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Quantification of Cellular Folate Species by LC-MS after Stabilization by Derivatization.

ABSTRACT: Folate cofactors play a key role in one-carbon metabolism. Analysis of individual folate species is hampered by the low chemical stability and high interconvertibility of folates, which can lead to severe experimental bias. Here, we present a complete workflow that employs simultaneous extraction and stabilization of folates by derivatization. We perform reductive methylation employing stable isotope labeled reagents to retain information on the position and redox state of one-carbon units as well as the redox state of the pteridine ring. The derivatives are analyzed by a targeted LC(HILIC)-MS/MS method without the need for deconjugation, thereby also preserving the glutamation state of folates. The presented method does not only improve analyte coverage and sensitivity as compared to other published methods, it also greatly simplifies sample handling and storage. Finally, we report differences in the response of bacterial and mammalian systems to pharmacological inhibition of dihydrofolate reductase.

SUBMITTER: Schittmayer M

PROVIDER: S-EPMC6011177 | biostudies-literature | 2018 Jun

REPOSITORIES: biostudies-literature

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Quantification of Cellular Folate Species by LC-MS after Stabilization by Derivatization.

Schittmayer Matthias M Birner-Gruenberger Ruth R Zamboni Nicola N

Analytical chemistry 20180605 12

Folate cofactors play a key role in one-carbon metabolism. Analysis of individual folate species is hampered by the low chemical stability and high interconvertibility of folates, which can lead to severe experimental bias. Here, we present a complete workflow that employs simultaneous extraction and stabilization of folates by derivatization. We perform reductive methylation employing stable isotope labeled reagents to retain information on the position and redox state of one-carbon units as we ...[more]

PMID: 29792680

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Project description:Diacylglycerols (DAGs) are important lipid mediators in cellular signaling transduction and metabolism. Imbalanced production or consumption of DAGs has a negative impact on the physiological functions of the body. However, comprehensive monitoring of structurally diverse DAGs remains a daunting task due to the rapid metabolism and ion suppression characteristics in biofluids. These bottlenecks call for developing a method that enables sensitive quantification of DAGs in biological sample. In this work, a straightforward charge derivatization strategy was developed to insert a series of structure analogs charge label, i.e., N, N-dimethylglycine (DMG) and N, N-dimethylalanine (DMA), on the free hydroxyl group of the DAGs. Owing to the existence of tertiary amino groups in charge label, the mass spectrometry ionization response of the derivatized DAGs was significantly increased in comparison with traditional metal ion adducts. After charge derivatization, the specific neutral loss diagnostic ions (DMG, 103 Da and DMA, 117 Da) were captured by mass spectrometry. Then, the DMG/DMA-oriented paired multiple reaction monitoring methods based on the characteristic diagnostic ions of the derivatized DAGs have been developed as sensitive methods for the detection (detection limit = 16 aM) and quantification (quantification limit = 62.5 aM) of DAGs in serum. Moreover, the tagged 1,2-DAGs and 1,3-DAGs sn-isomers have been well separated on the reversed-phase column in combination with ultra-performance liquid chromatography. Finally, metabolic characterizations of the tagged DAGs were further explored in L-Arginine-induced acute pancreatitis mice and resveratrol treated model mice. The results indicated that 1,2-DAGs were increased in the serum of model mice relative to normal controls and resveratrol significantly altered this metabolic abnormality. The currently established DMG/DMA-oriented paired charge derivatization strategy is promising for depicting DAGs changes more accurately in metabolic studies of lipid-related diseases and accurately evaluating drug treatment strategies.

Dataset Information

Quantification of Cellular Folate Species by LC-MS after Stabilization by Derivatization.

Publications

Quantification of Cellular Folate Species by LC-MS after Stabilization by Derivatization.

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