4.7. Acetyl-CoA and Malonyl-CoA Quantification

K. phaffii cell pellets were resuspended in 300 µL of MeOH/ACN/H₂O (40:40:20; v:v:v) and the suspension was mixed with an equal volume of 0.25–0.5 mm glass beads (Roth A533.1). Cells were lysed employing a Bead Mill Max (VWR) at 6 m/s setting for 30 s. The mixture was centrifuged for 1 min at 20.000g × and the extract was removed from the glass beads. The precipitated protein was resolubilized in 1% SDS at 95 °C for 5 min and the total protein content was determined employing the Pierce BCA Protein Assay Kit for the normalization of samples. The extract was centrifuged at 20.000g × for 5 min to remove residual particles and then subjected to HILIC-MS employing a ZICpHILIC column (5 µm, 150 × 2.1 mm), with solvent A being 20 mM ammonium formate at pH = 9.6 and solvent B being ACN. The following gradient was employed: 0–2 min 10% A, 2–14 min linear increase to 40% A, 14–20 min linear increase to 95% A, 20–23 min 95% A followed by re-equilibration at 10% A for 7 min. The MS system used was a timsTOF Pro (Bruker Daltonics, Bremen, Germany) operated with a VIP HESI source set to 200 °C in the default 4D-Lipidomics method. The identity of analytes was confirmed by employing authentic standards (Sigma Aldrich) by matching retention times, collisional cross-sections and exact mass and fragmentation patterns.