Format:
Online-Ressource
ISSN:
1615-9314
Content:
The accurate quantification of the highly unstable intracellular cofactor nicotinamide adenine dinucleotide phosphate in its oxidized and reduced forms demands a thorough evaluation of the analytical workflow and dedicated methods reflecting their solution chemistry as well as the biological importance of their ratio. In this work, we present a workflow for the analysis of intracellular levels of oxidized and reduced nicotinamide adenine dinucleotide phosphate in the yeast Pichia pastoris, including hot aqueous extraction, chromatographic separation in reversed‐phase conditions employing a 100% wettable stationary phase, and subsequent tandem mass spectrometric analysis. A thorough evaluation and optimization of the sample preparation procedure resulted in excellent biological repeatabilities (on average 〈10%, N = 3) without employing an internal standardization approach. As a consequence, the methodology proved to be appropriate for the relative assessment of intracellular levels of oxidized and reduced nicotinamide adenine dinucleotide phosphate in different P. pastoris strains. The ratio of reduced versus oxidized nicotinamide adenine dinucleotide phosphate was significantly higher in an engineered strain overexpressing glucose‐6‐phosphate dehydrogenase than in the corresponding wildtype strain. Interestingly, a difference was also observed in the nicotinamide adenine dinucleotide phosphate pool size, which was significantly higher in the wildtype than in the modified strain.
In:
volume:37
In:
number:16
In:
year:2014
In:
pages:2185-2191
In:
extent:7
In:
Journal of separation science, Weinheim : Wiley-VCH, [2000]-, 37, Heft 16 (2014), 2185-2191 (gesamt 7), 1615-9314
Language:
English
DOI:
10.1002/jssc.201400290
URN:
urn:nbn:de:101:1-2023012006080046432929
URL:
https://doi.org/10.1002/jssc.201400290
URL:
https://nbn-resolving.org/urn:nbn:de:101:1-2023012006080046432929
URL:
https://d-nb.info/1278674853/34
URL:
https://doi.org/10.1002/jssc.201400290
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