Zic-HILIC MS/MS Method for NADomics Provides Novel Insights into Redox Homeostasis in <i>Escherichia coli</i> BL21 Under Microaerobic and Anaerobic Conditions
<b>Background:</b> Nicotinamide adenine dinucleotide (NAD<sup>+</sup>), 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...
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2024-11-01
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| author | Divyata Vilas Rane Laura García-Calvo Kåre Andre Kristiansen Per Bruheim |
| author_facet | Divyata Vilas Rane Laura García-Calvo Kåre Andre Kristiansen Per Bruheim |
| author_sort | Divyata Vilas Rane |
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| description | <b>Background:</b> Nicotinamide adenine dinucleotide (NAD<sup>+</sup>), 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. <b>Methods:</b> 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<sup>+</sup> derivatives and it’s reduced forms, with <sup>13</sup>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<sup>+</sup> biosynthesis and utilization. The analytical method was validated and applied to investigate <i>Escherichia coli</i> BL21 under varying oxygen supplies including aerobic, microaerobic, and anaerobic conditions. <b>Conclusions</b>: The intracellular absolute metabolite concentrations ranged over four orders of magnitude with NAD<sup>+</sup> 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 <i>E. coli</i> 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<sup>+</sup> metabolism and the potential significance of these metabolites in various biological contexts. |
| format | Article |
| id | doaj-art-34215f222a21415cb703da9335e10fa1 |
| institution | Kabale University |
| issn | 2218-1989 |
| language | English |
| publishDate | 2024-11-01 |
| publisher | MDPI AG |
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| series | Metabolites |
| spelling | doaj-art-34215f222a21415cb703da9335e10fa12024-11-26T18:13:08ZengMDPI AGMetabolites2218-19892024-11-01141160710.3390/metabo14110607Zic-HILIC MS/MS Method for NADomics Provides Novel Insights into Redox Homeostasis in <i>Escherichia coli</i> BL21 Under Microaerobic and Anaerobic ConditionsDivyata Vilas Rane0Laura García-Calvo1Kåre Andre Kristiansen2Per Bruheim3Department of Biotechnology and Food Science, NTNU Norwegian University of Science and Technology, N-7491 Trondheim, NorwayDepartment of Biotechnology and Food Science, NTNU Norwegian University of Science and Technology, N-7491 Trondheim, NorwayDepartment of Biotechnology and Food Science, NTNU Norwegian University of Science and Technology, N-7491 Trondheim, NorwayDepartment of Biotechnology and Food Science, NTNU Norwegian University of Science and Technology, N-7491 Trondheim, Norway<b>Background:</b> Nicotinamide adenine dinucleotide (NAD<sup>+</sup>), 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. <b>Methods:</b> 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<sup>+</sup> derivatives and it’s reduced forms, with <sup>13</sup>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<sup>+</sup> biosynthesis and utilization. The analytical method was validated and applied to investigate <i>Escherichia coli</i> BL21 under varying oxygen supplies including aerobic, microaerobic, and anaerobic conditions. <b>Conclusions</b>: The intracellular absolute metabolite concentrations ranged over four orders of magnitude with NAD<sup>+</sup> 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 <i>E. coli</i> 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<sup>+</sup> metabolism and the potential significance of these metabolites in various biological contexts.https://www.mdpi.com/2218-1989/14/11/607pyridine nucleotidesNAD<sup>+</sup>redox ratiozic-HILIC MS/MSisotope dilutionmatrix-matched |
| spellingShingle | Divyata Vilas Rane Laura García-Calvo Kåre Andre Kristiansen Per Bruheim Zic-HILIC MS/MS Method for NADomics Provides Novel Insights into Redox Homeostasis in <i>Escherichia coli</i> BL21 Under Microaerobic and Anaerobic Conditions Metabolites pyridine nucleotides NAD<sup>+</sup> redox ratio zic-HILIC MS/MS isotope dilution matrix-matched |
| title | Zic-HILIC MS/MS Method for NADomics Provides Novel Insights into Redox Homeostasis in <i>Escherichia coli</i> BL21 Under Microaerobic and Anaerobic Conditions |
| title_full | Zic-HILIC MS/MS Method for NADomics Provides Novel Insights into Redox Homeostasis in <i>Escherichia coli</i> BL21 Under Microaerobic and Anaerobic Conditions |
| title_fullStr | Zic-HILIC MS/MS Method for NADomics Provides Novel Insights into Redox Homeostasis in <i>Escherichia coli</i> BL21 Under Microaerobic and Anaerobic Conditions |
| title_full_unstemmed | Zic-HILIC MS/MS Method for NADomics Provides Novel Insights into Redox Homeostasis in <i>Escherichia coli</i> BL21 Under Microaerobic and Anaerobic Conditions |
| title_short | Zic-HILIC MS/MS Method for NADomics Provides Novel Insights into Redox Homeostasis in <i>Escherichia coli</i> BL21 Under Microaerobic and Anaerobic Conditions |
| title_sort | zic hilic ms ms method for nadomics provides novel insights into redox homeostasis in i escherichia coli i bl21 under microaerobic and anaerobic conditions |
| topic | pyridine nucleotides NAD<sup>+</sup> redox ratio zic-HILIC MS/MS isotope dilution matrix-matched |
| url | https://www.mdpi.com/2218-1989/14/11/607 |
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