Zic-HILIC MS/MS Method for NADomics Provides Novel Insights into Redox Homeostasis in BL21 Under Microaerobic and Anaerobic Conditions

Overview

Journal Metabolites

Publisher MDPI

Date 2024 Nov 26

PMID 39590843

Authors

Divyata Vilas Rane

Laura Garcia-Calvo

Kare Andre Kristiansen

Per Bruheim

Affiliations

Soon will be listed here.

Abstract

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. 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 C 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 BL21 under varying oxygen supplies including aerobic, microaerobic, and anaerobic conditions. : 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 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.

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