Lipidomic Analysis of Hypoxia-Treated Cells

UFH-001 cells (in 100 mm plates) were exposed to normoxic or hypoxic conditions (as described above) in the presence or absence of BCP (20 or 200 μM) for the times indicated. Cells were washed (3X) with ice cold sterile saline (.0.9% NaCl). The final wash was removed, and methanol (5 mL) was added to the cells and scraped into 25 mL separating flasks. Ten mL of chloroform was added to each flask, shaken, and then left at room temperature for 30 min. Five mL of ice-cold ddH₂O was added and mixed. This was placed in a cold room for 72 h to separate the phases. The chloroform phase was collected and dried under nitrogen. Dried samples were reconstituted in chloroform and a subsample was evaporated to dryness under N₂. Phospholipids were hydrolyzed with 250 μL 1M KOH in methanol at 50°C for 3 h, followed by 250 μL 6M HCl in MeOH for 15 min at 80°C. FAMEs and sterols were extracted with Diethyl Ether: Hexane (1:1). After evaporating to dryness, silylation of the sterols occurred with 50 μl BSTFA:TCMS (99:1) and 10 mL anhydrous pyridine for 30 min at 37°C. One mL of this solution was injected into an Agilent 7890B Gas Chromatogram in splitless mode with an inlet temperature of 300°C and a DB-5 analytical column (30 m length, 0.25 mm diameter, with a built-in 10 m DuraGuard pre-column) with a flow of 1.12 mL/min (average velocity 23.5 cm/sec). Thermal ramping initiated at 80°C for 1 min, and then ramped 20°C/min to 200°C and then 10°C/min to 325°C and held for 10 min. Analytes were detected with an Agilent 5977A Mass Spectrometer with an EI ion source with the MS in scanning mode (50–600 m/z) and transfer line and ion source temperatures set at 230°C and 150°C, respectively. Peaks within a sample were deconvoluted using MassHunter (Agilent) software and preliminary analyte identities were assigned based on comparison to the NIST14 library [73 (link)] and in-house mass spectral libraries [74 (link)]. Spectral match factor thresholds for identified metabolites was > 85%. Individual metabolites were aligned across samples using an in-house R script implemented in R (4.0.3) through RStudio (2021.09.2 Build 382) using a combination of nearest neighbor and mass spectral similarity indices.