In vitro and in vivo metabolomics: tapping the potential of secondary electrospray ionization – high resolution mass spectrometry

Background: Metabolomics research plays a crucial role towards the advanced understanding of physiological and pathological conditions in living organisms. Due to the high complexity of the human metabolome, strategic bottom-up approaches can help to break down this complex network of metabolites into smaller manageable pieces. Volatile organic compounds (VOCs) analysis has gained tremendous attention in the field of medical diagnostics. Characterization of VOCs enables researchers to gain insights into alterations of metabolic pathways and helps to identify potential biomarkers of diseases. Secondary electrospray ionization-high resolution mass spectrometry (SESI-HRMS) is a non-invasive powerful technique to study those compounds non-invasively in real-time while achieving extraordinary sensitivity and specificity. Given these attractive attributes, SESI-HRMS has the potential to advance metabolomics research by providing fundamental information on the temporal profile of metabolic processes.
Aim: The main aim of this thesis was the real-time analysis of VOCs in the headspace of mammalian cells, bacterial cultures, clinical samples and mice in the context of metabolomics studies by means of newly developed experimental set-ups combined with SESI-HRMS.
Methods: In-house-designed airtight containers were used to measure VOCs emitted by cells, bacteria, clinical samples and mice. Synthetic or medical-grade air was used to pervade the containers and guided emitted VOCs towards the SESI-HRMS for real-time analysis. Data were evaluated by applying study-tailored univariate and multivariate statistical methods.
Results: Volatile metabolic signatures were successfully monitored non-invasively and in real-time over several hours with high consistency, and minimal external intervention and sample preparation. Distinct metabolic signatures were obtained during headspace analysis of immature and activated dendritic cells. Furthermore, feasibility of SESI-HRMS to detect dynamic isotopic incorporation in volatile metabolites in vitro and in vivo was demonstrated. Moreover, VOCs emitted by Staphylococcus aureus (SA) and Streptococcus pneumoniae (SP) cultures were detected within minutes of measurement, down to 1000 colony forming units, and enabled distinction of the bacterial pathogens at species and strain level. As a proof-of-principle, clinical samples were fingerprinted within a few minutes which allowed, to a certain degree, the differentiation between samples containing living or dead bacteria. For mice with a mild SA or SP infection, we found distinct volatile metabolites which were either up- or downregulated when comparing pre-infected and infected state.
Conclusion: Through this work it was demonstrated that SESI-HRMS in tandem with tailored engineered experimental set-ups is extremely useful for capturing dynamic insights in the volatile metabolome of different organisms. Such platforms in conjunction with SESI-HRMS complement existing analytical techniques for the performance of metabolomics studies. Metabolic insights in increasingly complex biological systems can be gained, which advances biomarker discovery and paves the way for further translational research as well as the development of diagnostic tools.