All the BLCA specimens were procured by a prior written informed consent under institute review board (IRB) approved protocols. Metabolites were extracted from BLCA tissues, and mouse liver pool was used as a quality control and followed the extraction procedure described8 (link),12 (link)–14 . Briefly, 10mg of tissue was used for the metabolic extraction. The extraction step starts with addition of 750μL ice-cold methanol: water (4:1) containing 20μL spiked internal standards (ISTDs). After homogenization, ice-cold chloroform and water were added in a 3:1 ratio for a final proportion of 4:3:2 methanol:chloroform:water. The organic and aqueous layers were collected, dried, and resuspended in methanol: water (1:1). The extract was deproteinized using a 3kDa molecular filter and the filtrate was dried under vacuum. The dried extracts were re-suspended in 100μL of injection solvent composed of 1:1 methanol: water and subjected to LC-MS.
Lipids were extracted using a modified Bligh-Dyer method15 (link). The extraction was carried out using 2:2:2 ratio of water: methanol: dichloromethane at room temparature after ISTDs into tissues and quality control pool12 (link). After homogenization of the samples, the organic layer was collected and completely dried under vacuum. Before MS analysis, the dried extract was resuspended in 100μL of buffer containing 10mM NH4Ac and subjected to LC/MS. The lipidome was separated using reverse-phase (RP) chromatography. To monitor the lipid extraction process, we used a standard pool of tissue samples from aliquots of the same samples.