Extracellular vesicles (EVs) act as key mediators of intercellular communication and are secreted and taken up by all cell types in the central nervous system (CNS). While detailed study of EV-based signaling is likely to significantly advance our understanding of human neurobiology, the technical challenges of isolating EVs from CNS tissues have limited their characterization using ‘omics’ technologies.
Now, researchers from Nanyang Technological University, Singapore have developed a new Protein Organic Solvent Precipitation (PROSPR) method that can efficiently isolate the EV repertoire from human biological samples. Subsequent LC-MS/MS-based proteomic profiling of EVs enriched from brain homogenates successfully identified 86 of the top 100 exosomal markers. Proteomic profiling of PROSPR-enriched CNS EVs indicated that > 75 % of the proteins identified matched previously reported exosomal and microvesicle cargoes, while also expanded the known human EV-associated proteome with 685 novel identifications. Similarly, lipidomic characterization of enriched CNS vesicles not only identified previously reported EV-specific lipid families (PS, SM, lysoPC, lysoPE) but also uncovered novel lipid isoforms not previously detected in human EVs. Finally, dedicated flow cytometry of PROSPR-CNS-EVs revealed that ~80 % of total microparticles observed were exosomes ranging in diameter from ≤100 nm to 300 nm.
Proteomics characterization of human PROSPR-CNS-EVs
a Venn diagram of EV proteins matched to records in the EV databases Exocarta. b List of 86 exosomal markers matched to top 100 exosomal markers in Exocarta. c Label-free quantitation of the top five most abundant proteins from whole brain proteome compared to their abundance in PROSPR-CNS-EVs. PROSPR-CNS-EV fractions contained on average less than three percent of whole brain amount of the top five highest abundant proteins. d Venn diagram of proteins identified from Ultra-CNS-EVs matched to PROSPR-CNS-EVs and EV protein records in Vesiclepedia and Exocarta. e Funrich site of expression analysis of novel identified EV proteins from PROSPR-CNS-EVs fractions. f Funrich cellular origin categories of novel identified proteins from PROSPR-CNS-EVs fractions.
These data demonstrate that the optimized use of PROSPR represents an easy-to-perform and inexpensive method of enriching EVs from human CNS tissues for detailed characterization by ‘omics’ technologies.