title: Gas chromatography-mass spectrometry with chemometric analysis for determining ¹²C and ¹³C labeled contributions in metabolomics and ¹³C flux analysis.
abstract: A novel method for the analysis of nearly co-eluting ¹²C and ¹³C isotopically labeled metabolites has been developed and evaluated for gas chromatography coupled to mass spectrometry (GC-MS) data. The method utilizes parallel factor analysis (PARAFAC) with two-dimensional GC-MS data when sample replicates are aligned and stacked in series to create a three-dimensional data cube for mathematical peak deconvolution and ¹²C and ¹³C contribution isolation, with the intent of increasing the accuracy and precision of quantitative metabolomics and ¹³C flux analysis. The platform is demonstrated with ¹³C-labeled metabolite extracts, generated via biosynthesis, added as an internal standard to unlabeled ¹²C metabolites extracted from the methanol-utilizing bacterium Methylobacterium extorquens AM1. Eleven representative metabolites that are common targets for flux analysis were chosen for validation. Good quantitative accuracy and precision were acquired for a 5.00 μM known metabolite concentration (for the 11 metabolites), with an average predicted concentration of 5.07 μM, and a RSD range of 1.2-13.0%. This study demonstrates the ability to reliably deconvolute ¹²C-unlabeled and ¹³C-labeled contributions for a given metabolite. Additionally, using this chemical analysis platform, a dynamic flux experiment is presented in which the incorporation of ¹³C-labeled cell extract can be detected in the methane-utilizing bacterium Methylosinus trichosporium OB3b and measured temporally.
authors.affiliation: Department of Chemical Engineering, University of Washington, Seattle, WA 98195-2180, USA.
authors.forename: Song
authors.initials: S
authors.lastname: Yang
authors.forename: Jeremy S
authors.initials: JS
authors.lastname: Nadeau
authors.forename: Elizabeth M
authors.initials: EM
authors.lastname: Humston-Fulmer
authors.forename: Jamin C
authors.initials: JC
authors.lastname: Hoggard
authors.forename: Mary E
authors.initials: ME
authors.lastname: Lidstrom
authors.forename: Robert E
authors.initials: RE
authors.lastname: Synovec
mesh_terms: D000596:Amino Acids / Q000378:metabolism; D002138:Calibration; D002247:Carbon Isotopes / Q000032:analysis* / Q000378:metabolism; D008401:Gas Chromatography-Mass Spectrometry / Q000379:methods*; D055432:Metabolomics / Q000379:methods*; D000432:Methanol / Q000378:metabolism; D020601:Methylobacterium extorquens / Q000378:metabolism; D015203:Reproducibility of Results
chemical_list: D000596:Amino Acids; D002247:Carbon Isotopes; D000432:Methanol