Comprehensive Metabolomic Profiling of Lung Tissue

Metabolite extraction from lung tissue for comprehensive metabolomic profiling was performed using the slightly modified protocol of a previously established method^{56 (link)}. Briefly, frozen tissue samples were combined with a 500-μL aliquot of ice-cold methanol containing methionine sulfone (L-Met) and 2-morpholinoethanesulfonic acid (MES), serving as designated internal standards (IS) for cationic and anionic metabolites, respectively. This mixture was subjected to homogenization using a Finger Masher manual homogenizer (AM79330, Sarstedt, Tokyo, Japan). To this homogenate, half the volume of ultrapure water (LC/MS grade, obtained from Wako) and 0.4 times the initial volume of chloroform (Nacalai Tesque, Kyoto, Japan) were added. The resulting mixture was centrifuged at 15,000 × g for 90 min at a temperature of 4 °C. After centrifugation, the resulting aqueous phase was subjected to filtration using an ultrafiltration tube (Ultrafree MC-PLHCC, Human Metabolome Technologies, Yamagata, Japan). The filtrate was then concentrated by nitrogen flow-assisted evaporation on a heating block (DTU-28N, TAITEC, Koshigaya City, Japan). The concentrated filtrate was resuspended in 50 μL ultrapure water for subsequent LC-MS/MS and IC-HR-MS analytical procedures.