fig4

Figure 4. Untargeted lipidomic comparison between EVs isolated from blood and plasma using the PROSPR enrichment protocol. (A) Venn diagram showing the number of lipid species identified by untargeted lipidomics in blood and plasma EVs; (B) Volcano plot displaying the differential abundance of lipid species between blood and plasma EVs. Lipids with significantly higher abundance in blood EVs compared to plasma (log₂FC > 1.5, P < 0.05) are shown in red; those with significantly lower abundance in blood EVs (log₂FC < -1.5, P < 0.05) in blue; lipids with statistically significant but smaller fold changes (|log₂FC| < 1.5) in orange; and non-significant lipids (P ≥ 0.05) in black; (C) PCA based on lipidomic profiles of blood (red) and plasma (blue) EVs, revealing sample group separation; (D) Heatmap and hierarchical clustering of the most discriminating lipid species between blood and plasma EVs. Clustering was performed using Euclidean distance and Ward’s linkage method. In cases where multiple lipid classes are shown as a putataive compound, the detected feature could not be unambiguously assigned to a single lipid species; (E) Quantification of lipid classes identified in blood (red) and plasma (blue) EVs; (F) Circular plot showing the lipid family and subclass composition of lipid species identified in blood EVs; (G) Circular plot showing the lipid family and subclass composition of lipid species identified in plasma EVs. In F and G, the inner ring denotes the main lipid family; the outer ring indicates specific lipid subclasses. Statistical significance was evaluated using unpaired t-test (P < 0.05). EVs: Extracellular vesicles; PCA: principal component analysis; GL: glycerolipids; GP: glycerophospholipids; SP: sphingolipids; TG: triradylglycerols; CL: cardiolipin; DG: diacylglycerols; PC: phosphatidylcholine; PE: phosphatidylethanolamine; PA: phosphatidic acid; PS: phosphatidylserine; PG: phosphatidylglycerol; PE-NMe₂: dimethyl phosphatidylethanolamine; PE-NMe: monomethyl phosphatidylethanolamine; PI: phosphatidylinositol; Cer: ceramides; EPC: ether-linked phosphatidylcholine; GlcCer: glucosylceramide; SM: sphingomyelin; FA: fatty acids; MAG: monoacylglycerol; Chol: cholesterol.