Abstract
Metabolite annotation and identification are primary challenges in untargeted metabolomics experiments. Rigorous workflows for reliable annotation of mass features with chemical structures or compound classes are needed to enhance the power of untargeted mass spectrometry. High-resolution mass spectrometry considerably improves the confidence in assigning elemental formulas to mass features in comparison to nominal mass spectrometry, and embedding of fragmentation methods enables more reliable metabolite annotations and facilitates metabolite classification. However, the analysis of mass fragmentation spectra can be a time-consuming step and requires expert knowledge. This study demonstrates how characteristic fragmentations, specific to compound classes, can be used to systematically analyze their presence in complex biological extracts like urine that have undergone untargeted mass spectrometry combined with data dependent or targeted fragmentation. Human urine extracts were analyzed using normal phase liquid chromatography (hydrophilic interaction chromatography) coupled to an Ion Trap-Orbitrap hybrid instrument. Subsequently, mass chromatograms and collision-induced dissociation and higher-energy collisional dissociation (HCD) fragments were annotated using the freely available MAGMa software1. Acylcarnitines play a central role in energy metabolism by transporting fatty acids into the mitochondrial matrix. By filtering on a combination of a mass fragment and neutral loss designed based on the MAGMa fragment annotations, we were able to classify and annotate 50 acylcarnitines in human urine extracts, based on high-resolution mass spectrometry HCD fragmentation spectra at different energies for all of them. Of these annotated acylcarnitines, 31 are not described in HMDB yet and for only 4 annotated acylcarnitines the fragmentation spectra could be matched to reference spectra. Therefore, we conclude that the use of mass fragmentation filters within the context of untargeted metabolomics experiments is a valuable tool to enhance the annotation of small metabolites.
Highlights
Mass spectrometry in conjunction with liquid chromatography has been successfully used for two decades to profile extracts of complex biological samples
We employed a generic metabolite extraction method (Vincent and Barrett, 2015) with untargeted small metabolite pHILIC and HILIC profiling approaches (Creek et al, 2011; Zhang et al, 2012) in combination with higher-energy collisional dissociation (HCD)-MSMS and collision induced dissociation (CID)-MSn fragmentations to determine whether robust metabolite annotation of small polar metabolites could be established using accurate mass fragmentation spectra
We have focused on seeking to enhance the annotation of acylcarnitines in the complex mixture human urine
Summary
Mass spectrometry in conjunction with liquid chromatography has been successfully used for two decades to profile extracts of complex biological samples. Assigning molecular structures to detected mass signals has proven to be a primary challenge in metabolomics studies (Van Der Hooft et al, 2013). Modern mass spectrometers are capable of capturing the molecular masses of ionized metabolites at highresolution, providing scientists with an unprecedented insight in complex biological mixtures such as cell extracts, plasma, or. Many modern mass spectrometers can trap ionized metabolites in collision cells and generate fragments that can be analyzed at high-resolution too (Schuhmann et al, 2011; Van Der Hooft et al, 2011, 2012b). Analysis of the resulting fragments and neutral losses usually provides additional structural information about the fragmented mass as well as more constraints for its elemental formula
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