The project adopted a holistic approach of metabolomics and proteomics profiling using mass spectrometry. The study addressed the following primary research objectives: (i) targeted assessment of tryptophan pathways metabolites, (ii) targeted assessment of inflammatory proteins, (iii) non-targeted serum metabolomics, and (iv) non-targeted metabolomics analysis of serum-derived exosomes. Individual assays were applied to achieve these research objectives.
Blood-based assays were developed and optimized under the designed objectives, with pooled serum and plasma samples used for method development and validation. A rapid targeted method with a high coverage has been developed for the quantification of tryptophan pathway metabolites in serum and plasma using UHPLC-MS/MS. Tryptophan pathway metabolites play a key role in gut-microbiota mediated metabolic mechanisms and are indicative of diet and co-microbial functions, therefore the assay has high significance to MASLD and intervention, with regards to the nutritional influence on risk and treatment outcomes. Within the proteomic profiling, 33 inflammatory, immunological, and lipid-related proteins were quantified using a targeted UHPLC/SRM-MS quantitative assay. These quantitative measurements of metabolites and proteins under targeted assays are significant to determine the concentrations in the biosamples. The developed method has been applied for corresponding evaluations of available MASLD cohort samples, and further data analysis is in progress for determining conclusive outcome. However, differential expression of these metabolites and proteins could be significant to represent promising candidates for molecular biomarkers of the disease progression and understanding their role in biological processes like metabolism.
Non-targeted serum metabolomics was performed using GC-Orbitrap-HRMS, while non-targeted metabolomics of serum-derived exosomes involved application of a pilot workflow for exosome isolation, molecular characterization, and metabolomics profiling on a UHPLC-Orbitrap-HRMS instrument. These studies lead to identification and characterization of perturbed endogenous metabolites and associated metabolic pathways in analysed serum samples and help to distinguish variable features pertaining to distinct MASLD stages. Furthermore, the findings will expand current knowledge by identifying additional metabolites not previously reported as perturbed in this disease.
