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Current Metabolomics


ISSN (Print): 2213-235X
ISSN (Online): 2213-2368

13C Metabolomics: NMR and IROA for Unknown Identification

Author(s): Chaevien S. Clendinen, Gregory S. Stupp, Bing Wang, Timothy J. Garrett and Arthur S. Edison

Volume 4, Issue 2, 2016

Page: [116 - 120] Pages: 5

DOI: 10.2174/2213235X04666160407212156

open access plus


Background: Isotopic Ratio Outlier Analysis (IROA) is an untargeted metabolomics method that uses stable isotopic labeling and LC-HRMS for identification and relative quantification of metabolites in a biological sample under varying experimental conditions.

Objective: We demonstrate a method using high-sensitivity 13C NMR to identify an unknown metabolite isolated from fractionated material from an IROA LC-HRMS experiment.

Methods: IROA samples from the nematode Caenorhabditis elegans were fractionated using LC-HRMS using 5 repeated injections and collecting 30 sec fractions. These were concentrated and analyzed by 13C NMR.

Results: We isotopically labeled samples of C. elegans and collected 2 adjacent LC fractions. By HRMS, one contained at least 2 known metabolites, phenylalanine and inosine, and the other contained tryptophan and an unknown feature with a monoisotopic mass of m/z 380.0742 [M+H]+. With NMR, we were able to easily verify the known compounds, and we then identified the spin system networks responsible for the unknown resonances. After searching the BMRB database and comparing the molecular formula from LC-HRMS, we determined that the fragments were a modified anthranilate and a glucose modified by a phosphate. We then performed quantum chemical NMR chemical shift calculations to determine the most likely isomer, which was 3’-O-phospho-β-D-glucopyranosyl-anthranilate. This compound had previously been found in the same organism, validating our approach.

Conclusion: We were able to dereplicate previously known metabolites and identify a metabolite that was not in databases by matching resonances to NMR databases and using chemical shift calculations to determine the correct isomer. This approach is efficient and can be used to identify unknown compounds of interest using the same material used for IROA.

Keywords: NMR, IROA, Metabolomics, MS, Carbon-13, LC-MS, C. elegans.

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