Absolute quantification of ppGpp and pppGpp by double-spike isotope dilution ion chromatography–high-resolution mass spectrometry
| Autor: | Clément Patacq, Fabien Létisse, Nicolas Chaudet |
|---|---|
| Přispěvatelé: | Laboratoire d'Ingénierie des Systèmes Biologiques et des Procédés (LISBP), Centre National de la Recherche Scientifique (CNRS)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut National de la Recherche Agronomique (INRA), Département de Bioprocédés R&D, Sanofi Pasteur SA, Université Fédérale Toulouse Midi-Pyrénées, Bioprocess R&D department of Sanofi Pasteur, Association Nationale de la Technologique (ANRT), MetaboHUB-ANR-11-INBS-0010, ANR-11-INBS-0010,METABOHUB,Développement d'une infrastructure française distribuée pour la métabolomique dédiée à l'innovation(2011), Institut National de la Recherche Agronomique (INRA)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Centre National de la Recherche Scientifique (CNRS), Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS), Université de Toulouse (UT) |
| Jazyk: | angličtina |
| Rok vydání: | 2018 |
| Předmět: |
0301 basic medicine
Spectrometry Mass Electrospray Ionization Absolute quantification Ion chromatography mass spectrum analysis Guanosine Guanosine Tetraphosphate Isotope dilution 01 natural sciences Analytical Chemistry 03 medical and health sciences chemistry.chemical_compound Isotopes spectrométrie de masse Serine Chromatography résistance bactérienne nucléotide Hydrolysis 010401 analytical chemistry thin layer chromatography Guanosine Pentaphosphate adaptation au stress Nucleotidyltransferases Thin-layer chromatography 0104 chemical sciences Kinetics Double spike 030104 developmental biology chemistry Mutation bacteria escherichia coli Mass spectrum analysis [CHIM.OTHE]Chemical Sciences/Other chromatographie en couche mince Chromatography Liquid |
| Zdroj: | Analytical Chemistry Analytical Chemistry, American Chemical Society, 2018, 90 (18), pp.10715-10723. ⟨10.1021/acs.analchem.8b00829⟩ Analytical Chemistry, 2018, 90 (18), pp.10715-10723. ⟨10.1021/acs.analchem.8b00829⟩ |
| ISSN: | 0003-2700 1520-6882 |
| DOI: | 10.1021/acs.analchem.8b00829⟩ |
| Popis: | Guanosine 5'-diphosphate 3'-diphosphate (ppGpp) and guanosine S'-triphosphate 3'-diphosphate (pppGpp) play a central role in the adaptation of bacterial and plant cells to nutritional and environmental stresses and in bacterial resistance to antibiotics. These compounds have historically been detected and quantified by two-dimensional thin-layer chromatography of P-32-radiolabeled nucleotides. We report a new method to quantify ppGpp and pppGpp in complex biochemical matrix using ion chromatography coupled to high-resolution mass spectrometry. The method is based on isotopic dilution mass spectrometry (IDMS) using C-13 to accurately quantify the nucleotides. However, the loss of a phosphate group from pppGpp during the sample preparation process results in the erroneous quantification of ppGpp. This bias was corrected by adding an extra N-15 isotope dilution dimension. This double-spike IDMS method was applied to quantify the ppGpp and pppGpp in Escherichia coli and in a mutant strain deleted for gppA (encoding the ppGpp phosphohydrolase) before and after exposure of both strains to serine hydroxamate, known to trigger the accumulation of these nucleotides. |
| Databáze: | OpenAIRE |
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