Untargeted Profiling of Tracer-Derived Metabolites Using Stable Isotopic Labeling and Fast Polarity-Switching LC–ESI-HRMS

Abstract

An untargeted metabolomics workflow for the detection of metabolites derived from endogenous or exogenous tracer substances is presented. To this end, a recently developed stable isotope-assisted LC–HRMS-based metabolomics workflow for the global annotation of biological samples has been further developed and extended. For untargeted detection of metabolites arising from labeled tracer substances, isotope pattern recognition has been adjusted to account for nonlabeled moieties conjugated to the native and labeled tracer molecules. Furthermore, the workflow has been extended by (i) an optional ion intensity ratio check, (ii) the automated combination of positive and negative ionization mode mass spectra derived from fast polarity switching, and (iii) metabolic feature annotation. These extensions enable the automated, unbiased, and global detection of tracer-derived metabolites in complex biological samples. The workflow is demonstrated with the metabolism of 13C9-phenylalanine in wheat cell suspension cultures in the presence of the mycotoxin deoxynivalenol (DON). In total, 341 metabolic features (150 in positive and 191 in negative ionization mode) corresponding to 139 metabolites were detected. The benefit of fast polarity switching was evident, with 32 and 58 of these metabolites having exclusively been detected in the positive and negative modes, respectively. Moreover, for 19 of the remaining 49 phenylalanine-derived metabolites, the assignment of ion species and, thus, molecular weight was possible only by the use of complementary features of the two ion polarity modes. Statistical evaluation showed that treatment with DON increased or decreased the abundances of many detected metabolites.

Metabolite profiling of sheath blight disease resistance in rice: in the case of positive ion mode analysis by CE/TOF-MS

“…Based on the result, it suggested that CE/TOF-MS study can be used as an option to replace the function of stable isotope labelling as carbon flow tracer in the plant. However, Kluger et al (2014) and You et al (2014) explained that stable isotope labelling is more effective to trace the metabolite element in cells.…”

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

“…Orbitrap measurements were performed in positive and negative electrospray ionisation mode separately with a scan range from m/z 130 to 1300. All other mass spectrometric settings were concordant with those of Kluger et al [25]. Data were acquired and evaluated with Thermo Xcalibur 4.0.27.10 and 2.2 software (both Thermo Fisher Scientific), respectively.…”

“…The combination with stable isotopic labelling (SIL) offers to differentiate between biologically derived metabolites and unspecific signals coming from solvents, reagents, biological matrix, or instrument noise [23,24]. SIL-assisted metabolomics workflows were recently developed to study the metabolism of endogenous or exogenous tracer compounds in biological systems [25]. …”

“…The objective of this study was to elucidate the metabolism of HT2 and T2 in oats ( Avena sativa L.) using a SIL-assisted metabolomics workflow for qualitative screening based on plant-treatment with a mixture of native and 13 C-labelled toxin, LC-Orbitrap-MS analysis in positive and negative ion mode and automated data processing by MetExtract II software [25]. Further structural information of the formed toxin derivatives was gained by LC-HRMS/MS measurements with a LC-Q-TOF-system.…”

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

“…Subsequent LC-HRMS measurement and TracExtract version 2 [21] data processing resulted in 151 Phe-derived features, which were detected in the 13 C-Phe-treated sample. In order to increase the number of features on the reference list and thereby enhance m / z and Rt correction accuracy, plants of the same cultivar were also raised in a tailor made growth chamber with 13 C enrichment of 98.8% as well as under native 12 CO 2 atmosphere in parallel.…”