Quantitative isotope-dilution high-resolution-mass-apectrometry analysis of multiple intracellular metabolites in Clostridium autoethanogenum with uniformly 13C-labeled standards derived from Spirulina
We have investigated the applicability of commercially available lyophilized spirulina (Arthrospira platensis), a microorganism uniformly labeled with 13C, as a readily accessible source of multiple 13C-labeled metabolites suitable as internal standards for the quantitative determination of intracel...
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| Format: | Article |
| Language: | English |
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American Chemical Society
2018
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| Online Access: | https://eprints.nottingham.ac.uk/51056/ |
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| author | Schatschneider, Sarah Abdelrazig, Salah M.A. Safo, Laudina Henstra, Anne M. Millat, Thomas Kim, Dong-Hyun Winzer, Klaus Minton, Nigel P. Barrett, David A. |
| author_facet | Schatschneider, Sarah Abdelrazig, Salah M.A. Safo, Laudina Henstra, Anne M. Millat, Thomas Kim, Dong-Hyun Winzer, Klaus Minton, Nigel P. Barrett, David A. |
| author_sort | Schatschneider, Sarah |
| building | Nottingham Research Data Repository |
| collection | Online Access |
| description | We have investigated the applicability of commercially available lyophilized spirulina (Arthrospira platensis), a microorganism uniformly labeled with 13C, as a readily accessible source of multiple 13C-labeled metabolites suitable as internal standards for the quantitative determination of intracellular bacterial metabolites. Metabolites of interest were analyzed by hydrophilic-interaction liquid chromatography coupled with high-resolution mass spectrometry. Multiple internal standards obtained from uniformly (U)-13C-labeled extracts from spirulina were used to enable isotope-dilution mass spectrometry (IDMS) in the identification and quantification of intracellular metabolites. Extraction of the intracellular metabolites of Clostridium autoethanogenum using 2:1:1 chloroform/methanol/water was found to be the optimal method in comparison with freeze–thaw, homogenization, and sonication methods. The limits of quantification were ≤1 μM with excellent linearity for all of the calibration curves (R2 ≥ 0.99) for 74 metabolites. The precision and accuracy were found to be within relative standard deviations (RSDs) of 15% for 49 of the metabolites and within RSDs of 20% for all of the metabolites. The method was applied to study the effects of feeding different levels of carbon monoxide (as a carbon source) on the central metabolism and Wood–Ljungdahl pathway of C. autoethanogenum grown in continuous culture over 35 days. Using LC-IDMS with U-13C spirulina allowed the successful quantification of 52 metabolites in the samples, including amino acids, carboxylic acids, sugar phosphates, purines, and pyrimidines. The method provided absolute quantitative data on intracellular metabolites that was suitable for computational modeling to understand and optimize the C. autoethanogenum metabolic pathways active in gas fermentation. |
| first_indexed | 2025-11-14T20:19:13Z |
| format | Article |
| id | nottingham-51056 |
| institution | University of Nottingham Malaysia Campus |
| institution_category | Local University |
| language | English |
| last_indexed | 2025-11-14T20:19:13Z |
| publishDate | 2018 |
| publisher | American Chemical Society |
| recordtype | eprints |
| repository_type | Digital Repository |
| spelling | nottingham-510562020-05-08T09:15:31Z https://eprints.nottingham.ac.uk/51056/ Quantitative isotope-dilution high-resolution-mass-apectrometry analysis of multiple intracellular metabolites in Clostridium autoethanogenum with uniformly 13C-labeled standards derived from Spirulina Schatschneider, Sarah Abdelrazig, Salah M.A. Safo, Laudina Henstra, Anne M. Millat, Thomas Kim, Dong-Hyun Winzer, Klaus Minton, Nigel P. Barrett, David A. We have investigated the applicability of commercially available lyophilized spirulina (Arthrospira platensis), a microorganism uniformly labeled with 13C, as a readily accessible source of multiple 13C-labeled metabolites suitable as internal standards for the quantitative determination of intracellular bacterial metabolites. Metabolites of interest were analyzed by hydrophilic-interaction liquid chromatography coupled with high-resolution mass spectrometry. Multiple internal standards obtained from uniformly (U)-13C-labeled extracts from spirulina were used to enable isotope-dilution mass spectrometry (IDMS) in the identification and quantification of intracellular metabolites. Extraction of the intracellular metabolites of Clostridium autoethanogenum using 2:1:1 chloroform/methanol/water was found to be the optimal method in comparison with freeze–thaw, homogenization, and sonication methods. The limits of quantification were ≤1 μM with excellent linearity for all of the calibration curves (R2 ≥ 0.99) for 74 metabolites. The precision and accuracy were found to be within relative standard deviations (RSDs) of 15% for 49 of the metabolites and within RSDs of 20% for all of the metabolites. The method was applied to study the effects of feeding different levels of carbon monoxide (as a carbon source) on the central metabolism and Wood–Ljungdahl pathway of C. autoethanogenum grown in continuous culture over 35 days. Using LC-IDMS with U-13C spirulina allowed the successful quantification of 52 metabolites in the samples, including amino acids, carboxylic acids, sugar phosphates, purines, and pyrimidines. The method provided absolute quantitative data on intracellular metabolites that was suitable for computational modeling to understand and optimize the C. autoethanogenum metabolic pathways active in gas fermentation. American Chemical Society 2018-04-03 Article PeerReviewed application/pdf en cc_by https://eprints.nottingham.ac.uk/51056/9/isotope%20acs.analchem.pdf Schatschneider, Sarah, Abdelrazig, Salah M.A., Safo, Laudina, Henstra, Anne M., Millat, Thomas, Kim, Dong-Hyun, Winzer, Klaus, Minton, Nigel P. and Barrett, David A. (2018) Quantitative isotope-dilution high-resolution-mass-apectrometry analysis of multiple intracellular metabolites in Clostridium autoethanogenum with uniformly 13C-labeled standards derived from Spirulina. Analytical Chemistry, 90 (7). pp. 4470-4477. ISSN 1520-6882 Isotope dilution mass spectrometry; Spirulina; Arthrospira; Clostridia; Biofuels; Gas fermentation; Metabolites https://doi.org/10.1021/acs.analchem.7b04758 doi:10.1021/acs.analchem.7b04758 doi:10.1021/acs.analchem.7b04758 |
| spellingShingle | Isotope dilution mass spectrometry; Spirulina; Arthrospira; Clostridia; Biofuels; Gas fermentation; Metabolites Schatschneider, Sarah Abdelrazig, Salah M.A. Safo, Laudina Henstra, Anne M. Millat, Thomas Kim, Dong-Hyun Winzer, Klaus Minton, Nigel P. Barrett, David A. Quantitative isotope-dilution high-resolution-mass-apectrometry analysis of multiple intracellular metabolites in Clostridium autoethanogenum with uniformly 13C-labeled standards derived from Spirulina |
| title | Quantitative isotope-dilution high-resolution-mass-apectrometry analysis of multiple intracellular metabolites in Clostridium autoethanogenum with uniformly 13C-labeled standards derived from Spirulina |
| title_full | Quantitative isotope-dilution high-resolution-mass-apectrometry analysis of multiple intracellular metabolites in Clostridium autoethanogenum with uniformly 13C-labeled standards derived from Spirulina |
| title_fullStr | Quantitative isotope-dilution high-resolution-mass-apectrometry analysis of multiple intracellular metabolites in Clostridium autoethanogenum with uniformly 13C-labeled standards derived from Spirulina |
| title_full_unstemmed | Quantitative isotope-dilution high-resolution-mass-apectrometry analysis of multiple intracellular metabolites in Clostridium autoethanogenum with uniformly 13C-labeled standards derived from Spirulina |
| title_short | Quantitative isotope-dilution high-resolution-mass-apectrometry analysis of multiple intracellular metabolites in Clostridium autoethanogenum with uniformly 13C-labeled standards derived from Spirulina |
| title_sort | quantitative isotope-dilution high-resolution-mass-apectrometry analysis of multiple intracellular metabolites in clostridium autoethanogenum with uniformly 13c-labeled standards derived from spirulina |
| topic | Isotope dilution mass spectrometry; Spirulina; Arthrospira; Clostridia; Biofuels; Gas fermentation; Metabolites |
| url | https://eprints.nottingham.ac.uk/51056/ https://eprints.nottingham.ac.uk/51056/ https://eprints.nottingham.ac.uk/51056/ |