title: 13C Metabolomics: NMR and IROA for Unknown Identification.
abstract: 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-sensitivity13C 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 by13C 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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Biochemistry & Molecular Biology,; Department of Chemistry & Biochemistry, Georgia Institute of Technology, Atlanta GA, 30332, USA; Molecular & Experimental Medicine, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037.
authors.forename: Chaevien S
authors.initials: CS
authors.lastname: Clendinen
authors.affiliation: Department of Biochemistry & Molecular Biology,; Department of Chemistry & Biochemistry, Georgia Institute of Technology, Atlanta GA, 30332, USA; Molecular & Experimental Medicine, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037.
authors.forename: Gregory S
authors.initials: GS
authors.lastname: Stupp
authors.affiliation: Department of Biochemistry & Molecular Biology,; Department of Biochemistry & Molecular Biology.
authors.forename: Bing
authors.initials: B
authors.lastname: Wang
authors.affiliation: Department of Pathology, Immunology, and Laboratory Medicine,; Southeast Center for Integrated Metabolomics, University of Florida, Gainesville FL 32610-0245, USA.
authors.forename: Timothy J
authors.initials: TJ
authors.lastname: Garrett
authors.affiliation: Department of Biochemistry & Molecular Biology,; Southeast Center for Integrated Metabolomics, University of Florida, Gainesville FL 32610-0245, USA.
authors.forename: Arthur S
authors.initials: AS
authors.lastname: Edison
keywords: C.elegans; Carbon-13; IROA; LC-MS; MS; Metabolomics; NMR