Lipidomics analysis of multiple organs (BAT, Ileum, Jejunum, Muscle, Pancreas) upon in vivo application of KY0418_Negative mode

In Brief: Raw data here contains lipidomics analysis (negative ion mode) of mice organs collected post orally administered with vehicle or KY0418 (10 mpk) for 3 doses (BID). File names: #1-5: replicates of Vehicle group; #7-11: replicates of KY0418 group. A, BAT; I, Ileum; J, Jejunum; M, Muscle; P, Pancreas; QC, quality control samples. Tissue lipidomic profiling post KY0418-treatment (detailed information): For tissue collection, mice were subcutaneously implanted with LLC1 cells. One-week post tumor implantation, mice were grouped and orally administered with vehicle or KY0418 (10 mpk) for 3 doses (BID). Mice were then euthanized, and tissues were collected and aliquoted for H&E analysis or flash-frozen for subsequent lipidomic extraction and mass spectrometry analysis. To extract lipids, tissues (~20 mg) were placed into 2-mL tubes preloaded with two 2 mm and one 3 mm steel beads. To each tube, 1 mL of pre-chilled methanol/water solution (1:1, v/v) was added. The samples were then homogenized using a grinding machine (Jingxin Technologies) operating a bead beater at 40 Hz for 6 cycles (30 s on and a 30 s off) at 0°C. Following homogenization, the mixtures were centrifuged at 16,100 g for 20 min at 4°C. The residual pellet was gently dried to remove residual 50% methanol. To the dried pellet, 1 mL of prechilled methyl tert-butyl ether (MTBE)/methanol solution (1:1, v/v) containing 10 μL of internal standards (ISD, SPLASH LIPIDOMIX Internal Standard, Avanti Polar Lipids, Cat# 330707-1EA) was added. The samples were then homogenized, centrifuged, and dried under the same conditions as described for the aqueous extraction. The resulting residues were stored at –80°C until analysis. For lipidomic analysis, the lipid extracts were reconstituted in 100 μL of isopropanol/acetonitrile/water solution (1:2:1, v/v/v). The quality control (QC) samples were made from a pool of all aqueous or organic samples and run in duplicate after every 12 samples. Lipidomic analysis and data processing: For lipidomic analysis by LC-MS/MS, tissue lipid extracts (3 µL) were injected into a ACQUITY UPLC BEH C8 column (100 × 2.1 mm,1.7 μm, 130 Å, Waters, Cat# 186002878), and separated chromatographically over a 25 min gradient elution at a flow rate of 0.3 mL/min. The mobile phase in positive ion mode consisted of (A) water with 0.1% (v/v) FA and 10 mM ammonium acetate, and (B) ACN/IPA (80:20) with 0.1% (v/v) FA. In negative ion mode, FA was omitted from the mobile phase. The gradient program was as follows: 70-85% B at 1.5-5.5 min, 85 % B isocratic at 5.5-9.5 min, 85-90% B at 9.5-14 min, 90-100% B at 14-17 min, 100% B isocratic at 17-20 min, 100-70% B at 20-20.1 min, followed by 5 min re-equilibration at 70% B. The column temperature was maintained at 55°C. The ion source parameters were set as below: sheath gas 40 arb, auxiliary gas 10 arb, sweep gas 1 arb, spray voltage 3.5 kV in positive and 2.6 kV in negative mode, vaporizer temperature 320°C, ion transfer tube temperature 325°C, and S-lens RF level at 70%. Full MS scan range was 200-1,700 Da with a resolution of 120,000. The maximum injection time with 100 ms and the Standard AGC target was used. The intensity threshold was set at 1×104. To enable lipid identification and qualitative analysis, data-dependent MS2 (ddMS2) was acquired with the following parameters: Orbitrap resolution at 30,000, precursor isolation window at 1.5 m/z, HCD collision energy at 25, stepped mode at 30%, Standard AGC target, and a maximum injection time of 54 ms. Individual lipid species were quantified by referencing to spiked internal standards, which included d7-PC33:1(15:0/18:1), d7-PE33:1(15:0/18:1), d7-PG33:1(15:0/18:1), d7-PS33:1(15:0/18:1), d7-PI33:1(15:0/18:1), d7-PA33:1(15:0/18:1), d7-LPC18:1, d7-LPE18:1, d7-Chol Ester 18:1, d7-MG18:1, d7-DG33:1(15:0/18:1), d7-TG48:1 (15:0/18:1/15:0), d9-SM18:1 and d7-cholesterol from Avanti Polar Lipids.