| Title: |
Zic-HILIC MS/MS Method for NADomics Provides Novel Insights into Redox Homeostasis in Escherichia coli BL21 Under Microaerobic and Anaerobic Conditions. |
| Authors: |
Rane, Divyata Vilas, García-Calvo, Laura, Kristiansen, Kåre Andre, Bruheim, Per |
| Source: |
Metabolites (2218-1989); Nov2024, Vol. 14 Issue 11, p607, 22p |
| Subject Terms: |
PYRIDINE nucleotides, ESCHERICHIA coli, ISOTOPE dilution analysis, OXIDATION-reduction reaction, MASS spectrometry, NAD (Coenzyme) |
| Abstract: |
Background: Nicotinamide adenine dinucleotide (NAD+), its precursors, and its derivatives (collectively NADome) play a crucial role in cellular processes and maintain redox homeostasis. Understanding the dynamics of these metabolic pools and redox reactions can provide valuable insights into metabolic functions, especially cellular regulation and stress response mechanisms. The accurate quantification of these metabolites is challenging due to the interconversion between the redox forms. Methods: Our laboratory previously developed a zwitterionic hydrophilic interaction liquid chromatography (zic-HILIC)–tandem mass spectrometry method for the quantification of five essential pyridine nucleotides, including NAD+ derivatives and it's reduced forms, with 13C isotope dilution and matrix-matched calibration. In this study, we have improved the performance of the chromatographic method and expanded its scope to twelve analytes for a comprehensive view of NAD+ biosynthesis and utilization. The analytical method was validated and applied to investigate Escherichia coli BL21 under varying oxygen supplies including aerobic, microaerobic, and anaerobic conditions. Conclusions: The intracellular absolute metabolite concentrations ranged over four orders of magnitude with NAD+ as the highest abundant, while its precursors were much less abundant. The composition of the NADome at oxygen-limited conditions aligned more with that in the anaerobic conditions rather than in the aerobic phase. Overall, the NADome was quite homeostatic and E. coli rapidly, but in a minor way, adapted the metabolic activity to the challenging shift in the growth conditions and achieved redox balance. Our findings demonstrate that the zic-HILIC-MS/MS method is sensitive, accurate, robust, and high-throughput, providing valuable insights into NAD+ metabolism and the potential significance of these metabolites in various biological contexts. [ABSTRACT FROM AUTHOR] |
|
Copyright of Metabolites (2218-1989) is the property of MDPI and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.) |
| Database: |
Complementary Index |
