MetExtract II: A Software Suite for Stable Isotope-Assisted Untargeted Metabolomics

Abstract

Stable isotope labeling (SIL) techniques have the potential to enhance different aspects of liquid chromatography-high-resolution mass spectrometry (LC-HRMS)-based untargeted metabolomics methods including metabolite detection, annotation of unknown metabolites, and comparative quantification. In this work, we present MetExtract II, a software toolbox for detection of biologically derived compounds. It exploits SIL-specific isotope patterns and elution profiles in LC-HRMS(/MS) data. The toolbox consists of three complementary modules: M1 (AllExtract) uses mixtures of uniformly highly isotope-enriched and native biological samples for selective detection of the entire accessible metabolome. M2 (TracExtract) is particularly suited to probe the metabolism of endogenous or exogenous secondary metabolites and facilitates the untargeted screening of tracer derivatives from concurrently metabolized native and uniformly labeled tracer substances. With M3 (FragExtract), tandem mass spectrometry (MS/MS) fragments of corresponding native and uniformly labeled ions are evaluated and automatically assigned with putative sum formulas. Generated results can be graphically illustrated and exported as a comprehensive data matrix that contains all detected pairs of native and labeled metabolite ions that can be used for database queries, metabolome-wide internal standardization, and statistical analysis. The software, associated documentation, and sample data sets are freely available for noncommercial use at http://metabolomics-ifa.boku.ac.at/metextractII .

Figure 1

Illustration of implemented data processing steps in the presented software modules AllExtract, TracExtract, and FragExtract.

Figure 2

LC-HRMS(/MS) data illustrating deoxynivalenol 3-glucoside (DON-3-Glc, C₂₁H₃₀O₁₁). (A) Theoretical isotopologue patterns of native (M, [¹²C₂₁¹H₃₀¹⁶O₁₁ + ¹H]⁺; m/z 459.1862, native ¹²C enrichment of 98.93%) and U-¹³C-labeled (M′, [¹³C₂₁¹H₃₀¹⁶O₁₁ + ¹H]⁺; m/z 480.2566, uniform ¹³C enrichment of 99.1%) metabolite ions. Other isotopologue signals (e.g., ¹⁸O or ²H) are not depicted as their abundance is too low. (B) Isotope patterns of native and partly ¹³C-labeled (M′, [¹³C₁₅¹²C₆¹H₃₀¹⁶O₁₁ + ¹H]⁺; m/z 474.2365) biotransformation product ions. In M′, only the 15 carbon atoms of DON are ¹³C, while the remaining six carbon atoms of Glc are ¹²C. The m/z difference (Δm) between M and M′ corresponds to the total number of labeled atoms in the respective ions (C_n). (C, D) Section of simulated MS/MS spectra of native and U-¹³C-labeled [DON-3-Glc + H]⁺ precursor ions. Mass increments between corresponding fragment ions (F_x and F_x′) reflect the number of ¹³C atoms per MS/MS fragment. Isotopologue signals of native and labeled precursor ions, as well as their fragments, will be present only if a corresponding broad mass isolation window is used in MS/MS analysis. (E) Coeluting chromatographic peaks of native and partly ¹³C-labeled [DON-3-Glc + H]⁺ (data provided by Kluger et al.).