A Pseudo-Targeted Method for Global Profiling of Cholesteryl Esters by Liquid Chromatography-Zeno High-Resolution Multiple Reaction Monitoring Mass Spectrometry

Abstract Cholesteryl esters (CEs) play critical roles in diverse physiological processes, yet their comprehensive analysis remains challenging due to the limited availability of authentic standards and the low abundance of many CE species. To address this, we developed a pseudo-targeted screening strategy that integrates virtual database construction with retention time prediction for the global analysis of CEs using optimized liquid chromatography-Zeno high-resolution multiple reaction monitoring (LC-Zeno MRM-HR). Compared to conventional data-dependent acquisition (DDA), our method demonstrated superior sensitivity and specificity. Application of this strategy to biological samples resulted in the identification of 80 CE species. Among these, 18 are reported here for the first time, and 9 were unequivocally validated using authentic standards, underscoring the method’s extended coverage and reliable identification accuracy. We further applied this method to analyze CE metabolism in hepatocellular carcinoma (HCC) cells. Pharmacological inhibition of SOAT1 (by avasimibe) and NCEH1 (by JW480) revealed their opposing roles: SOAT1 inhibition depleted CEs, while NCEH1 inhibition promoted their accumulation, consistent with established biology understanding and affirming the utility of our approach. Finally, leveraging the analytical power of this strategy, we conducted a multi-tissue survey across brain, heart, lung, kidney, stomach, fat, and plasma. This revealed distinct, organ-specific CE profiles that correlate with local physiology, establishing a foundational blueprint for tissue-specific CE distribution.