Dottorato in SCIENZE FARMACEUTICHE

Titolo della tesi: Metabolic survey of complex matrices: untargeted investigation by mass spectrometry

Metabolomics is having a growing impact on numerous disciplines including food and plant sciences, drug development, toxicology, environmental science, and medicine. The analysis of complex natural mixtures provides comprehensive information on the chemical pattern of a biological system and concur to identify new metabolites. At the same time, the metabolome comprises a large number of entities which differ in their nature and quantity. The simultaneous determination of all metabolites in a sample by employing a single analytical method represents a complex challenge; therefore, various and often complementary tools are commonly used to discover the rich pattern of biomolecular composition. The employment of an adequate multimethodological approach plays a key role for a successful metabolomic study, both in the targeted and untargeted method. The latter one aims is to maximize the coverage of detected hits and hence provide the opportunity to observe unexpected changes. Due to its high sensitivity, resolving power and mass accuracy, Fourier transform ion-cyclotron resonance mass spectrometry (FT-ICR MS) is one of the most suitable techniques able to provide an extensive coverage of thousands of metabolites from complex samples and distinguish them based on the accurate mass and naturally abundant elemental isotopes determination. The huge amount of data so obtained requires the application of databases and visualization tools to simplify the interpretation of the information obtained. In the first part of the present thesis, untargeted metabolomic studies have been addressed to provide a thoroughly characterization of foodstuff, of both vegetable and animal origin. The electrospray ionization (ESI) source coupled to FT-ICR MS enabled to explore the metabolic profile of hydroalcoholic and organic extracts of Goji berries and leaves, “Rosso di Sulmona” garlic, and a spray-dried Acheta domesticus powder. These investigations unveiled the presence of healthy components with nutraceutical potential, providing important information that links food with human health and well-being. Also, untargeted research on agrifood by-products, such as Goji leaves and garlic tunicas, unveiled a broad range of natural bioactive compounds making them promising for their (re)using in several fields. In addition, the untargeted approach based on ESI FT-ICR MS analysis has been selected to achieve a maximum metabolome exploration of cell free supernatants and membrane vesicles produced by Lactobacillus reuteri DSM 17938 in planktonic and biofilm phenotypes, in the attempt to ascribe, for the first time, the antimicrobial properties to their chemical diversity. Several putative compounds have been identified as possibly involved in antimicrobial activity. In this way, the metabolic information obtained could help to provide a biological interpretation of their health effects. Lastly, the complementary evidence gathered by two ionization sources, ESI and atmospheric-pressure chemical ionization (APCI) coupled with MS, was employed for the detection of several metal-based CO release molecules (CO-RMs). Collision induced dissociation (CID) was instead the MS/MS technique of choice to gain structural elucidation on CO loss. Also, MS experiments have been carried out to test their stability in solution over the time by monitoring their ion abundances in presence of an internal reference.