An LC-MS chemical derivatization method for the measurement of five different one-carbon states of cellular tetrahydrofolate

Chen, Li; Ducker, Gregory S.; Lu, Wenyun; Teng, Xin; Rabinowitz, Joshua D.

doi:10.1007/s00216-017-0514-4

Cited by 39 publications

(44 citation statements)

References 44 publications

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“…however, we were able to detect four more analogs of FA. 33 The new APCI-MS approach was less sensitive when compared with the ESI-MS/MS, but the LOQs for most of the intermediates were comparable to the work of Chen et al 43 Nevertheless using the APCI-based method, we retrieved the concentration of DHF, which was not possible using the ESI ion source. We speculate that APCI analyses can be less susceptible to matrix effects (including ion suppression) when compared with ESI, which may explain the better ionization of DHF.…”

Section: Discussionmentioning

confidence: 74%

“…The quantification of the folate pool in biological samples remains a challenge due to the complexity of the sample matrix. Work of Chen et al required a solid phase extraction (SPE) step using cyclohexy‐based chemistry cartridges for the enrichment of FA derivatives in cultured human cells . A study from Pawlosky et al used C18 Sep‐Pak cartridges, whereas Nandania et al used strong anion exchange cartridges for the enrichment in human whole blood .…”

Section: Discussionmentioning

confidence: 99%

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Comparison of ESI‐MS/MS and APCI‐MS methods for the quantification of folic acid analogs in C. elegans

et al. 2019

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Folic acid (FA) plays a vital role in central metabolism, including the one carbon cycle, nucleotide, and amino acid biosynthesis. The development of sensitive, accurate analytical methods to measure FA intermediates in tissues is critical to understand their biological roles in diverse physiological and pathological contexts. Here, we developed a highly sensitive method for the simultaneous quantification of FA intermediates in the nematode Caenorhabditis elegans as a model to dissect metabolic networks. The method was further validated by analyzing the worm folate pool upon RNAi knockdown of the dihydrofolate reductase gene dhfr‐1. Comparative mass spectrometry behavior of the FA analogs using two different ion sources, electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI), revealed ESI‐MS/MS to be more sensitive, but APCI‐MS provided more detailed structure inferences, which can elucidate chemical investigation and synthesis of FA analogs. Finally, we report on the use of in vitro oxidation coupled with high‐resolution mass spectrometry as a tool to discover new endogenous FA derivatives in the nematode.

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Section: Discussionmentioning

confidence: 74%

Section: Discussionmentioning

confidence: 99%

Comparison of ESI‐MS/MS and APCI‐MS methods for the quantification of folic acid analogs in C. elegans

et al. 2019

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“…Thus, we sought to examine the effects of MTR knockout on folate species required for nucleotide synthesis. The folate precursor for purine synthesis, 10-formyl THF, is indistinguishable from a closely related species 5,10-methenyl THF by LC/MS due to known interconversion after cell lysis 32,33 . We observed that MTR knockout results in depletion of the combined levels of 5,10-methenyl THF and 10-formyl THF ( Figure 2D-E), suggesting that purine synthesis could be affected.…”

Section: Resultsmentioning

confidence: 99%

Methionine synthase is essential for cancer cell proliferation in physiological folate environments

Sullivan

Darnell

Reilly

et al. 2020

Preprint

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Targeting folate metabolism can be an effective way to treat cancer. The enzyme methionine synthase catalyzes a key reaction in both folate and methionine metabolism. Early work suggested that inhibiting methionine synthase might restrain tumor growth, though the mechanism remains unclear. We find that due to its specific role in processing folates, methionine synthase is required for cancer proliferation. However, widely used cell culture conditions obscure the proliferative and metabolic consequences of methionine synthase inhibition. Complete dependence on methionine synthase only arises when 5-methyl tetrahydrofolate, the major folate found in circulation, is the predominant folate source provided to cells. In these physiological folate conditions, methionine synthase activity is necessary to maintain intracellular levels of nucleotides, but not methionine. These data reveal that the extracellular environment can alter the essentiality of methionine synthase and suggest that this enzyme plays a crucial cell-autonomous role in supporting nucleotide synthesis and cell proliferation in physiological contexts. Fellowship. A.M.D. was supported by a Jane Coffin Childs Postdoctoral Fellowship. M.G.V.H. acknowledges support from R35CA242379, R01CA201276, the Ludwig Center at MIT, the MIT Center for Precision Cancer Medicine, SU2C, the Emerald Foundation, the Lustgarten Foundation, and a Faculty Scholars Grant from HHMI. AUTHOR CONTRIBUTIONS Conceptualization, M.R.S., M.G.V.H.; Methodology, M.R.S., M.F.R., C.A.L.; Formal Analysis, M.R.S., A.M.D.; Investigation, M.R.S., A.M.D., M.F.R.; Resources, C.A.L.; Visualization, M.R.S., A.M.D.; Writing -Original Draft, M.R.S.; Writing -Review & Editing, M.R.S., A.M.D., M.G.V.H.; Funding Acquisition, M.R.S., A.M.D., M.G.V.H.

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“…The methodology employed in a previous study (46) was most successful when cell densities were high and compounds that blocked folate metabolism were used to increase the concentration of the analytes. This technique has recently been supplanted by an approach that uses derivatization agents to trap unstable methylene-THF intermediates as stable methyl-THF intermediates (47). As the derivatization step may lead to dechlorination, this method was not used in the present study.…”

Section: Discussionmentioning

confidence: 99%

Proteogenomics Reveals Novel Reductive Dehalogenases and Methyltransferases Expressed during Anaerobic Dichloromethane Metabolism

Kleindienst

Chourey

Gao

et al. 2019

Appl Environ Microbiol

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Dichloromethane (DCM) is susceptible to microbial degradation under anoxic conditions and is metabolized via the Wood-Ljungdahl pathway; however, mechanistic understanding of carbon-chlorine bond cleavage is lacking. The microbial consortium RM contains the DCM degrader "Candidatus Dichloromethanomonas elyunquensis" strain RM, which strictly requires DCM as a growth substrate. Proteomic workflows applied to DCM-grown consortium RM biomass revealed a total of 1,705 nonredundant proteins, 521 of which could be assigned to strain RM. In the presence of DCM, strain RM expressed a complete set of Wood-Ljungdahl pathway enzymes, as well as proteins implicated in chemotaxis, motility, sporulation, and vitamin/cofactor synthesis. Four corrinoid-dependent methyltransferases were among the most abundant proteins. Notably, two of three putative reductive dehalogenases (RDases) encoded within strain RM's genome were also detected in high abundance.Expressed RDase 1 and RDase 2 shared 30% amino acid identity, and RDase 1 was most similar to an RDase of Dehalococcoides mccartyi strain WBC-2 (AOV99960, 52% amino acid identity), while RDase 2 was most similar to an RDase of Dehalobacter sp. strain UNSWDHB (EQB22800, 72% amino acid identity). Although the involvement of RDases in anaerobic DCM metabolism has yet to be experimentally verified, the proteome characterization results implicated the possible participation of one or more reductive dechlorination steps and methyl group transfer reactions, leading to a revised proposal for an anaerobic DCM degradation pathway. IMPORTANCE Naturally produced and anthropogenically released DCM can reside in anoxic environments, yet little is known about the diversity of organisms, enzymes, and mechanisms involved in carbon-chlorine bond cleavage in the absence of oxygen. A proteogenomic approach identified two RDases and four corrinoid-dependent methyltransferases expressed by the DCM degrader "Candidatus Dichloromethanomonas elyunquensis" strain RM, suggesting that reductive dechlorination and methyl group transfer play roles in anaerobic DCM degradation. These findings suggest that the characterized DCM-degrading bacterium Dehalobacterium formicoaceticum and "Candidatus Dichloromethanomonas elyunquensis" strain RM utilize distinct strategies for Downloaded from carbon-chlorine bond cleavage, indicating that multiple pathways evolved for anaerobic DCM metabolism. The specific proteins (e.g., RDases and methyltransferases) identified in strain RM may have value as biomarkers for monitoring anaerobic DCM degradation in natural and contaminated environments.

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An LC-MS chemical derivatization method for the measurement of five different one-carbon states of cellular tetrahydrofolate

Cited by 39 publications

References 44 publications

Comparison of ESI‐MS/MS and APCI‐MS methods for the quantification of folic acid analogs in C. elegans

Comparison of ESI‐MS/MS and APCI‐MS methods for the quantification of folic acid analogs in C. elegans

Methionine synthase is essential for cancer cell proliferation in physiological folate environments

Proteogenomics Reveals Novel Reductive Dehalogenases and Methyltransferases Expressed during Anaerobic Dichloromethane Metabolism

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