Fully validated assay for the quantification of endogenous nucleoside mono- and triphosphates using online extraction coupled with liquid chromatography–tandem mass spectrometry

Machon, Christelle; Jordheim, Lars Petter; Puy, Jean-Yves; Lefèbvre, Isabelle; Dumontet, Charles; Guitton, Jérôme

doi:10.1007/s00216-014-7711-1

Cited by 32 publications

(37 citation statements)

References 38 publications

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“…ATP and GTP were quantified by liquid chromatography– tandem mass spectrometry (LC‐MS/MS) by using an assay developed in our laboratory (26). The method is based on online extraction coupled to separation of the compounds on a Hypercarb analytical column (Thermo Fisher Scientific Life Sciences).…”

Section: Methodsmentioning

confidence: 99%

Metastasis suppressor NM23 limits oxidative stress in mammals by preventing activation of stress‐activated protein kinases/JNKs through its nucleoside diphosphate kinase activity

et al. 2017

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(nonmetastatic expressed 1) gene, which encodes nucleoside diphosphate kinase (NDPK) A [also known as nonmetastatic clone 23 (NM23)-H1 in humans and NM23-M1 in mice], is a suppressor of metastasis, but several lines of evidence-mostly from plants-also implicate it in the regulation of the oxidative stress response. Here, our aim was to investigate the physiologic relevance of NDPK A with respect to the oxidative stress response in mammals and to study its molecular basis. -knockout mice died sooner, suffered greater hepatocyte injury, and had lower superoxide dismutase activity than did wild-type (WT) mice in response to paraquat-induced acute oxidative stress. Deletion of reduced total NDPK activity and exacerbated activation of the stress-related MAPK, JNK, in the liver in response to paraquat. In a mouse transformed hepatocyte cell line and in primary cultures of normal human keratinocytes, MAPK activation in response to HO and UVB, respectively, was dampened by expression of NM23-M1/NM23-H1, dependent on its NDPK catalytic activity. Furthermore, excess or depletion of NM23-M1/NM23-H1 NDPK activity did not affect the intracellular bulk concentration of nucleoside di- and triphosphates. -deficient mouse embryo fibroblasts grew poorly in culture, were more sensitive to stress than WT fibroblasts, and did not immortalize, which suggested that they senesce earlier than do WT fibroblasts. Collectively, these results indicate that the NDPK activity of NM23-M1/NM23-H1 protects cells from acute oxidative stress by inhibiting activation of JNK in mammal models.-Peuchant, E., Bats, M.-L., Moranvillier, I., Lepoivre, M., Guitton, J., Wendum, D., Lacombe, M.-L., Moreau-Gaudry, F., Boissan, M., Dabernat, S. Metastasis suppressor NM23 limits oxidative stress in mammals by preventing activation of stress-activated protein kinases/JNKs through its nucleoside diphosphate kinase activity.

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Section: Methodsmentioning

confidence: 99%

Metastasis suppressor NM23 limits oxidative stress in mammals by preventing activation of stress‐activated protein kinases/JNKs through its nucleoside diphosphate kinase activity

et al. 2017

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“…Matrix effect (ME), recovery (RE) and processing efficiency (PE) were determined following the experiments described by Matuszewski, Constanzer, and Chavez-Eng (2003). Since the biological matrix (cellular lysate) already contained the analytes (endogenous dNTPs), to investigate the ME, RE and PE of the desaltation and concentration process, we used the internal standards peak area (stable labeled isotopes of each analyte) as previously described by Machon et al (2014). Six different lots of PBMC lysate matrix were extracted, and three levels of stable labeled internal standard working solutions were prepared at concentrations of 150, 600 and 2000 fmol/sample.…”

Section: Validationmentioning

confidence: 99%

Development and validation of an LC–MS/MS quantitative method for endogenous deoxynucleoside triphosphates in cellular lysate

Chen

McAllister

Klein

et al. 2016

Biomedical Chromatography

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The endogenous deoxynucleoside triphosphate (dNTP) pool includes deoxyadenosine triphosphate (dATP), deoxycytidine triphosphate (dCTP), deoxyguanosine triphosphate (dGTP), and thymidine triphosphate (TTP). The endogenous dNTP pool is regulated by complex enzymatic pathways that can be targeted by drugs, such as antimetabolites. In addition, these components compete with antiviral nucleos(t)ide analog triphosphates, contributing to the mechanism of pharmacologic action. This communication describes the development and validation of a sensitive method to quantify the intracellular dNTP pool in cellular lysates. The extraction process was optimized for dNTPs using an indirect strategy—the separation of mono-, di-, and triphosphate moieties by strong anion exchange, dephosphorylation of target fractions to molar equivalent nucleosides—followed by sensitive quantitation using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The validated analytical range was 50 to 2500 fmol/sample for each dNTP. The assay was used to quantify dNTPs in peripheral blood mononuclear cells (PBMC) from forty clinical research participants (n=279 samples). Median (interquartile range) concentrations were 143 (116, 169) for dATP, 737 (605, 887) for dCTP, 237 (200, 290) for dGTP, and 315 (220, 456) for TTP, respectively, in femtomole per million cells. This method allows for future studies of endogenous dNTP disposition in subjects receiving antimetabolites, nucleos(t)ide analogues, or for other clinical scenarios.

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“…Ion pairing reverse phase (IP-RP) chromatographic conditions have been described for the LC-MS analysis of nucleotides, 93,94 although primarily standard nucleotides were investigated. Similarly, while HILIC coupled to ESI-MS has been described for the analysis of standard nucleotides, 90 it is unclear if that approach is applicable to mixtures of modified nucleotides.…”

Section: Intact Rnasmentioning

confidence: 99%

The identification and characterization of non-coding and coding RNAs and their modified nucleosides by mass spectrometry

Gaston

Limbach

2014

RNA Biology

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The analysis of ribonucleic acids (RNA) by mass spectrometry has been a valuable analytical approach for more than 25 years. In fact, mass spectrometry has become a method of choice for the analysis of modified nucleosides from RNA isolated out of biological samples. This review summarizes recent progress that has been made in both nucleoside and oligonucleotide mass spectral analysis. Applications of mass spectrometry in the identification, characterization and quantification of modified nucleosides are discussed. At the oligonucleotide level, advances in modern mass spectrometry approaches combined with the standard RNA modification mapping protocol enable the characterization of RNAs of varying lengths ranging from low molecular weight short interfering RNAs (siRNAs) to the extremely large 23 S rRNAs. New variations and improvements to this protocol are reviewed, including top-down strategies, as these developments now enable qualitative and quantitative measurements of RNA modification patterns in a variety of biological systems.

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Fully validated assay for the quantification of endogenous nucleoside mono- and triphosphates using online extraction coupled with liquid chromatography–tandem mass spectrometry

Cited by 32 publications

References 38 publications

Metastasis suppressor NM23 limits oxidative stress in mammals by preventing activation of stress‐activated protein kinases/JNKs through its nucleoside diphosphate kinase activity

Metastasis suppressor NM23 limits oxidative stress in mammals by preventing activation of stress‐activated protein kinases/JNKs through its nucleoside diphosphate kinase activity

Development and validation of an LC–MS/MS quantitative method for endogenous deoxynucleoside triphosphates in cellular lysate

The identification and characterization of non-coding and coding RNAs and their modified nucleosides by mass spectrometry

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