High-resolution targeted mass spectrometry for comprehensive quantification of sphingolipids: clinical applications and characterization of extracellular vesicles

Abstract

Sphingolipids (SL), a class of membrane lipids, play important roles in numerous biological processes. Their significant structural diversity poses challenges for accurate quantification. To address this, liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) has emerged as a powerful tool for sphingolipidomics, capable of profiling these lipids comprehensively. In this study, we utilized LC-MS/MS with high-resolution mass spectrometry (MRM^HR) to develop a targeted method for the identification and quantification of various SL species. This method, based on validated parameters such as precursor/fragment ions (m/z) and retention time, demonstrated high sensitivity and accuracy, successfully identifying SL species across 12 distinct classes. Its open-panel design also facilitates the analysis of new SL-species targets. Notably, using this approach, we identified 40 SL species in plasma samples from COVID-19 patients, and we determined the influence of matrix metalloproteinase-3 (MMP-3) expression on the positive downstream of SL metabolism. Beyond plasma analysis, this method has potential applications in other biomedical contexts, such as extracellular vesicles (EVs), describing the cargo of sphingosine-1-phosphate (S1P) on macrophage-derived EVs. The establishment of this targeted workflow enabling precise quantification of a wide range of SL species, holds promise for identifying novel biomarkers and therapeutic targets.