Equisplash mix

EPIs (2 × 10⁸) were resuspended in 500 μl ice-cold PBS and a small aliquot (30 μl) was removed for protein quantification (Pierce BCA Protein Assay Kit; Thermo Fisher Scientific). For lipid quantification, EquiSPLASH mix (Avanti Polar Lipids) deuterated standards were spiked into the first extraction solvent mixture (chloroform:methanol:PBS, 1:2:0.8, v/v/v). For every 50 μg of protein, 1 μl EquiSPLASH mix (Avanti Polar Lipids) was added before cell extraction. Samples were vortexed vigorously for 1 min and centrifuged at 1200g for 10 min, with the resulting supernatant transferred to a fresh Supelco PTFE vial. Remaining parasite pellets were further subjected to three sequential extractions with chloroform:methanol (2:1, v/v), pooled, and dried under constant N₂ gas. Finally, lipids were separated by modified Folch’s partition (49 ). Folch’s lower phase was separated, pooled, dried under N₂ gas, and stored at −20 °C. Folch’s lower phase were redissolved in 100 μl chloroform:methanol (1:2, v/v) for UHPLC-LC-MS/MS analysis. Five microliters of each sample were combined into a single pool for quality control (QC).

Control or CARM1 knockout A1847 cells were washed with cold PBS, scraped into cold methanol, and spiked with EquiSPLASH mix (Avanti Polar Lipids). Lipids were extracted using a modified Folch extraction (2:1:1 chloroform:methanol:0.88% sodium chloride) and analyzed by LC/MS-MS. LipidSearch 4.2 software (Thermo Fisher Scientific) detected lipid species based on MS-MS spectra with product ion mass tolerances and 5 ppm precursor. The identification of lipid species was filtered on the basis of the expected identification quality and main adduct. The peak areas were utilized for quantification and were adjusted by EquiSPLASH lipids to represent the respective classes. In addition, the values were normalized to the protein content in each sample. The quantification of lipid classes was performed by summing peak areas of all species in the same class. Regarding the saturation analysis, the FAs incorporated into lipids with high-confidence identifications (grades A and B from LipidSearch) were classified in accordance with the number of carbon double-bonds as saturated (0), monounsaturated (1 (link)), or polyunsaturated (>1). The quantification of each lipid species with FA level identification was weighted by the number of FAs of each type present in the specie.