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An Isotopic Ratio Outlier Analysis Approach for Global Metabolomics of Biosynthetically Talented Actinomycetes.

ABSTRACT: Actinomycetes are powerhouses of natural product biosynthesis. Full realization of this biosynthetic potential requires approaches for recognizing novel metabolites and determining mediators of metabolite production. Herein, we develop an isotopic ratio outlier analysis (IROA) ultra-high performance liquid chromatography-mass spectrometry (UHPLC/MS) global metabolomics strategy for actinomycetes that facilitates recognition of novel metabolites and evaluation of production mediators. We demonstrate this approach by determining impacts of the iron chelator 2,2'-bipyridyl on the Nocardiopsis dassonvillei metabolome. Experimental and control cultures produced metabolites with isotopic carbon signatures that were distinct from corresponding "standard" culture metabolites, which were used as internal standards for LC/MS. This provided an isotopic MS peak pair for each metabolite, which revealed the number of carbon atoms and relative concentrations of metabolites and distinguished biosynthetic products from artifacts. Principal component analysis (PCA) and random forest (RF) differentiated bipyridyl-treated samples from controls. RF mean decrease accuracy (MDA) values supported perturbation of metabolites from multiple amino acid pathways and novel natural products. Evaluation of bipyridyl impacts on the nocazine/XR334 diketopiperazine (DKP) pathway revealed upregulation of amino acid precursors and downregulation of late stage intermediates and products. These results establish IROA as a tool in the actinomycete natural product chemistry arsenal and support broad metabolic consequences of bipyridyl.

SUBMITTER: Carey J

PROVIDER: S-EPMC6780544 | biostudies-other | 2019 Sep

REPOSITORIES: biostudies-other

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An Isotopic Ratio Outlier Analysis Approach for Global Metabolomics of Biosynthetically Talented Actinomycetes.

Carey Jordan J Nguyen Thanh T Korchak Jennifer J Beecher Christopher C de Jong Felice F Lane Amy L AL

Metabolites 20190910 9

Actinomycetes are powerhouses of natural product biosynthesis. Full realization of this biosynthetic potential requires approaches for recognizing novel metabolites and determining mediators of metabolite production. Herein, we develop an isotopic ratio outlier analysis (IROA) ultra-high performance liquid chromatography-mass spectrometry (UHPLC/MS) global metabolomics strategy for actinomycetes that facilitates recognition of novel metabolites and evaluation of production mediators. We demonstr ...[more]

PMID: 31510039

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Project description:Studies of isotopically labeled compounds have been fundamental to understanding metabolic pathways and fluxes. They have traditionally, however, been used in conjunction with targeted analyses that identify and quantify a limited number of labeled downstream metabolites. Here we describe an alternative workflow that leverages recent advances in untargeted metabolomic technologies to track the fates of isotopically labeled metabolites in a global, unbiased manner. This untargeted approach can be applied to discover novel biochemical pathways and characterize changes in the fates of labeled metabolites as a function of altered biological conditions such as disease. To facilitate the data analysis, we introduce X(13)CMS, an extension of the widely used mass spectrometry-based metabolomic software package XCMS. X(13)CMS uses the XCMS platform to detect metabolite peaks and perform retention-time alignment in liquid chromatography/mass spectrometry (LC/MS) data. With the use of the XCMS output, the program then identifies isotopologue groups that correspond to isotopically labeled compounds. The retrieval of these groups is done without any a priori knowledge besides the following input parameters: (i) the mass difference between the unlabeled and labeled isotopes, (ii) the mass accuracy of the instrument used in the analysis, and (iii) the estimated retention-time reproducibility of the chromatographic method. Despite its name, X(13)CMS can be used to track any isotopic label. Additionally, it detects differential labeling patterns in biological samples collected from parallel control and experimental conditions. We validated the ability of X(13)CMS to accurately retrieve labeled metabolites from complex biological matrices both with targeted LC/MS/MS analysis of a subset of the hits identified by the program and with labeled standards spiked into cell extracts. We demonstrate the full functionality of X(13)CMS with an analysis of cultured rat astrocytes treated with uniformly labeled (U-)(13)C-glucose during lipopolysaccharide (LPS) challenge. Our results show that out of 223 isotopologue groups enriched from U-(13)C-glucose, 95 have statistically significant differential labeling patterns in astrocytes challenged with LPS compared to unchallenged control cells. Only two of these groups overlap with the 32 differentially regulated peaks identified by XCMS, indicating that X(13)CMS uncovers different and complementary information from untargeted metabolomic studies. Like XCMS, X(13)CMS is implemented in R. It is available from our laboratory website at http://pattilab.wustl.edu/x13cms.php .

Dataset Information

An Isotopic Ratio Outlier Analysis Approach for Global Metabolomics of Biosynthetically Talented Actinomycetes.

Publications

An Isotopic Ratio Outlier Analysis Approach for Global Metabolomics of Biosynthetically Talented Actinomycetes.

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