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Automated LC-HRMS(/MS) approach for the annotation of fragment ions derived from stable isotope labeling-assisted untargeted metabolomics.

Neumann NK, Lehner SM, Kluger B, Bueschl C, Sedelmaier K, Lemmens M, Krska R, Schuhmacher R - Anal. Chem. (2014)

Bottom Line: Furthermore, the developed approach is exemplified with nine unknown biochemical compounds contained in F. graminearum samples derived from an untargeted metabolomics experiment.The mass difference between the corresponding fragment ions present in the MS/MS spectra of the native and U-(13)C-labeled compound enabled the assignment of the number of carbon atoms to each fragment signal and allowed the generation of meaningful putative molecular formulas for each fragment ion, which in turn also helped determine the elemental composition of the precursor ion.Compared to laborious manual analysis of the MS/MS spectra, the presented algorithm marks an important step toward efficient fragment signal elucidation and structure annotation of metabolites in future untargeted metabolomics studies.

View Article: PubMed Central - PubMed

Affiliation: Center for Analytical Chemistry, ‡Institute for Biotechnology in Plant Production, Department for Agrobiotechnology (IFA-Tulln), University of Natural Resources and Life Sciences Vienna (BOKU) , Konrad Lorenz Strasse 20, 3430 Tulln, Austria.

ABSTRACT
Structure elucidation of biological compounds is still a major bottleneck of untargeted LC-HRMS approaches in metabolomics research. The aim of the present study was to combine stable isotope labeling and tandem mass spectrometry for the automated interpretation of the elemental composition of fragment ions and thereby facilitate the structural characterization of metabolites. The software tool FragExtract was developed and evaluated with LC-HRMS/MS spectra of both native (12)C- and uniformly (13)C (U-(13)C)-labeled analytical standards of 10 fungal substances in pure solvent and spiked into fungal culture filtrate of Fusarium graminearum respectively. Furthermore, the developed approach is exemplified with nine unknown biochemical compounds contained in F. graminearum samples derived from an untargeted metabolomics experiment. The mass difference between the corresponding fragment ions present in the MS/MS spectra of the native and U-(13)C-labeled compound enabled the assignment of the number of carbon atoms to each fragment signal and allowed the generation of meaningful putative molecular formulas for each fragment ion, which in turn also helped determine the elemental composition of the precursor ion. Compared to laborious manual analysis of the MS/MS spectra, the presented algorithm marks an important step toward efficient fragment signal elucidation and structure annotation of metabolites in future untargeted metabolomics studies. Moreover, as demonstrated for a fungal culture sample, FragExtract also assists the characterization of unknown metabolites, which are not contained in databases, and thus exhibits a significant contribution to untargeted metabolomics research.

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MS/MS spectra of 3AcDONstandard spiked into culture filtrates.The upper spectrum shows the fragment signals of the native 3AcDON(m/z 339.1438) at the initial concentrationof 1 mg/L (a) and at approximately 0.1 mg/L (b). Combined view ofthe EIC [M + H]+ of 3AcDON in the full scan mode and EICsof selected MS/MS signals (0.1 mg/L) (c).
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fig2: MS/MS spectra of 3AcDONstandard spiked into culture filtrates.The upper spectrum shows the fragment signals of the native 3AcDON(m/z 339.1438) at the initial concentrationof 1 mg/L (a) and at approximately 0.1 mg/L (b). Combined view ofthe EIC [M + H]+ of 3AcDON in the full scan mode and EICsof selected MS/MS signals (0.1 mg/L) (c).

Mentions: Figure 2 shows LC-HRMS/MS spectra of native 3AcDON standardin spiked F. graminearum culture filtratesat the highest concentration tested (1 mg/L, Figure 2a) and the lowest concentration for which at least one fragmentsignal could still be annotated (0.1 mg/L, Figure 2b). In Figure 2c, the extracted ionchromatogram (EIC) of the precursor ion 3AcDON and EICs of selectedMS/MS signals are presented. For the LC-MS/MS spectra at 1 mg/L, wefound similar results compared to pure standards (i.e., no unspecificMS/MS signals fit the predefined criteria). At a concentration of0.1 mg/L, only one fragment signal (m/z 231.0996) was automatically annotated, which in contrast to theother fragments found at this concentration showed a similar chromatographicpeak shape and retention time compared to the full scan EIC of theprecursor ion 3AcDON (m/z 339.1438).The selected other fragments however could be classified as backgroundsignals or pseudo ions (m/z 224.848and 109.767) on the basis of their chromatographic behavior or represented“spike” signals (m/z 320.055 and 271.779), observed only once in a single MS/MS spectrum.Lowering the compound’s concentrations obviously leads to lowerprecursor intensities and hence, less fragment signals, which canbe automatically recognized by the software (TableS-2). It was shown for all 10 analytical standards spiked tothe culture filtrate that even at the lowest tested concentrationlevels neither the presence of matrix compounds nor pseudo ions alteredthe algorithm’s ability to filter unspecific MS/MS signals(Figures S-5 to S-56).


Automated LC-HRMS(/MS) approach for the annotation of fragment ions derived from stable isotope labeling-assisted untargeted metabolomics.

Neumann NK, Lehner SM, Kluger B, Bueschl C, Sedelmaier K, Lemmens M, Krska R, Schuhmacher R - Anal. Chem. (2014)

MS/MS spectra of 3AcDONstandard spiked into culture filtrates.The upper spectrum shows the fragment signals of the native 3AcDON(m/z 339.1438) at the initial concentrationof 1 mg/L (a) and at approximately 0.1 mg/L (b). Combined view ofthe EIC [M + H]+ of 3AcDON in the full scan mode and EICsof selected MS/MS signals (0.1 mg/L) (c).
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4126838&req=5

fig2: MS/MS spectra of 3AcDONstandard spiked into culture filtrates.The upper spectrum shows the fragment signals of the native 3AcDON(m/z 339.1438) at the initial concentrationof 1 mg/L (a) and at approximately 0.1 mg/L (b). Combined view ofthe EIC [M + H]+ of 3AcDON in the full scan mode and EICsof selected MS/MS signals (0.1 mg/L) (c).
Mentions: Figure 2 shows LC-HRMS/MS spectra of native 3AcDON standardin spiked F. graminearum culture filtratesat the highest concentration tested (1 mg/L, Figure 2a) and the lowest concentration for which at least one fragmentsignal could still be annotated (0.1 mg/L, Figure 2b). In Figure 2c, the extracted ionchromatogram (EIC) of the precursor ion 3AcDON and EICs of selectedMS/MS signals are presented. For the LC-MS/MS spectra at 1 mg/L, wefound similar results compared to pure standards (i.e., no unspecificMS/MS signals fit the predefined criteria). At a concentration of0.1 mg/L, only one fragment signal (m/z 231.0996) was automatically annotated, which in contrast to theother fragments found at this concentration showed a similar chromatographicpeak shape and retention time compared to the full scan EIC of theprecursor ion 3AcDON (m/z 339.1438).The selected other fragments however could be classified as backgroundsignals or pseudo ions (m/z 224.848and 109.767) on the basis of their chromatographic behavior or represented“spike” signals (m/z 320.055 and 271.779), observed only once in a single MS/MS spectrum.Lowering the compound’s concentrations obviously leads to lowerprecursor intensities and hence, less fragment signals, which canbe automatically recognized by the software (TableS-2). It was shown for all 10 analytical standards spiked tothe culture filtrate that even at the lowest tested concentrationlevels neither the presence of matrix compounds nor pseudo ions alteredthe algorithm’s ability to filter unspecific MS/MS signals(Figures S-5 to S-56).

Bottom Line: Furthermore, the developed approach is exemplified with nine unknown biochemical compounds contained in F. graminearum samples derived from an untargeted metabolomics experiment.The mass difference between the corresponding fragment ions present in the MS/MS spectra of the native and U-(13)C-labeled compound enabled the assignment of the number of carbon atoms to each fragment signal and allowed the generation of meaningful putative molecular formulas for each fragment ion, which in turn also helped determine the elemental composition of the precursor ion.Compared to laborious manual analysis of the MS/MS spectra, the presented algorithm marks an important step toward efficient fragment signal elucidation and structure annotation of metabolites in future untargeted metabolomics studies.

View Article: PubMed Central - PubMed

Affiliation: Center for Analytical Chemistry, ‡Institute for Biotechnology in Plant Production, Department for Agrobiotechnology (IFA-Tulln), University of Natural Resources and Life Sciences Vienna (BOKU) , Konrad Lorenz Strasse 20, 3430 Tulln, Austria.

ABSTRACT
Structure elucidation of biological compounds is still a major bottleneck of untargeted LC-HRMS approaches in metabolomics research. The aim of the present study was to combine stable isotope labeling and tandem mass spectrometry for the automated interpretation of the elemental composition of fragment ions and thereby facilitate the structural characterization of metabolites. The software tool FragExtract was developed and evaluated with LC-HRMS/MS spectra of both native (12)C- and uniformly (13)C (U-(13)C)-labeled analytical standards of 10 fungal substances in pure solvent and spiked into fungal culture filtrate of Fusarium graminearum respectively. Furthermore, the developed approach is exemplified with nine unknown biochemical compounds contained in F. graminearum samples derived from an untargeted metabolomics experiment. The mass difference between the corresponding fragment ions present in the MS/MS spectra of the native and U-(13)C-labeled compound enabled the assignment of the number of carbon atoms to each fragment signal and allowed the generation of meaningful putative molecular formulas for each fragment ion, which in turn also helped determine the elemental composition of the precursor ion. Compared to laborious manual analysis of the MS/MS spectra, the presented algorithm marks an important step toward efficient fragment signal elucidation and structure annotation of metabolites in future untargeted metabolomics studies. Moreover, as demonstrated for a fungal culture sample, FragExtract also assists the characterization of unknown metabolites, which are not contained in databases, and thus exhibits a significant contribution to untargeted metabolomics research.

Show MeSH