Measurement of Intracellular Coenzyme A Esters

In order to measure the content of intracellular coenzyme A esters, mycelia obtained form 5 mL of fermentation broth were washed twice with deionized water and centrifuged at 8,000 × g for 10 min. The wet hyphae were suspended in 300 μL of 15% trichloroacetic acid and lysed by vortexing for 3 min at 4∘C with 150 μL of glass beads. After centrifugation for 10 min at 8,000 × g and 4°C, the supernatant was passed through an OASIS HLB SPE cartridge (waters, United States) under vacuum to extract coenzyme A esters, as reported previously (Mo et al., 2012a). The analysis of coenzyme A esters was performed using Ultra-high performance liquid chromatography (UPLC, Waters, United States)-electrospray ionization (ESI)-tandem mass spectrometry (MS; Xveo TQ-XS, Waters, United States), equipped with an ACQUITY UPLC HSS T3 column (100 mm × 2.1 mm, 1.7 μm; Waters, United States), as previously reported, with some modifications (Park et al., 2007). The mobile phase was composed of 5 mM ammonium acetate and 0.05% acetic acid in water (A), and 80% acetonitrile with the same additive concentration (B). The gradient elution program included: 0–0.8 min, 90% A/10% B; 0.8–2 min, a linear gradient from 90% A/10%B to 60% A/40% B; 2–6 min, a linear gradient from 60% A/40% B to 20% A/80% B; 6–8 min, 20% A/80% B; 8–12 min, and a linear gradient from 20% A/80% B to 90% A/10% B. The quantification was done in multiple reaction monitoring (MRM) mode with two mass ions: m/z parent > m/z daughter (acetyl-CoA, 810 > 303; malonyl-CoA, 854 > 347; propionyl-CoA, 824 > 317; methylmalonyl-CoA, 868 > 361).