Chemical synthesis and mass spectrometric detection of microbiome-derived N-acyl amides
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Tiivistelmä
N-acyl amides (NAAs) are a structurally and functionally diverse group of endogenous cell signalling molecules, known for regulating several physiological responses in the body. Resent research has accumulated evidence for gut microbiome-derived NAAs that could influence the host physiology via gut-brain signalling. In the field of metabolomics, liquid chromatography-mass spectrometry represents the most extensively utilized analysis method. However, it has not been as widely applied in faecal analysis, which is the optimal biological sample matrix when investigating gut microbiome-derived metabolites, such as NAAs. The aims of this thesis were to chemically synthesize two NAA standards, histamine-C5:0 and Arg-C18:0, and to detect NAAs from human faecal samples using targeted mass spectrometric analysis.
Successful synthesis of histamine-C5:0 and Arg-C18:0 was confirmed with liquid chromatography-tandem mass spectrometry (LC-MS/MS) and nuclear magnetic resonance spectroscopy. The newly synthesized NAAs together with NAA standard mixtures provided by a collaborator were used as qualitative standards in faecal analysis. NAA profiling was conducted using a newly developed targeted LC-MS/MS method, with high selectivity based on retention time as well as predefined precursor ion and fragment ion spectrum matching.
In this study 32 NAAs were identified in human faecal samples with targeted LC-MS/MS. While there were no statistically significant differences in the relative abundances of NAAs between the sexes, these results demonstrated the utility of synthetic NAA standards together with optimized targeted LC-MS/MS method in detection of microbiome-derived NAAs in faecal samples, which have remained underexplored due to their biological complexity.