SignificanceTumor cells reprogram their metabolism to support cell growth, proliferation, and differentiation, thus driving cancer progression. Profiling of the metabolic signatures in heterogeneous tumors facilitates the understanding of tumor metabolism and introduces potential metabolic vulnerabilities that might be targeted therapeutically. We proposed a spatially resolved metabolomics method for high-throughput discovery of tumor-associated metabolite and enzyme alterations using ambient mass spectrometry imaging. Metabolic pathway-related metabolites and metabolic enzymes that are associated with tumor metabolism were efficiently discovered and visualized in heterogeneous esophageal cancer tissues. Spatially resolved metabolic alterations hold the key to defining the dependencies of metabolism that are most limiting for cancer growth and exploring metabolic targeted strategies for better cancer treatment.

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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.