Strategy for Global Profiling and Identification of 2- and 3-Hydroxy Fatty Acids in Plasma by UPLC-MS/MS.

2-Hydroxy fatty acids (2-OHFAs) and 3-hydroxy fatty acids (3-OHFAs) with the same carbon backbone are isomers, both of which are closely related to diseases involving fatty acid oxidation disorder. However, the comprehensive profiling of 2- and 3-OHFAs remains an ongoing challenge due to their high structure similarity, few structure-informative product ions, and limited availability of standards. Here, we developed a new strategy to profile and identify 2- and 3-OHFAs according to structure-dependent retention time prediction models using ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Both accurate MS and MS/MS spectra were collected for peak annotation by comparison with an in-house database of theoretically possible 2- and 3-OHFAs. The structures were further confirmed by the validated structure-dependent retention time prediction models, taking advantage of the correlation between the retention time, carbon chain length and number of double bonds, as well as the hydroxyl position-induced isomeric retention time shift rule. With the use of this strategy, 18 2-OHFAs and 32 3-OHFAs were identified in the pooled plasma, of which 7 2-OHFAs and 20 3-OHFAs were identified for the first time in this work, furthering our understanding of OHFA metabolism. Subsequent quantitation method was developed by scheduled multiple reaction monitoring (MRM) and then applied to investigate the alteration of 2- and 3-OHFAs in esophageal squamous cell carcinoma (ESCC) patients. Finally, a potential biomarker panel consisting of six OHFAs with good diagnostic performance was achieved. Our study provides a new strategy for isomer identification and analysis, showing great potential for targeted metabolomics in clinical biomarker discovery.

Spatially resolved metabolomics to discover tumor-associated metabolic alterations.

Characterization of tumor metabolism with spatial information contributes to our understanding of complex cancer metabolic reprogramming, facilitating the discovery of potential metabolic vulnerabilities that might be targeted for tumor therapy. However, given the metabolic variability and flexibility of tumors, it is still challenging to characterize global metabolic alterations in heterogeneous cancer. Here, we propose a spatially resolved metabolomics approach to discover tumor-associated metabolites and metabolic enzymes directly in their native state. A variety of metabolites localized in different metabolic pathways were mapped by airflow-assisted desorption electrospray ionization mass spectrometry imaging (AFADESI-MSI) in tissues from 256 esophageal cancer patients. In combination with in situ metabolomics analysis, this method provided clues into tumor-associated metabolic pathways, including proline biosynthesis, glutamine metabolism, uridine metabolism, histidine metabolism, fatty acid biosynthesis, and polyamine biosynthesis. Six abnormally expressed metabolic enzymes that are closely associated with the altered metabolic pathways were further discovered in esophageal squamous cell carcinoma (ESCC). Notably, pyrroline-5-carboxylate reductase 2 (PYCR2) and uridine phosphorylase 1 (UPase1) were found to be altered in ESCC. The spatially resolved metabolomics reveal what occurs in cancer at the molecular level, from metabolites to enzymes, and thus provide insights into the understanding of cancer metabolic reprogramming.