatography (HPLC)–electrospray ionization (ESI)–tandem MS (MS/MS) employing an API 4000 QTrap mass spectrometer (ABSciex, Darmstadt, Germany) hyphenated to a 1200 SL HPLC (Agilent, Boeblingen, Germany). Negative mode ionization and multiple reaction monitoring with a transition for each isotopolog was performed. A Discovery HS F5-3 HPLC column (2.1 × 150 mm, 3 μm; Supelco, Bellefonte, PA, USA) with a Security Guard column (C18, Phenomenex, Aschaffenburg, Germany) and gradient elution with 0.1% formic acid in water (v/v) as mobile phase A and acetonitrile as mobile phase B was used.

For the measurement of amino acids and organic acids from stable isotope tracer experiments, the said methods were extended to cover the transitions of all expected isotopologs.

In addition to HPLC–MS/MS analysis, intracellular citrate was also analysed by gas chromatography (GC)–MS after derivatization. A 50 µL aliquot of the sample extract was dried followed by methoximation and silylation and subsequent GC–MS analysis employing the derivatization protocol and instrumental setup previously described⁶⁰. An injection volume of 1 µL and splitless injection was used.

Nucleobases were analysed by HPLC–quad time of flight–MS after hydrolysis of nucleic acids. The pellet obtained after metabolite extraction described above was hydrolyzed with 200 µL 2 M HCl at 120 °C for 4 h. After hydrolysis, 1 M NaOH was used to neutralize the solution. The neutralized solution was then dried with a vacuum evaporator and the residue was re-solved in 100 μL of water for liquid chromatography–MS analysis. A Maxis Impact QTOF-MS (Bruker Daltonics, Bremen, Germany) hyphenated to an Thermo Scientific Dionex Ultimate 3000 UHPLC system (Idstein, Germany) through an ESI source operated in positive ion mode was used. Separation was carried out on a Waters Atlantis T3 reversed-phase column (2.1 × 150 mm, 3 µm) with mobile phase A (0.1 % formic acid in H₂O, v/v) and B (0.1% formic acid in acetonitrile, v/v) and gradient elution. The column was kept at 35 °C and a flow rate of 0.3 mL/min was employed.

MS data were acquired in a mass range of 50–1000 m/z with an acquisition rate of 2 spectra/s. Mass calibration was performed using sodium formate clusters (10 mM sodium formate in 50:50 (v/v) water/ isopropanol). For data analysis, the peak area of the [M + H]⁺ peak of each possible isotopolog was integrated.

Correction for natural stable isotope abundance and tracer impurity in the tracing experiments was performed using an in-house tool to correct MS/MS data and IsoCor for full-MS data⁶⁵.