Mass-spectrometry based analytical methods to study the role of metabolism in regulating ferroptosis. (A) The role of cysteine metabolism. Stable isotope-labeled serine and glutamine tracing approach are used to evaluate trans-sulfuration pathway and GCLC activity, respectively. The global metabolomics approach is used to analysis of γ-Glu-AAs. The cysteine and GSH is quantified after alkylating thiol group due to its high reactivity. (B) The role of radical-trapping antioxidant metabolism. CoQ10H2 and CoQ10 is quantified with an antioxidant to protect the auto-oxidation of CoQ10H2 during sample preparation and instrumental analysis. Also, the global metabolomics approaches are used to determine metabolic consequence of DHODH and GCH1 alteration, respectively. (C) The role of poly-unsaturated fatty acid metabolism. LC-MS/MS-based analysis including global lipidomics and targeted oxidized lipid analysis are applied to study role of poly-unsaturated lipid regulatory enzymes. RTA: radical-trapping antioxidant, (Cys)2: cystine, Cys: cysteine, Ser: serine, TSS: trans-sulfuration, Gln: Glutamine, GCLC: glutamate-cysteine ligase catalytic subunit, γ-Glu-Cys: γ-glutamyl-cysteine, GSH: glutathione, GSSG: oxidized glutathione, AAs: amino acids, γ-Glu-AAs: γ-glutamyl-amino acids, GPX4: glutathione peroxidase 4, PLOOH: phospholipid hydroperoxide, PLOH: phospholipid alcohol, PLOO･: phospholipid peroxyl radical, CoQ10: coenzyme Q10, CoQ10H2: dihydrocoenzyme Q10, FSP1: ferroptosis suppressor protein 1, DHODH: dihydroorotate dehydrogenase, BH4: tetrahydrobiopterin, BH2: dihydrobiopterin, GCH1: guanosine triphosphate cyclohydrolase 1, PUFA: poly-unsaturated fatty acid, PUFA-PLs: phospholipids containing polyunsaturated fatty acid, ASCL4: acyl-CoA synthetase long-chain family member 4, LPCAT3: lysophosphatidylcholine acyltransferase 3, LOX: lipoxygenase.