Lipidomics analysis of multiple organs upon in vivo application of WMK-1

C57BL/6 mice were treated with vehilce or 40 mpk WMK-1 for two days (QD, through i.p. injection). Tissues, including liver, spleen, kidney, lung, and heart were collected and loaded for lipidomics and metabolomics analysis via LC-MC/MS. For lipidomic analysis, 3 µL of the prepared tissue lipidomics samples were loaded into a ACQUITY UPLC BEH C8 column (100 × 2.1 mm,1.7 μm, 130 Å, Waters, Cat# 186002878), and separated chromatographically with a 25-min gradient elution at a flow rate of 0.3 mL/min. The mobile phase for the positive ion acquisition mode contains H2O with 0.1% (v/v) FA and 10 mM ammonium acetate for Phase A, and ACN/IPA (80:20) with 0.1% (v/v) FA for Phase B. The mobile phase for the negative ion mode is similar to the ones for the positive ion mode except excluding formic acid in the buffer. The LipidSearch software (v4.2.21, Thermo Fisher Scientific) was used for lipidomics peak detection, peak alignment, and compound annotation. Specifically, we used a 0.5-min retention time interval for peak detection with the isotope compositions recalculated. 5 ppm and 8 ppm deviations were employed as tolerance ranges for precursor and product ions, respectively, during Database searches. The minimum relative fragment ion intensity threshold was set at 1%. Isomer peaks were filtered out. In parallel, the precursor ion quantitation was performed with a 5 ppm tolerance and a 0.5-min retention time range. Overall, we targeted 88 sub-classes across phospholipids, sphingolipids, neutral lipids, derivatized lipids (biotinylation, diazomethane), and fatty acyl groups in our compound search procedure. The positive ion mode included adducts of [M+H]+, [M+NH4]+, [M+Na]+, and [M+H-H2O]+, while the negative ion mode included adducts of [M-H]-, [M-CH3]- and [M+HCOO]-. The peak alignment was performed with retention time tolerance = 0.6 min, and lipids with identification grades scoring as A, B, C, D were included for downstream analysis. The ISDs used in our experiments were listed below: for the positive ion mode, 15:0/18:1(d7)-PC ([M+H]+, m/z 753.6134, RT 8.74 min), 15:0/18:1(d7)-PE ([M+H]+, m/z 711.5664, RT 8.96 min), d7- PI33:1(15:0/18:1) ([M+NH4]+, m/z 847.6036, RT 6.84 min), 15:0-18:1(d7)-PG ([M+NH4]+, m/z 759.5875, RT 8.46 min), d7-LPC18:1 ([M+H]+, m/z 529.3994, RT 2.02 min), d7-LPE18:1 ([M+H]+, m/z 487.3524, RT 2.06 min), d7-Chol Ester 18:1 ([M+NH4]+, m/z 675.6779, RT 5.60 min), 18:1- 15:0-18:1(d7)-15:0 TG ([M+NH4]+, m/z 829.7985, RT 19.34 min), and d18:1-18:1(d9) SM ([M+H]+, m/z 738.6470, RT 7.86 min). For the negative ion mode, 15:0-18:1(d7) PC ([M+CH3COO]-, m/z 811.6199, RT 8.59 min), 15:0- 18:1(d7) PE ([M-H]-, m/z 709.5519, RT 8.71 min), d18:1-18:1(d9) SM ([M+CH3COO]-, m/z 796.6536, RT 7.86 min), d7-LPC18:1 ([M+CH3COO]-, m/z 587.4059, RT 2.00 min), d7-LPE18:1 ([M-H]-, m/z 485.3379, RT 2.02 min), and d7-MG18:1 ([M+CH3COO]-, m/z 422.3504, RT 3.36 min).