Isotopic Ratio Outlier Analysis Global Metabolomics of Caenorhabditis elegans

Abstract

We demonstrate the global metabolic analysis of Caenorhabditis elegans stress responses using a mass spectrometry-based technique called Isotopic Ratio Outlier Analysis (IROA). In an IROA protocol, control and experimental samples are isotopically labeled with 95% and 5% 13C, and the two sample populations are mixed together for uniform extraction, sample preparation, and LC-MS analysis. This labeling strategy provides several advantages over conventional approaches: 1) compounds arising from biosynthesis are easily distinguished from artifacts, 2) errors from sample extraction and preparation are minimized because the control and experiment are combined into a single sample, 3) measurement of both the molecular weight and the exact number of carbon atoms in each molecule provides extremely accurate molecular formulae, and 4) relative concentrations of all metabolites are easily determined. A heat shock perturbation was conducted on C. elegans to demonstrate this approach. We identified many compounds that significantly changed upon heat shock, including several from the purine metabolism pathway, which we use to demonstrate the approach. The metabolomic response information by IROA may be interpreted in the context of a wealth of genetic and proteomic information available for C. elegans. Furthermore, the IROA protocol can be applied to any organism that can be isotopically labeled, making it a powerful new tool in a global metabolomics pipeline.

LC-HRMS Based Non-Targeted Metabolomic Profiling of Wheat (<I>Triticum aestivum</I> L.) under Post-Anthesis Drought Stress

“…The IS was an isotopically labeled wheat leaf that had been grown in an atmosphere of 13 C labeled carbon dioxide resulting in a uniform and universal labeling of approximately 97% (IROA Technologies) [24]. Next, 500 µl of methanol/10mM aqueous ammonium acetate (50:50) was added to the dried powder and vortexed for 1 min at room temperature.…”

“…The objectives of this study were: 1) to elucidate the differential metabolite accumulation in wheat leaves under post-anthesis drought stress conditions over 24 hours time period, and the involvement of those metabolites in different pathways in relation to drought tolerance and 2) to identify potential metabolite biomarkers associated with drought stress in wheat. To achieve this objective, we employed a non-targeted LC-HRMS Isotopic Ratio Outlier Analysis (IROA) Global Metabolomics method [24] for identifying metabolites from the leaf tissue of the drought stressed and control wheat plants at 8 time points of 24-hour period.…”

A roadmap for the XCMS family of software solutions in metabolomics

“…[21,22] While these strategies have proven effective, we should point out that their application is limited to samples which can be cultured with labels (clinical specimens remain a challenge). One approach for removing artifacts, known as credentialing, was designed to be interoperable with the XCMS software.…”

“…[20] Additionally, the diffreport does not provide a reliable approximation of metabolites detected due to adducts, isotopes, fragments, and artifacts. [21,22] Indeed, depending on experimental conditions, more than 50% of the features on a diffreport can be a result of fragments and artifacts. [23] As the field of metabolomics has evolved over the last decade, there has been a major push to better annotate the XCMS diffreport.…”

Defining the Metabolome: Size, Flux, and Regulation

“…To identify and remove these artifactual features, isotopic labeling methods such as credentialing and isotope-ratio outlier analysis have been developed. (Mahieu et al, 2014; Stupp et al, 2013) In brief, identical samples with and without 13 C labels are mixed. Features of biological origin consequently produce a unique isotopic pattern in the mass spectra, whereas artifactual features do not.…”