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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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FragExtract results for LC-MS/MS spectra of native andU-13C 3AcDON standard. The numbers in the table (a) correspondto theannotated fragment signals highlighted in orange and blue in the originalproduct ion spectra of the native and the U-13C-labeledprecursor masses (m/z 339.1438 and m/z 356.2009) (b). For verification ofchromatographic peak shape and coelution of the fragment ions, theextracted ion chromatograms of fragments that were annotated by FragExtractare plotted by the software (c).
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fig1: FragExtract results for LC-MS/MS spectra of native andU-13C 3AcDON standard. The numbers in the table (a) correspondto theannotated fragment signals highlighted in orange and blue in the originalproduct ion spectra of the native and the U-13C-labeledprecursor masses (m/z 339.1438 and m/z 356.2009) (b). For verification ofchromatographic peak shape and coelution of the fragment ions, theextracted ion chromatograms of fragments that were annotated by FragExtractare plotted by the software (c).

Mentions: The presented approach aims at the automated evaluation of high-resolutiontandem mass spectra, is based on the use of highly U-13C-enriched labeled compounds or labeled biological samples, and relieson the successive LC-MS/MS recordings of 12C and U-13C-labeled substances. As native and U-13C-labeledcompounds show the same fragmentation behavior in tandem MS, the resultingfragmentation pattern in the product ion spectra ultimately looksthe same, only shifted toward higher masses of the U-13C-labeled compound, as for example shown for 3AcDON in Figure 1b.


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)

FragExtract results for LC-MS/MS spectra of native andU-13C 3AcDON standard. The numbers in the table (a) correspondto theannotated fragment signals highlighted in orange and blue in the originalproduct ion spectra of the native and the U-13C-labeledprecursor masses (m/z 339.1438 and m/z 356.2009) (b). For verification ofchromatographic peak shape and coelution of the fragment ions, theextracted ion chromatograms of fragments that were annotated by FragExtractare plotted by the software (c).
© Copyright Policy
Related In: Results  -  Collection

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

fig1: FragExtract results for LC-MS/MS spectra of native andU-13C 3AcDON standard. The numbers in the table (a) correspondto theannotated fragment signals highlighted in orange and blue in the originalproduct ion spectra of the native and the U-13C-labeledprecursor masses (m/z 339.1438 and m/z 356.2009) (b). For verification ofchromatographic peak shape and coelution of the fragment ions, theextracted ion chromatograms of fragments that were annotated by FragExtractare plotted by the software (c).
Mentions: The presented approach aims at the automated evaluation of high-resolutiontandem mass spectra, is based on the use of highly U-13C-enriched labeled compounds or labeled biological samples, and relieson the successive LC-MS/MS recordings of 12C and U-13C-labeled substances. As native and U-13C-labeledcompounds show the same fragmentation behavior in tandem MS, the resultingfragmentation pattern in the product ion spectra ultimately looksthe same, only shifted toward higher masses of the U-13C-labeled compound, as for example shown for 3AcDON in Figure 1b.

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