Fragment Formula Calculator (FFC): Determination of Chemical Formulas for Fragment Ions in Mass Spectrometric Data

Abstract

The accurate determination of mass isotopomer distributions (MID) is of great significance for stable isotope-labeling experiments. Most commonly, MIDs are derived from gas chromatography/electron ionization mass spectrometry (GC/EI-MS) measurements. The analysis of fragment ions formed during EI, which contain only specific parts of the original molecule can provide valuable information on the positional distribution of the label. The chemical formula of a fragment ion is usually applied to derive the correction matrix for accurate MID calculation. Hence, the correct assignment of chemical formulas to fragment ions is of crucial importance for correct MIDs. Moreover, the positional distribution of stable isotopes within a fragment ion is of high interest for stable isotope-assisted metabolomics techniques. For example, 13C-metabolic flux analyses (13C-MFA) are dependent on the exact knowledge of the number and position of retained carbon atoms of the unfragmented molecule. Fragment ions containing different carbon atoms are of special interest, since they can carry different flux information. However, the process of mass spectral fragmentation is complex, and identifying the substructures and chemical formulas for these fragment ions is nontrivial. For that reason, we developed an algorithm, based on a systematic bond cleavage, to determine chemical formulas and retained atoms for EI derived fragment ions. Here, we present the fragment formula calculator (FFC) algorithm that can calculate chemical formulas for fragment ions where the chemical bonding (e.g., Lewis structures) of the intact molecule is known. The proposed algorithm is able to cope with general molecular rearrangement reactions occurring during EI in GC/MS measurements. The FFC algorithm is able to integrate stable isotope labeling experiments into the analysis and can automatically exclude candidate formulas that do not fit the observed labeling patterns.1 We applied the FFC algorithm to create a fragment ion repository that contains the chemical formulas and retained carbon atoms of a wide range of trimethylsilyl and tertbutyldimethylsilyl derivatized compounds. In total, we report the chemical formulas and backbone carbon compositions for 160 fragment ions of 43 alkylsilyl-derivatives of primary metabolites. Finally, we implemented the FFC algorithm in an easy-to-use graphical user interface and made it publicly available at http://www.ffc.lu.

Exploring the Process of Energy Generation in Pathophysiology by Targeted Metabolomics: Performance of a Simple and Quantitative Method

“…In this case, to stabilize this structure, the CH 2 • radical should be linked to the oxygen from the oxyamine group (Figure 2c). Special attention is required in the identification of phosphate compounds (Figure 2d) because they yield three characteristic fragments [30] and retention time is crucial. Quantifier and qualifier ions used to identify phosphate compounds are summarized in Supplemental Table 2.…”

Non-targeted Tracer Fate Detection

“…IsoMETLIN can make use of MS/MS spectra to resolve individual isotopomers. Such information can also be ob-tained from NTFD by analyzing specific mass spectrometric fragments [9] or combinations thereof [25]. MzMatch-ISO [18] and X 13 CMS [14] are both R packages that allow for the non-targeted determination of MIDs and can be regarded as the LC-MS equivalents of NTFD.…”

“…MIDs of individual fragments provide valuable information on elemental composition and origin of the respective atoms. Moreover, the MID can reveal overlapping fragments of the same mass [9]. For this purpose, we embedded the NTFD algorithm into the Fragment Formula Calculator (FFC) software [9].…”

“…Moreover, the MID can reveal overlapping fragments of the same mass [9]. For this purpose, we embedded the NTFD algorithm into the Fragment Formula Calculator (FFC) software [9]. FFC determines elemental composition and potential substructures of mass spectrometric fragment ions by a graph-based combinatorial approach.…”

“…However, to determine the theoretical MID, knowledge of the elemental composition of the compound of interest is required [9]. For this reason, usually only a predefined list of compounds is analyzed for isotopic enrichment.…”