Untargeted UPLC-MSProfiling Pipeline to Expand TissueMetabolome Coverage: Application to Cardiovascular Disease

摘要

Metabolic profiling studies aim to achieve broad metabolome coverage in specific biological samples. However, wide metabolome coverage has proven difficult to achieve, mostly because of the diverse physicochemical properties of small molecules, obligating analysts to seek multiplatform and multimethod approaches. Challenges are even greater when it comes to applications to tissue samples, where tissue lysis and metabolite extraction can induce significant systematic variation in composition. We have developed a pipeline for obtaining the aqueous and organic compounds from diseased arterial tissue using two consecutive extractions, followed by a different untargeted UPLC-MS analysis method for each extract. Methods were rationally chosen and optimized to address the different physicochemical properties of each extract: hydrophilic interaction liquid chromatography (HILIC) for the aqueous extract and reversed-phase chromatography for the organic. This pipeline can be generic for tissue analysis as demonstrated by applications to different tissue types. The experimental setup and fast turnaroundtime of the two methods contributed toward obtaining highly reproduciblefeatures with exceptional chromatographic performance (CV % < 0.5%),making this pipeline suitable for metabolic profiling applications.We structurally assigned 226 metabolites from a range of chemicalclasses (e.g., carnitines, α-amino acids, purines, pyrimidines,phospholipids, sphingolipids, free fatty acids, and glycerolipids)which were mapped to their corresponding pathways, biological functionsand known disease mechanisms. The combination of the two untargetedUPLC-MS methods showed high metabolite complementarity. We demonstratethe application of this pipeline to cardiovascular disease, wherewe show that the analyzed diseased groups (n = 120)of arterial tissue could be distinguished based on their metabolicprofiles.