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Anal Chem. 2016 Mar 1;88(5):2747-54. doi: 10.1021/acs.analchem.5b04263. Epub 2016 Feb 17.

Isotopic Ratio Outlier Analysis of the S. cerevisiae Metabolome Using Accurate Mass Gas Chromatography/Time-of-Flight Mass Spectrometry: A New Method for Discovery.

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Stable Isotope and Metabolomics Core Facility, Diabetes Center, Department of Medicine, Albert Einstein College of Medicine , Bronx, New York 10461, United States.
Department of Biochemistry, Albert Einstein College of Medicine , Bronx, New York 10461, United States.
IROA Technologies , Ann Arbor, Michigan 48105, United States.
Department of Chemistry, University of Florida , Gainesville, Florida 32611, United States.
Department of Pathology, Immunology, and Laboratory Medicine, University of Florida , Gainesville, Florida 32611, United States.


Isotopic ratio outlier analysis (IROA) is a (13)C metabolomics profiling method that eliminates sample to sample variance, discriminates against noise and artifacts, and improves identification of compounds, previously done with accurate mass liquid chromatography/mass spectrometry (LC/MS). This is the first report using IROA technology in combination with accurate mass gas chromatography/time-of-flight mass spectrometry (GC/TOF-MS), here used to examine the S. cerevisiae metabolome. S. cerevisiae was grown in YNB media, containing randomized 95% (13)C, or 5%(13)C glucose as the single carbon source, in order that the isotopomer pattern of all metabolites would mirror the labeled glucose. When these IROA experiments are combined, the abundance of the heavy isotopologues in the 5%(13)C extracts, or light isotopologues in the 95%(13)C extracts, follows the binomial distribution, showing mirrored peak pairs for the molecular ion. The mass difference between the (12)C monoisotopic and the (13)C monoisotopic equals the number of carbons in the molecules. The IROA-GC/MS protocol developed, using both chemical and electron ionization, extends the information acquired from the isotopic peak patterns for formulas generation. The process that can be formulated as an algorithm, in which the number of carbons, as well as the number of methoximations and silylations are used as search constraints. In electron impact (EI/IROA) spectra, the artifactual peaks are identified and easily removed, which has the potential to generate "clean" EI libraries. The combination of chemical ionization (CI) IROA and EI/IROA affords a metabolite identification procedure that enables the identification of coeluting metabolites, and allowed us to characterize 126 metabolites in the current study.

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