Lipidomics

For lipidomics analysis, cells were grown to about OD₆₀₀ 0.8 and lipids were extracted with chloroform:methanol (2:1). Yeast lipid extracts were prepared using a standard chloroform-methanol mixture, spiked with appropriate internal standards, and analyzed using a 6490 Triple Quadrupole LC/MS system (Agilent Technologies, Santa Clara, CA) [97]. Glycerophospholipids and sphingolipids were separated with normal-phase HPLC as described before [97], with a few changes. An Agilent Zorbax Rx-Sil column (inner diameter 2.1 × 100 mm) was used under the following conditions: mobile phase A (chloroform:methanol:1 M ammonium hydroxide, 89.9:10:0.1, v/v) and mobile phase B (chloroform:methanol:water:ammonium hydroxide, 55:39.9:5:0.1, v/v); 95% A for 2 min, linear gradient to 30% A over 18 min and held for 3 min, and linear gradient to 95% A over 2 min and held for 6 min. Sterols and glycerolipids were separated with reverse-phase HPLC using an isocratic mobile phase as before [97], except with an Agilent Zorbax Eclipse XDB-C18 column (4.6 × 100 mm).

Quantification of lipid species was accomplished using multiple reaction monitoring (MRM) transitions [97, 98] in conjunction with the referencing of appropriate internal standards: PA 17:0/14:1, PC 17:0/20:4, PE 17:0/14:1, PG 17:0/20:4, PI 17:0/20:4, PS 17:0/14:1, LPC 17:0, LPE 14:0, Cer d18:1/17:0, D7-cholesterol, cholesteryl ester (CE) 17:0, 4ME 16:0 diether DG, D5-TG 16:0/18:0/16:0 (Avanti Polar Lipids, Alabaster, AL). Quality and batch controls [99] were included to assess instrument stability and reproducibility and allow for correction of drift and other systematic noise, e.g., biases correlated with analysis order and/or sample preparation. Values are represented as mole fraction with respect to total lipid (mole percentage) [97]. All lipid species and subclasses were analyzed with one-way ANOVA followed by a post hoc Bonferroni test.