Absolute Quantification of ppGpp and pppGpp by Double-Spike Isotope Dilution Ion Chromatography–High-Resolution Mass Spectrometry

Guanosine 5′-diphosphate 3′-diphosphate (ppGpp) and guanosine 5′-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 detect...

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Bibliographic Details
Published in:Analytical chemistry (Washington) 2018-09, Vol.90 (18), p.10715-10723
Main Authors: Patacq, Clément, Chaudet, Nicolas, Létisse, Fabien
Format: Article
Language:English
Subjects:
Antibiotics
Biochemistry
Cells
Chemical Sciences
Chemistry
Dilution
E coli
Environmental stress
Guanosine
High resolution
Historical account
Ion chromatography
Isotopes
Mass spectrometry
Mass spectroscopy
Nucleotides
Other
Phosphohydrolase
Plant cells
Sample preparation
Serine
Spectroscopy
Thin layer chromatography
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Summary:Guanosine 5′-diphosphate 3′-diphosphate (ppGpp) and guanosine 5′-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 32P-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 13C 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 15N 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.
ISSN:0003-2700
1520-6882
DOI:10.1021/acs.analchem.8b00829