Stable Isotope–Assisted Plant Metabolomics: Combination of Global and Tracer-Based Labeling for Enhanced Untargeted Profiling and Compound Annotation

Abstract

Untargeted approaches and thus biological interpretation of metabolomics results are still hampered by the reliable assignment of the global metabolome as well as classification and (putative) identification of metabolites. In this work we present an liquid chromatography-mass spectrometry (LC-MS)–based stable isotope assisted approach that combines global metabolome and tracer based isotope labeling for improved characterization of (unknown) metabolites and their classification into tracer derived submetabolomes. To this end, wheat plants were cultivated in a customized growth chamber, which was kept at 400 ± 50 ppm 13CO2 to produce highly enriched uniformly 13C-labeled sample material. Additionally, native plants were grown in the greenhouse and treated with either 13C9-labeled phenylalanine (Phe) or 13C11-labeled tryptophan (Trp) to study their metabolism and biochemical pathways. After sample preparation, liquid chromatography-high resolution mass spectrometry (LC-HRMS) analysis and automated data evaluation, the results of the global metabolome- and tracer-labeling approaches were combined. A total of 1,729 plant metabolites were detected out of which 122 respective 58 metabolites account for the Phe- and Trp-derived submetabolomes. Besides m/z and retention time, also the total number of carbon atoms as well as those of the incorporated tracer moieties were obtained for the detected metabolite ions. With this information at hand characterization of unknown compounds was improved as the additional knowledge from the tracer approaches considerably reduced the number of plausible sum formulas and structures of the detected metabolites. Finally, the number of putative structure formulas was further reduced by isotope-assisted annotation tandem mass spectrometry (MS/MS) derived product ion spectra of the detected metabolites. A major innovation of this paper is the classification of the metabolites into submetabolomes which turned out to be valuable information for effective filtering of database hits based on characteristic structural subparts. This allows the generation of a final list of true plant metabolites, which can be characterized at different levels of specificity.

Enhanced Metabolome Coverage and Evaluation of Matrix Effects by the Use of Experimental-Condition-Matched 13C-Labeled Biological Samples in Isotope-Assisted LC-HRMS Metabolomics

“…However, to illustrate the biological relevance, the additionally detected treatment-specific metabolites shall briefly be summarized here. More than one-third of the 62 putative stress-specific compounds (toxin-stressed additional metabolites) are secondary metabolites, most of which were also found in previous studies [38,39], using the same LC-HRMS method. They largely derive from the aromatic amino acids phenylalanine tryptophan and tyrosine [38][39][40].…”

“…More than one-third of the 62 putative stress-specific compounds (toxin-stressed additional metabolites) are secondary metabolites, most of which were also found in previous studies [38,39], using the same LC-HRMS method. They largely derive from the aromatic amino acids phenylalanine tryptophan and tyrosine [38][39][40]. For example, feruloyltryptamine is one of the detected toxin-treatment-specific metabolites, which was identified (identification with confidence level 1 based on the scoring system from [41]).…”

“…Although features of treatment-specific metabolites might in principle also be detected without SIA application, they would still largely be hidden among numerous non-biology-derived compounds, as has been demonstrated earlier [1]. Furthermore, the presented approach supplements metabolomics studies as it facilitates annotation and structure elucidation of detected metabolic features by taking advantage of the specific isotope pattern of native and labeled compound analogs from MS and MS/MS spectra (for more details, see also [39]).…”

Stable Isotope-Assisted Plant Metabolomics: Investigation of Phenylalanine-Related Metabolic Response in Wheat Upon Treatment With the Fusarium Virulence Factor Deoxynivalenol

“…Two individual labeling experiments were carried out to generate a reference list of all detectable metabolites of the phenylalanine-submetabolome. The method of combining global with tracer-based stable isotope labeling is described in detail in the associated manuscript of this combined publication (Doppler et al, 2019).…”

“…Moreover, since DON is also known to show elicitor like properties including the induction of secondary metabolism in host plants (Gunnaiah et al, 2012), we wanted to investigate the effect of the pure mycotoxin on the metabolism of the central defense precursor phenylalanine (Phe) in wheat. To this end, a global and a tracer-based stable isotope-assisted workflow were combined to follow the Fhb1 -related defense response of wheat spikes upon treatment with DON over a time period of 96 h. Methodical details on the metabolomics workflow as well as its benefits resulting from the combination of different labeling techniques can be found in the related publication (Doppler et al, 2019).…”