¹³C Metabolomics: NMR and IROA for Unknown Identification

Area of Science:

• Metabolomics
• Structural Biology
• Analytical Chemistry

Background:

• Isotopic Ratio Outlier Analysis (IROA) is an untargeted metabolomics technique utilizing stable isotopic labeling and LC-HRMS for metabolite identification and quantification.
• IROA enables the analysis of biological samples under various experimental conditions.

Purpose of the Study:

• To demonstrate a high-sensitivity 13C NMR method for identifying an unknown metabolite.
• The metabolite was isolated from fractionated material obtained from an IROA LC-HRMS experiment.

Main Methods:

• Samples of Caenorhabditis elegans were subjected to IROA and fractionated using LC-HRMS.
• Fractions were analyzed by 13C NMR, and resonance assignments were compared to the BMRB database.
• Quantum chemical NMR chemical shift calculations were performed to determine the correct isomer.

Main Results:

• LC-HRMS identified known metabolites (phenylalanine, inosine, tryptophan) and an unknown feature (m/z 380.0742 [M+H]+).
• NMR analysis confirmed known compounds and identified spin system networks for the unknown feature.
• The unknown compound was identified as 3'-O-phospho-β-D-glucopyranosyl-anthranilate, a known metabolite in C. elegans.

Conclusions:

• The study successfully dereplicated known metabolites and identified a novel compound using NMR databases and chemical shift calculations.
• This integrated approach is efficient for identifying unknown compounds from IROA-generated material.