Proteomic and Lipidomic Profiling of Immune Cell-Derived Subpopulations of Extracellular Vesicles

Extracellular vesicles (EVs) are heterogeneous and play important roles in intercellular communication, contributing to physiological and pathological processes. Since few markers currently exist to differentiate subtypes of EVs, this study aimed to determine proteomic and lipidomic differences among four EV subpopulations. Large and small EVs (L-EVs and S-EVs) were isolated from human mast cells (HMC-1) and monocytes (THP-1) by differential ultracentrifugation and then further separated by density cushions into two different densities [low-density (LD) and high-density (HD)]. L-EVs were pelleted at 16,500 × g, and S-EVs were pelleted at 118,000 × g. LD EVs were collected at 1.079-1.146 g/mL, while HD EVs were collected at 1.146-1.185 g/mL. The morphology, size and yield of EVs were determined by TEM and western blot. The proteome and lipidome of the EV subpopulations were determined with mass spectrometry. A total of 5364 proteins were quantified, and L-EVs LD were enriched in mitochondrial proteins such as TIMM/TOMM and MICOS proteins, while L-EVs HD were enriched in cytoskeleton- and cytokinesis-associated proteins, such as KIF proteins. S-EVs LD were enriched in tetraspanins, ADAM10 and ESCRT machinery proteins, while S-EVs HD were enriched in proteins commonly viewed as contaminants, such as histones, complement factors and collagen. Proteins involved in membrane trafficking between the plasma membrane and organelles, such as adaptor protein complexes, the conserved oligomeric Golgi complex, the trafficking protein particle complex, sortin-nexins, TBC1 domain proteins and coatomer subunits, were expressed at similar levels across all EV subtypes. Furthermore, 107 lipids were quantified, and phosphatidylethanolamine (PE) was less abundant in L-EVs LD as compared to the other EV subtypes, while ceramides were enriched in L-EVs as compared to S-EVs.This study demonstrates that there is a core proteome and lipidome that is similar across all four EV subtypes, but importantly, it also shows that a portion of the proteome and lipidome differs in EV subpopulations separated based on size and density. We suggest that these could be important markers in future EV studies and that they may reflect a different biogenesis and EV function.