Quantitation of endogenous nucleoside triphosphates and nucleosides in human cells by liquid chromatography tandem mass spectrometry

Thomas, Dominique; Herold, Nikolas; Keppler, Oliver T.; Geißlinger, Gerd; Ferreiròs, Nerea

doi:10.1007/s00216-015-8588-3

Cited by 32 publications

(40 citation statements)

References 44 publications

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“…The precursor nucleoside analogs (unphosphorylated) used for all cell culture assays were purchased from the following sources: Tocris (cytarabine, fludarabine, clofarabine, and cladribine), Accord Healthcare GmbH (gemcitabine), and Jena Bioscience (vidarabine and nelarabine). All nucleotide standards, internal standards, and dNs for the LC-MS/MS analysis were obtained from Sigma-Aldrich, Silantes, or Alsachim (44). Cytarabine-13 C 3 was purchased from Santa Cruz and used for LC-MS/MS analysis.…”

Section: Methodsmentioning

confidence: 99%

The structural basis for cancer drug interactions with the catalytic and allosteric sites of SAMHD1

Knecht

Buzovetsky

Schneider

et al. 2018

Proc. Natl. Acad. Sci. U.S.A.

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SAMHD1 is a deoxynucleoside triphosphate triphosphohydrolase (dNTPase) that depletes cellular dNTPs in noncycling cells to promote genome stability and to inhibit retroviral and herpes viral replication. In addition to being substrates, cellular nucleotides also allosterically regulate SAMHD1 activity. Recently, it was shown that high expression levels of SAMHD1 are also correlated with significantly worse patient responses to nucleotide analog drugs important for treating a variety of cancers, including acute myeloid leukemia (AML). In this study, we used biochemical, structural, and cellular methods to examine the interactions of various cancer drugs with SAMHD1. We found that both the catalytic and the allosteric sites of SAMHD1 are sensitive to sugar modifications of the nucleotide analogs, with the allosteric site being significantly more restrictive. We crystallized cladribine-TP, clofarabine-TP, fludarabine-TP, vidarabine-TP, cytarabine-TP, and gemcitabine-TP in the catalytic pocket of SAMHD1. We found that all of these drugs are substrates of SAMHD1 and that the efficacy of most of these drugs is affected by SAMHD1 activity. Of the nucleotide analogs tested, only cladribine-TP with a deoxyribose sugar efficiently induced the catalytically active SAMHD1 tetramer. Together, these results establish a detailed framework for understanding the substrate specificity and allosteric activation of SAMHD1 with regard to nucleotide analogs, which can be used to improve current cancer and antiviral therapies.

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Section: Methodsmentioning

confidence: 99%

The structural basis for cancer drug interactions with the catalytic and allosteric sites of SAMHD1

Knecht

Buzovetsky

Schneider

et al. 2018

Proc. Natl. Acad. Sci. U.S.A.

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“…Newer methods utilize mass spectrometry as a more selective and sensitive detection method. Quantification has been performed in multiple matrices and for multiple nucleotides in these methods, (18–21) however, most utilize direct analysis techniques where phosphate fractions are separated on an HPLC column using ion pairing based mobile phases. This separation method may cause ion suppression from the mobile phase and also affects the column chromatography for measuring other molecules with HPLC.…”

Section: Introductionmentioning

confidence: 99%

A LC-MS/MS Method for Quantifying Adenosine, Guanosine and Inosine Nucleotides in Human Cells

et al. 2016

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Purpose To develop and validate a method for the simultaneous measurement of adenosine, guanosine, and inosine derived from mono (MP) and triphosphate (TP) forms in peripheral blood mononuclear cells (PBMCs), red blood cells (RBCs) and dried blood spots (DBS). Methods Solid phase extraction of cell lysates followed by dephosphorylation to molar equivalent nucleoside and LC-MS/MS quantification. Results The assay was linear for each of the three quantification ranges: 10–2000, 1.0–200 and 0.25–50 pmol/sample for adenosine, guanosine, and inosine, respectively. Intraassay (n=6) and interassay (n=18) precision (%CV) were within 1.7% to 16% while accuracy (%deviation) was within −11.5 % to 14.7 % for all three analytes. Nucleotide monophosphates were less concentrated than triphosphates (except for inosine) and levels in PBMCs were higher than RBCs for all three nucleotides (10, 55, and 5.6 fold for ATP, GTP and ITP, respectively). DBS samples had an average (SD) of −26% (22.6%) lower TP and 184% (173%) higher MP levels compared to paired RBC lysates, suggesting hydrolysis of the TP in DBS. Conclusion This method was accurate and precise for physiologically relevant concentrations of adenosine, guanosine and inosine nucleotides in mono- and triphosphate forms, providing a bioanalytical tool for quantitation of nucleotides for clinical studies.

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“…The concentrations of ATP, adenosine and cAMP in the samples were analyzed by liquid chromatographyelectrospray ionization-tandem mass spectrometry as previously described [23]. Briefly, the analytes were extracted from 50 µl cell culture supernatant by protein precipitation with methanol and the supernatant was divided in two fractions.…”

Section: Determination Of Adenosine and Atp By Lc-ms/msmentioning

confidence: 99%

“…Adenosine and cAMP were analyzed using an Atlantis T3 column (100 x 2.1 mm, Waters). Adenosine was analyzed similarly as described previously [23], cAMP was quantified using 13 C 5 -cAMP as internal standard. The precursor-to-product ion transitions used as quantifier for cAMP was m/z 328.0 à 134.0.…”

Section: Determination Of Adenosine and Atp By Lc-ms/msmentioning

confidence: 99%

“…For the chromatographic analysis of the ATP, an anion exchange HPLC column (BioBasic AX, 150 x 2.1 mm, Thermo) and a 5500 QTrap (Sciex) were used as analyzers, operating as triple quadrupole in positive multiple reaction monitoring (MRM) mode. The analysis of ATP was performed as previously described [23]. Adenosine and cAMP were analyzed using an Atlantis T3 column (100 x 2.1 mm, Waters).…”

Section: Determination Of Adenosine and Atp By Lc-ms/msmentioning

confidence: 99%

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Activation of Adenylyl Cyclase Causes Stimulation of Adenosine Receptors

Pleli

Ferreiròs

Thomas

et al. 2018

Cell Physiol Biochem

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Background/Aims: Signaling of G_s protein-coupled receptors (GsPCRs) is accomplished by stimulation of adenylyl cyclase, causing an increase of the intracellular cAMP concentration, activation of the intracellular cAMP effectors protein kinase A (PKA) and Epac, and an efflux of cAMP, the function of which is still unclear. Methods: Activation of adenylyl cyclase by GsPCR agonists or cholera toxin was monitored by measurement of the intracellular cAMP concentration by ELISA, anti-phospho-PKA substrate motif phosphorylation by immunoblotting, and an Epac-FRET assay in the presence and absence of adenosine receptor antagonists or ecto-nucleotide phosphodiesterase/pyrophosphatase2 (eNPP2) inhibitors. The production of AMP from cAMP by recombinant eNPP2 was measured by HPLC. Extracellular adenosine was determined by LC-MS/MS, extracellular ATP by luciferase and LC-MS/MS. The expression of eNPP isoenzymes 1-3 was examined by RT-PCR. The expression of multidrug resistance protein 4 was suppressed by siRNA. Results: Here we show that the activation of GsPCRs and the GsPCRs-independent activation of G_s proteins and adenylyl cyclase by cholera toxin induce stimulation of cell surface adenosine receptors (A_2A or A_2B adenosine receptors). In PC12 cells stimulation of adenylyl cyclase by GsPCR or cholera toxin caused activation of A_2A adenosine receptors by an autocrine signaling pathway involving cAMP efflux through multidrug resistance protein 4 and hydrolysis of released cAMP to AMP by eNPP2. In contrast, in PC3 cells cholera toxin- and GsPCR-induced stimulation of adenylyl cyclase resulted in the activation of A_2B adenosine receptors. Conclusion: Our findings show that stimulation of adenylyl cyclase causes a remarkable activation of cell surface adenosine receptors.

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Quantitation of endogenous nucleoside triphosphates and nucleosides in human cells by liquid chromatography tandem mass spectrometry

Cited by 32 publications

References 44 publications

The structural basis for cancer drug interactions with the catalytic and allosteric sites of SAMHD1

The structural basis for cancer drug interactions with the catalytic and allosteric sites of SAMHD1

A LC-MS/MS Method for Quantifying Adenosine, Guanosine and Inosine Nucleotides in Human Cells

Activation of Adenylyl Cyclase Causes Stimulation of Adenosine Receptors

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