Sterol Quantification by GC-MS

Lipid extraction and GC-MS was conducted as previously described [59 (link)]. Pellets with 5x10⁸ cells were used for lipid extraction. The dried total samples were resuspended in 100 μL chloroform added to 100 μL of BSTFA reagent (Thermo Scientific) and incubated at 70°C for 1 hour prior to GC-MS (Agilent 7890B GC–MS, Agilent 5977A MSD) analysis [60 (link)]. The retention time and mass spectral patterns of a sterol standard were used as references for lipid analysis. Sterol standards used in this study include cholesterol (Avanti 700100), ergosterol (Cayman 19850), 4,4-Dimethyl zymosterol (Avanti 700073), Zymosterol (Avanti 700068), and Eburicol (Smolecule S633611). The relative amount of sterols A and B were estimated based on the relative percentage of the sterol to ergosterol peak areas in each sample. Cholesterol was added as an internal standard for these analyses prior to lipid extraction.