In‐source fragmentation and correlation analysis as tools for metabolite identification exemplified with CE‐TOF untargeted metabolomics

Abstract

The role of non-targeted metabolomics with its discovery power is constantly growing in many different fields of science. However, its biggest advantage of uncovering the unexpected is turning into one of its biggest bottlenecks, particularly in metabolite identification. Among different methods for metabolite identification or ID confirmation, tandem MS analysis plays a very important role. However, this method is limited to only certain types of MS analysers, making for example TOF-MS inaccessible for this type of metabolite identification. To overcome this, in-source fragmentation has been used to fragment molecules and obtain product ions. Since the molecule of interest is not isolated prior to its fragmentation, the acquired spectrum contains many different signals arising from the fragmentation of all compounds present in the sample. Therefore, to assign product ions to their precursors, a novel use of correlation analysis was tested with r ≥0.9 as an assignation of a product ion belonging to the precursor. This method and chosen cut-off was tested on three different sample complexity levels: conducting the analysis on a single standard, mix of co-eluting standards and on a plasma sample. Obtained results clearly proved the effectiveness of the proposed methodology for metabolite ID confirmation. Moreover, the proposed strategy can be successfully applied for semi-quantification of co-eluting molecules with the same monoisotopic mass but that differ in fragmentation pattern. The proposed methodology can greatly improve the robustness and throughput of identification in metabolomics studies by use of TOF-MS, which is crucial to obtain meaningful and trustful results.

Capillary Electrophoresis Mass Spectrometry as A Tool for Untargeted Metabolomics

“…These multisignals, called in the literature metabolic features [109] . Nowadays also in-source fragmentation is considered as a significant source of additional signals appearing in CE-MS data [110].…”

“…This is particularly important in the case of isomeric metabolites where different isoforms reveal different biological properties. Spiking the sample it is even possible to determine if a particular peak (EIC) corresponds to just a single molecule or it is a composite amount of two isomeric metabolites [110]. Although within global distribution this method was applied in only approximately 12% of cases (considering only the usage of standards this percentage rise up to 22%) (Figure 7), it is placed in the second position just after separate injection of standards.…”

CE–MS for anionic metabolic profiling: An overview of methodological developments

“…However, it should be noted that this CE-MS method employed a relatively high fragmentor voltage (380 V), which might induce in-source fragmentation due to energetic collisions that occur in the ESI source. This effect was earlier studied by Godzien et al, who evaluated the impact of the enhancement of fragmentor voltage (150-230 V) on in-source fragmentation [35].…”

Phenotyping of gut microbiota: Focus on capillary electrophoresis

“…Once identified the mass features that can be considered as potential markers, the final step is to link their accurate masses with certain structures using a public or an in house database, so that a metabolite annotation can be performed at different confidence levels . In this sense, for CE‐TOF‐MS a really interesting research avenue is opened by Godzien et al (2015) who successfully correlated ( r 2 ≥ 0.9) the electrospray (ESI) in‐source fragmentation pattern with the MS/MS identification signature. This procedure has been tested on three different sample complexity levels, e.g.…”