Empower3 software suite

To measure the malate shuttle-associated metabolites, we adapted a previously published method^{52 (link)}. In brief, 10⁶ cells were extracted in 100 µl ice-cold methanol with sonication on ice. For derivatization, 50 µl extract was mixed with 25 µl 140 mM 3-nitrophenylhydrazine hydrochloride, 25 µl methanol and 100 µl 50 mM ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride, and incubated for 20 min at 60 °C. Samples were separated by reversed-phase chromatography on an Acquity H-class UPLC system coupled to a QDa mass detector (Waters) with an Acquity HSS T3 column (100 mm × 2.1 mm, 1.8 µm, Waters), which was heated to 40 °C. Separation of derivatives was achieved by increasing the concentration of 0.1% formic acid in acetonitrile (B) in 0.1% formic acid in water (A) at 550 µl min⁻¹ as follows: 2 min 15% B, 2.01 min 31% B, 5 min 54% B, 5.01 min 90% B, hold for 2 min and return to 15% B in 2 min. Mass signals for the following compounds were detected in single ion record mode by using negative detector polarity and 0.8 kV capillary voltage: malate (403.3 m/z; 25 V CV), succinate (387.3 m/z; 25 CV), fumarate (385.3 m/z; 30 V), citrate (443.3 m/z; 10 V), pyruvate (357.3 m/z; 15 V) and α-ketoglutarate (550.2 m/z; 25 CV). Data acquisition and processing were performed with the Empower3 software suite (Waters).

To analyze the content of branched-chain α-ketoacids, 100 μl of yeast conditioned medium was mixed with 200 μl cold 1M perchloric acid. Insoluble materials were removed by centrifugation, 150 μl of the resulting supernatant were mixed with an equal volume of 25 mM o-phenylendiamine solution and derivatized by incubation at 50°C for 30 min. After centrifugation the derivatized keto-acids were separated by reversed phase chromatography on an Acquity HSS T3 column (100 mm x 2.1 mm, 1.7 μm, Waters) connected to an Acquity H-class UPLC system. Prior to separation, the column was heated to 40°C and equilibrated with 5 column volumes of solvent A (0.1% formic acid in 10% acetonitrile) at a flow rate of 0.55 ml/min. Separation of ketoacid derivates was achieved by increasing the concentration of solvent B (acetonitrile) in solvent A as follows: 2 min 2% B, 5 min 18% B, 5.2 min 22% B, 9 min 40% B, 9.1min 80% B and hold for 2 min, then return to 2% B in 2 min. The separated derivatives were detected by fluorescence (Acquity FLR detector, Waters, excitation: 350 nm, emission: 410 nm). Data acquisition and processing were performed with the Empower3 software suite (Waters).

TCA cycle intermediates were quantified in MCEC treated with 2 mM acetoacetate or 2 mM D‐β‐hydroxybutyrate (Sigma‐Aldrich) or the respective controls for 24 h. Samples were extracted in methanol with sonication and the separation protocol was adapted from Uran et al (2007 (link)). In brief, samples were analyzed on an Acquity HSS T3 column (Waters) heated to 40°C connected to an Acquity H‐class UPLC system (Waters). The UPLC system was coupled to a QDa mass detector (Waters) in single ion record mode, using negative detector polarity and 0.8 kV capillary voltage. Data acquisition and processing was performed with the Empower3 software suite (Waters).