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 12C- and uniformly 13C (U-13C)-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-13C-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.