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Article Dans Une Revue Talanta Année : 2019

Monitoring of phosphorylation using immobilized kinases by on-line enzyme bioreactors hyphenated with High-Resolution Mass Spectrometry

Résumé

Nucleosides analogues are the cornerstone of the treatment of several human diseases. They are especially at the forefront of antiviral therapy. Their therapeutic efficiency depends on their capacity to be converted to the active nucleoside triphosphate form through successive phosphorylation steps catalyzed by nucleoside/nucleotide kinases. In this context, it is mandatory to develop a rapid, reliable and sensitive enzyme activity test to evaluate their metabolic pathways. In this study, we report a proof of concept to directly monitor on-line nucleotide multiple phosphorylation. The methodology was developed by on-line enzyme bioreactors hyphenated with High-Resolution Mass Spectrometry detection. Human Thymidylate Kinase (hTMPK) and human Nucleoside Diphosphate Kinase (hNDPK) were covalently immobilized on functionalized silica beads, and packed into micro-bioreactors (40 μL). By continuous infusion of substrate into the bioreactors, the conversion of thymidine monophosphate (dTMP) into its di- (dTDP) and tri-phosphorylated (dTTP) forms was visualized by monitoring their Extracted Ion Chromatogram (EIC) of their [M - H]$^-$ ions. Both bioreactors were found to be robust and durable over 60 days (storage at 4 °C in ammonium acetate buffer), after 20 uses and more than 750 min of reaction, making them suitable for routine analysis. Each on-line conversion step was shown rapid (<5 min), efficient (conversion efficiency > 55%), precise and repeatable (CV < 3% for run-to-run analysis). The feasibility of the on-line multi-step conversion from dTMP to dTTP was also proved. In the context of selective antiviral therapy, this proof of concept was then applied to the monitoring of specificity of conversion of two synthesized Acyclic Nucleosides Phosphonates (ANPs), regarding human Thymidylate Kinase (hTMPK) and vaccina virus Thymidylate Kinase (vvTMPK).
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hal-03466045 , version 1 (20-12-2021)

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Paternité - Pas d'utilisation commerciale

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Justine Ferey, David da Silva, Cyril Colas, Pierre Lafite, Dimitrios Topalis, et al.. Monitoring of phosphorylation using immobilized kinases by on-line enzyme bioreactors hyphenated with High-Resolution Mass Spectrometry. Talanta, 2019, 205, pp.120120. ⟨10.1016/j.talanta.2019.120120⟩. ⟨hal-03466045⟩
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