Ferroptosis is a type of programmed cell death that is morphologically, biochemically, and genetically distinct from other forms of regulated cell death. It is characterized by iron-dependent accumulation of lipid peroxides and disruption of cellular membrane integrity. Since the discovery of ferroptosis (1), many ferroptosis-related metabolic alterations have been studied, suggesting possible intertwinement of diverse metabolic pathways in ferroptosis induction.
Much evidence has shown that Cyst(e)ine metabolism plays a crucial role in ferroptosis prevention. Inhibition of cystine uptake and cysteine depletion can induce ferroptosis by depleting intracellular glutathione (GSH) and accumulating glutamic acid (1, 2). While GSH works as a co-factor of glutathione peroxidase 4 (GPX4) to detoxify lipid peroxidation (3), glutamate accelerates ferroptosis by accumulating reactive oxygen species (2). In the same context, glutamate-cysteine ligase catalytic subunit (GCLC), a rate-limiting GSH synthetic enzyme, also protects against ferroptosis by converting glutamate into non-toxic gamma-glutamyl peptides (2). On the other hand, there are other parallel ferroptosis protective enzymes. FSP1 in the plasma membrane and mitochondrial DHODH can reduce coenzyme Q10 (CoQ10) producing dihydro-CoQ10 (CoQ10H2), a solid radical-trapping antioxidant (RTA) (4-6). GTP cyclohydrolase 1 has also been shown to suppress ferroptosis, producing another RTA of tetrahydrobiopterin and contributing to CoQ10 synthesis (7). Enzymes involved in the mevalonate pathway can also protect against ferroptosis by generating CoQ10 and GPX4 (8). In contrast, many ferroptosis-inducible metabolic enzymes including LPCAT3, ASCL4, FADS1, and ELOVL5 can accumulate poly-unsaturated fatty acid (PUFA)-containing phospholipids (9-11) as well as LOXs and POR oxidizing PUFA containing lipids (9, 12, 13). Since all these factors can regulate ferroptosis, precise metabolic alterations need to be evaluated to find the primary source of ferroptosis under various biological conditions.
Modern mass spectrometry based analytical techniques play a crucial role in the discovery of metabolic alteration regulating ferroptosis by extensive analysis of metabolites such as the sulfhydryl residue containing cysteine metabolites, RTAs, and membrane phospholipids. This review discusses the journey of elucidating central ferroptosis mechanisms with present cutting-edge analytical methods.
Cyst(e)ine metabolism: Since the discovery of ferroptosis, the role of cyst(e)ine metabolism in ferroptosis regulation has been emphasized. Dixon
Cysteine is a rate-limiting building block for the synthesis of GSH, a potent antioxidant and the cofactor of GPX4 which prevents ferroptosis. Therefore, measuring intracellular cysteine and GSH would be important to understand the precise cause of ferroptosis. Notably, as both cysteine and GSH are easily oxidized outside cells due to their high reduction potential, their thiol group needs to be protected during sample preparation and instrumental analysis. Hence, several agents have been used to derivatize the sulfhydryl residue of cysteine and GSH. These reagents could be alkylating agents conjugated to a chromophore or fluorophore include monobromobimane, 4-fluoro-7-sulfamoylbenzofurazan, 7-fluoro-benzo-2-oxa-1,3-diazole-4-sulphonate, 5-iodoacetamidofluorescein, 2-chloro-1-methylpyridinium iodide and 2-chloro-1-methylquinolinium tetrafluoroborate (14, 15); or maleimide-thiol conjugation such as N-ethyl maleimide (NEM), propionoyl, hexanoyl, and 4-methylcyclohexylcarbonyl maleimide (16). Thiol derivatization assures the accuracy of the quantitative method by preventing thiol oxidation and increases the method’s sensitivity by enhancing MS ionization efficiency (17). Recently, NEM has been used to alkylate the sulfhydryl residue of cysteine and GSH followed by LC-MS analysis to quantify the depletion of intracellular cysteine and GSH under a cystine deprived condition (2, 18).
System xCT regulates not only cystine uptake, but also glutamate exportation. Therefore, xCT inhibition can result in the accumulation of glutamate that accelerates ferroptosis by ROS accumulation (2). Importantly, the mass spectrometry-based metabolomics approach plays a pivotal role not only in the quantification of glutamate, but also in the discovery of unexpected glutamate metabolism detoxifying glutamate stress under a cystine-depleted condition. With a non-targeted metabolite profiling approach, dramatic accumulation of γ-glutamyl-peptides in cystine-starved cells was observed (Fig. 1A). By applying a stable isotope-labeled glutamine tracing approach, it was further confirmed that γ-glutamyl-peptides could be directly synthesized from glutamate and other amino acids through GCLC, not from the canonical GSH degradation pathway (Fig. 1A) (2). These results further emphasize that simultaneous targeting of cystine uptake and the production of γ-glutamyl-peptides provide a synergistic therapeutic potential for anti-cancer therapy.
Radical-trapping antioxidant metabolism: Recent studies have discovered the function of coenzyme Q10 (CoQ10) metabolism to prevent ferroptosis independent of GPX4, a primary enzyme that can prevent ferroptosis. Both cytosolic FSP1 (4, 5) and DHODH in mitochondria (6) can reduce CoQ10 to di-hydro CoQ10 (CoQ10H2), an element suppressing ferroptosis by quenching radicals.
Interestingly, two independent research groups have discovered that FSP1 protein is a novel ferroptosis preventive protein by applying different assays of genetic suppressor screening (4) or CRISPR screening (5) approach to GPX KO or GPX inhibitor-treated cells, respectively. On the other hand, chromatography coupled to mass spectrometry-based global metabolomics approach has shown that GPX4 inhibitor-treated cells consistently accumulate uridine while depleting its precursor of carbamoyl aspartate, suggesting the potential role of DHODH, an enzyme localized on the mitochondrial inner membrane, in preventing ferroptosis (Fig. 1B) (6). These findings imply that in addition to the genetic screening approach, metabolomics analysis might be helpful for finding novel ferroptosis regulatory factors.
To validate the function of ferroptosis preventive enzymes, the ratio of CoQ10 to CoQ10H2 must be measured. Like the sulfhydryl residue containing metabolite analysis, oxidation of CoQ10H2 to CoQ10 during sample preparation and instrumental analysis can change the ultimate CoQ10/CoQ10H2 ratio and express an unreliable result. Therefore, antioxidants such as butyl-hydroxytoluene (BHT) or tert-butyl-hydroquinone were added to extraction solvent (5). Due to electroactive groups of CoQ10, the electrochemical detector showed advantage in detecting these metabolites (5). Alternatively, a mass spectrometer detector can be used to measure CoQ10 and CoQ10H2 quantity with stable isotope-labeled internal standard (Fig. 1B) (6).
CRISPR/dCas9 overexpression screening approach has shown that GCH1 is another GPX4-independent ferroptosis suppressive protein (7). Applying FT-ICR-MS-based global metabolite profiling followed by a UPLC-QTOF-MS-based targeted metabolomics approach has further confirmed that GCH1 overexpressed cells can protect against ferroptosis by producing tetrahydrobiopterin (BH4) which works as a radical-trapping antioxidant and a cofactor for CoQ10 synthesis (Fig. 1B) (7). Consistently, CRISPR Screening combined with global metabolomics approach revealed that the BH4 synthesis metabolic pathway could protect against ferroptosis by working as an RTA and by synergizing with Vitamin E (19). Moreover, They further showed that methotrexate could synergize ferroptosis induction by inhibiting DHFR, which catalyzes the regeneration of BH4 (19), emphasizing the therapeutic potential by inducing synergistic ferroptosis of cancer cells.
Poly-unsaturated fatty acid metabolism: Oxidation of polyunsaturated fatty acid (PUFA)-containing lipids is a prominent phenotype of ferroptosis. It is known that many PUFA-containing lipid regulatory enzymes are ferroptosis inducible (9-11, 20). Combining the gene expression analysis in the GPX4 deleted cells resistant against ferroptosis and the genome-wide CRISPR-based genetic screening assay, Doll
In addition to studying lipid metabolism, oxidized lipids have been analyzed to validate metabolic pathways that regulate ferroptosis. For instance, Yang
Overall, MS-based lipid analytical platform is a crucial tool to precisely evaluate ferroptosis phenotype in various biological and pathological conditions.
This mini-review described a mass spectrometry-based analysis to understand ferroptosis regulatory metabolism. In addition to this, there might be other ferroptosis-regulatory metabolic pathways that need to be evaluated. For instance, coenzyme A (CoA) is known to contribute to ferroptosis regulation by being involved in HMG-CoA in the mevalonate pathway, which is essential for CoQ10 synthesis (23). However, the precise role of CoA derivatives has not been well understood. Furthermore, although lipid oxidation is a hallmark of ferroptosis, the function of lipid oxidative fragments still needs to be evaluated. Therefore, developing methods for quantitative and comprehensive analysis of CoA derivatives and oxidized lipids is needed to understand the new functional metabolism that plays a crucial role in ferroptosis. Another essential technique would be the matrix-assisted laser desorption/ionization (MALDI) based mass imaging and isotope tracing, which can be used in tissue-specific metabolism analysis (24). This technique will let us know spatial metabolic alterations that regulate ferroptosis in multiple organs. Overall, the development of mass spectrometry approach will provide a better chance to understand the novel mechanism of ferroptosis.
This research was supported by the New Faculty Startup Fund from Seoul National University and the National Research Foundation of Korea (NRF-2022M3A9I2017587, NRF-2022R1C1C1003619).
The authors have no conflicting interests.