Lischnig, A.; Karimi, N.; Larsson, P.; Ekstrom, K.; Crescitelli, R.; Olin, A.-C.; Lasser, C.

Introduction: Extracellular vesicles (EVs) are a heterogeneous group of membrane-enclosed vesicles released by cells. They play important roles in intercellular communication and contribute to several physiological and pathological processes. Cells release subpopulations of EVs with distinct biogenesis and functions, however, we currently have few markers to differentiate them. This study, therefore, aimed to determine proteomic and lipidomic differences among four EV subpopulations of varying sizes and densities. Methods: Large and small EVs (L-EVs and S-EVs) were isolated from two immune cell lines by differential ultracentrifugation at 16,500 x g and 118,000 x g, respectively. The crude EVs were then further separated by density cushion centrifugation. EVs were isolated from the interphase between 1.079-1.146 and 1.146-1.185 g/ml, hereafter referred to as low density (LD) and high density (HD), respectively. This resulted in four subpopulations of EVs: L-EVs LD, L-EVs HD, S-EVs LD, and S-EVs HD. The morphology, size, and yield of EVs were determined by nanoparticle tracking analysis, electron microscopy, and western blot. The proteome and lipidome of the four subpopulations of EVs were determined with mass spectrometry. Results: A total of 5364 proteins were quantified in the dataset. L-EV and S-EVs as well as LD and HD were well separated. Briefly, L-EVs LD were enriched in mitochondrial proteins such as the TIMM/TOMM complex, MICOS, and ATP5 proteins. In contrast, L-EVs HD were enriched in proteins associated with the cytoskeleton, such as KIF proteins. Furthermore, S-EVs LD were enriched in tetraspanins and ESCRT machinery proteins, while S-EVs HD were enriched in histones, CCT proteins, and proteins from the complement pathway. Proteins such as flotillins, RABs, annexins, and integrins were enriched in two or several subpopulations. Furthermore, 107 lipids were quantified, and the most abundant lipids in EVs were phosphatidylcholine (PC), sphingomyelin, and phosphatidylethanolamine (PE). The most profound difference was that PE was less abundant in L-EVs LD as compared to the other EV subtypes, and ceramides were enriched in L-EVs as compared to S-EVs. Conclusion: This study demonstrates that the proteome and lipidome differ in EV subpopulations separated based on size and density. Furthermore, it validates several protein groups that have previously been suggested to be enriched in either S-EVs or L-EVs.