Comprehensive lipidomic analysis for the absolute quantification of the platelet lipidome. (A) Platelet isolation and purification from mice and human models. (B) Ex vivo stimulation using different stimuli. (C) Lipid extraction and internal standard addition applying to lipid-category tailored analysis. (D) Lipid analysis by shotgun or targeted lipidomic analysis. (E) Data analysis and structural characterization by LipidXplorer or Skyline, followed by absolute quantification of all detected lipid species. (F) Establishment of a correlation and anticorrelation lipid-lipid matrix to form a lipid network. Elucidation of knockout (KO) models and perturbation-specific differences identified from absolute quantification of lipids. (G) Validation by additional ex vivo experiments, including functional assays.