MetExtract: a new software tool for the automated comprehensive extraction of metabolite-derived LC/MS signals in metabolomics research

Motivation: Liquid chromatography–mass spectrometry (LC/MS) is a key technique in metabolomics. Since the efficient assignment of MS signals to true biological metabolites becomes feasible in combination with in vivo stable isotopic labelling, our aim was to provide a new software tool for this purpose

Stable Isotope-Assisted Evaluation of Different Extraction Solvents for Untargeted Metabolomics of Plants

The evaluation of extraction protocols for untargeted metabolomics approaches is still difficult. We have applied a novel stable isotope-assisted workflow for untargeted LC-HRMS-based plant metabolomics , which allows for the first time every detected feature to be considered for method evaluation. The efficiency and complementarity of commonly used extraction solvents, namely 1 + 3 (v/v) mixtures of water and selected organic solvents (methanol, acetonitrile or methanol/acetonitrile 1 + 1 (v/v)), with and without the addition of 0.1% (v/v) formic acid were compared. Four different wheat organs were sampled, extracted and analysed by LC-HRMS. Data evaluation was performed with the in-house-developed MetExtract II software and R. With all tested solvents a total of 871 metabolites were extracted in ear, 785 in stem, 733 in leaf and 517 in root samples, respectively. Between 48% (stem) and 57% (ear) of the metabolites detected in a particular organ were found with all extraction mixtures, and 127 of 996 metabolites were consistently shared between all extraction agent/organ combinations. In aqueous methanol, acidification with formic acid led to pronounced pH dependency regarding the precision of metabolite abundance and the number of detectable metabolites, whereas extracts of acetonitrile-containing mixtures were less affected. Moreover, methanol and acetonitrile have been found to be complementary with respect to extraction efficiency. Interestingly, the beneficial properties of both solvents can be combined by the use of a water-methanol-acetonitrile mixture for global metabolite extraction instead of aqueous methanol or aqueous acetonitrile alone

Methanol Generates Numerous Artifacts during Sample Extraction and Storage of Extracts in Metabolomics Research

Many metabolomics studies use mixtures of (acidified) methanol and water for sample extraction. In the present study, we investigated if the extraction with methanol can result in artifacts. To this end, wheat leaves were extracted with mixtures of native and deuterium-labeled methanol and water, with or without 0.1% formic acid. Subsequently, the extracts were analyzed immediately or after storage at 10 °C, −20 °C or −80 °C with an HPLC-HESI-QExactive HF-Orbitrap instrument. Our results showed that 88 (8%) of the >1100 detected compounds were derived from the reaction with methanol and either formed during sample extraction or short-term storage. Artifacts were found for various substance classes such as flavonoids, carotenoids, tetrapyrrols, fatty acids and other carboxylic acids that are typically investigated in metabolomics studies. 58 of 88 artifacts were common between the two tested extraction variants. Remarkably, 34 of 73 (acidified extraction solvent) and 33 of 73 (non-acidified extraction solvent) artifacts were formed de novo as none of these meth(ox)ylated metabolites were found after extraction of native leaf samples with CD3OH/H2O. Moreover, sample extracts stored at 10 °C for several days, as can typically be the case during longer measurement sequences, led to an increase in both the number and abundance of methylated artifacts. In contrast, frozen sample extracts were relatively stable during a storage period of one week. Our study shows that caution has to be exercised if methanol is used as the extraction solvent as the detected metabolites might be artifacts rather than natural constituents of the biological system. In addition, we recommend storing sample extracts in deep freezers immediately after extraction until measurement

Metabolism of HT-2 Toxin and T-2 Toxin in Oats

The Fusarium mycotoxins HT-2 toxin (HT2) and T-2 toxin (T2) are frequent contaminants in oats. These toxins, but also their plant metabolites, may contribute to toxicological effects. This work describes the use of 13C-assisted liquid chromatography–high-resolution mass spectrometry for the first comprehensive study on the biotransformation of HT2 and T2 in oats. Using this approach, 16 HT2 and 17 T2 metabolites were annotated including novel glycosylated and hydroxylated forms of the toxins, hydrolysis products, and conjugates with acetic acid, putative malic acid, malonic acid, and ferulic acid. Further targeted quantitative analysis was performed to study toxin metabolism over time, as well as toxin and conjugate mobility within non-treated plant tissues. As a result, HT2-3-O-β-d-glucoside was identified as the major detoxification product of both parent toxins, which was rapidly formed (to an extent of 74% in HT2-treated and 48% in T2-treated oats within one day after treatment) and further metabolised. Mobility of the parent toxins appeared to be negligible, while HT2-3-O-β-d-glucoside was partly transported (up to approximately 4%) through panicle side branches and stem. Our findings demonstrate that the presented combination of untargeted and targeted analysis is well suited for the comprehensive elucidation of mycotoxin metabolism in plants

MetMatch: A Semi-Automated Software Tool for the Comparison and Alignment of LC-HRMS Data from Different Metabolomics Experiments

Due to its unsurpassed sensitivity and selectivity, LC-HRMS is one of the major analytical techniques in metabolomics research. However, limited stability of experimental and instrument parameters may cause shifts and drifts of retention time and mass accuracy or the formation of different ion species, thus complicating conclusive interpretation of the raw data, especially when generated in different analytical batches. Here, a novel software tool for the semi-automated alignment of different measurement sequences is presented. The tool is implemented in the Java programming language, it features an intuitive user interface and its main goal is to facilitate the comparison of data obtained from different metabolomics experiments. Based on a feature list (i.e., processed LC-HRMS chromatograms with mass-to-charge ratio (m/z) values and retention times) that serves as a reference, the tool recognizes both m/z and retention time shifts of single or multiple analytical datafiles/batches of interest. MetMatch is also designed to account for differently formed ion species of detected metabolites. Corresponding ions and metabolites are matched and chromatographic peak areas, m/z values and retention times are combined into a single data matrix. The convenient user interface allows for easy manipulation of processing results and graphical illustration of the raw data as well as the automatically matched ions and metabolites. The software tool is exemplified with LC-HRMS data from untargeted metabolomics experiments investigating phenylalanine-derived metabolites in wheat and T-2 toxin/HT-2 toxin detoxification products in barley

Stable Isotope-Assisted Metabolomics for Deciphering Xenobiotic Metabolism in Mammalian Cell Culture

We present a workflow based on stable isotope-assisted metabolomics and the bioinformatics tool MetExtract II for deciphering xenobiotic metabolites produced by human cells. Its potential was demonstrated by the investigation of the metabolism of deoxynivalenol (DON), an abundant food contaminatn, in a liver cracinoma cell line (HeoG2) and a model for colon carcinoma (HT29). Detected known metabolites included DON-3-sulfate, DON-10-sulfonate, and DON-10-glutathione as well as DON-cysteine. Conjugation with amino acids and antibiotics was confirmed for the first time. The approach allows the untargeted elucidation of human xenobiotic products in tissue culture.<br /

Gramiketides, Novel Polyketide Derivatives of Fusarium graminearum, Are Produced during the Infection of Wheat

The plant pathogen Fusarium graminearum is a proficient producer of mycotoxins and other in part still unknown secondary metabolites, some of which might act as virulence factors on wheat. The PKS15 gene is expressed only in planta, so far hampering the identification of an associated metabolite. Here we combined the activation of silent gene clusters by chromatin manipulation (kmt6) with blocking the metabolic flow into the competing biosynthesis of the two major mycotoxins deoxynivalenol and zearalenone. Using an untargeted metabolomics approach, two closely related metabolites were found in triple mutants (kmt6 tri5 pks4,13) deficient in production of the major mycotoxins deoxynivalenol and zearalenone, but not in strains with an additional deletion in PKS15 (kmt6 tri5 pks4,13 pks15). Characterization of the metabolites, by LC-HRMS/MS in combination with a stable isotope-assisted tracer approach, revealed that they are likely hybrid polyketides comprising a polyketide part consisting of malonate-derived acetate units and a structurally deviating part. We propose the names gramiketide A and B for the two metabolites. In a biological experiment, both gramiketides were formed during infection of wheat ears with wild-type but not with pks15 mutants. The formation of the two gramiketides during infection correlated with that of the well-known virulence factor deoxynivalenol, suggesting that they might play a role in virulence