Skylite: Skyline-Based Lipid Isomer Retention Time Evaluation for Lipidomics in Metabolic Dysfunction-Associated Steatohepatitis

Metabolic dysfunction-associated steatotic liver disease (MASLD) is the most prevalent liver disorder worldwide and can progress to steatohepatitis. Elevated de novo lipogenesis (DNL) is a key contributor to hepatic steatosis. Fatty acid (FA) desaturation produces several unsaturated lipid isomers that are structurally very similar but have diverse biological functions. However, due to their structural similarity, many conventional mass spectrometry approaches cannot detect such metabolic alterations. Thus, we introduce the Skylite (Skyline-based lipid isomer retention time evaluation) workflow using conventional liquid chromatography-mass spectrometry (LC-MS) to identify important isomer features. Retention times of isomeric phosphatidylcholines are compared with the well-characterized human plasma reference standard NIST 1950. Retention time trends correlate well with fixed-charge derivatized FA in liquid chromatography and ozone-induced dissociation mass spectrometry data. The interpretation is supported by double bond diagnostic fragments in LC-MS/MS experiments of epoxidized hydrolyzed fatty acids. We investigate hepatic lipid profiles, focusing on esterified fatty acids in two mouse models of metabolic dysfunction-associated steatohepatitis (MASH). Out of 37 phosphatidylcholine sum compositions, the workflow identifies 123 lipid features. Importantly, CCl₄-induced and melanocortin-4 receptor knockout mice on a western diet (WD) have significantly higher levels of mead acid, branched-chain fatty acid, and <i>n</i>-7 PUFA incorporated into phosphatidylcholines. While the MASH mouse liver tissues contain notable amounts of <i>n</i>-7 PUFA, no <i>n</i>-10 PUFA were detected, potentially indicating a unique desaturation pattern. The screening for altered lipid isomer profiles bridges the gap between high-throughput analyses and specialized structure-resolved techniques.