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. 2016 May 23;11(1):41.
doi: 10.1186/s13024-016-0108-1.

Enrichment of extracellular vesicles from tissues of the central nervous system by PROSPR

Xavier Gallart-Palau  1 , Aida Serra  1 , Siu Kwan Sze  2
Affiliations

Enrichment of extracellular vesicles from tissues of the central nervous system by PROSPR

Xavier Gallart-Palau et al. Mol Neurodegener. .

Abstract

Background: 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. We therefore developed a new Protein Organic Solvent Precipitation (PROSPR) method that can efficiently isolate the EV repertoire from human biological samples.

Results: In the current report, we present a novel experimental workflow that outlines the process of sample extraction and enrichment of CNS-derived EVs using PROSPR. 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.

Conclusions: 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. We predict that widespread use of the methodology described herein will greatly accelerate the study of EVs biology in neuroscience.

Keywords: Exosomes; Extracellular vesicles; Human brain; Lipidomics; Microvesicles; Proteomics; Tissue extraction.

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Figures

Fig. 1
Fig. 1
Lipidomics characterization of PROSPR-CNS-EVs. a Profile of lipid classes identified in EVs lipidome including percentage of identification of EV-characteristic lipids* [18]. b Characterized lipid isoforms were compared with whole brain lipidome (+ identifies those lipids found in whole brain lipidome) and matched to records in the EV databases Exocarta [18], Vesiclepedia [19] and EVpedia [21]. Matched lipid isoforms are shown in colors according to their EVs databases match whereas non-matched isoforms are shown in black
Fig. 2
Fig. 2
Proteomics characterization of human PROSPR-CNS-EVs. a Venn diagram of EV proteins matched to records in the EV databases Exocarta [19], Vesiclepedia [21] and EVpedia [22]. Of the total 2901 proteins identified, 61.8 % were associated with exosome markers and cargoes, 75.4 % were associated with microvesicle markers and cargoes and 23.6 % were novel identifications in brain EVs. b List of 86 exosomal markers matched to top 100 exosomal markers in Exocarta. Top 10 of common exosomal markers were indicated in red. 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 [21] and Exocarta [19]. Unique 126 EV proteins found in common between Ultra-CNS-EVs and PROSPR-CNS-EVs were compared to whole brain proteome and 72 of these CNS-EV markers were also identified in whole brain whereas 54 were only identified in EVs enriched fractions. Complete list of these proteins is reported in the Additional file 6: Dataset 5 e Funrich [24] 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
Fig. 3
Fig. 3
Characterization of PROSPR-CNS-EVs by dedicated flow cytometry (dFC). Microparticle gates for EV size distribution analysis were set after the testing of commercial latex beads from three standard EV sizes. a Gate determination of 100 nm beads. b Gate determination of 300 nm beads. c Gate determination of 500 nm beads. d dFC measurement of PE signal from phospholipid-specific stained EVs. The gate for phospholipid-stained vesicles (R8) shows that 83.32 ± 0.65 % of the total material in PROSPR-CNS-EVs was positively stained. e Size distribution of EVs by gated analysis of stained particles from PROSPR-CNS-EVs. Lower exosomal range (≤ 100 nm) represented 74.65 ± 2.68 % of total identified microparticles. Higher exosomal range (≤ 300 nm) represented the 8.51 ± 1.11 % of total identified microparticles. Lower microvesicle range (≤ 500 nm) represented the 3.49 ± 0.17 % of total identified microparticles. Higher microvesicle range (> 500 nm) represented the 13.34 ± 1.43 % of total identified microparticles
Fig. 4
Fig. 4
Analysis of PROSPR-CNS-EVs enriched from mouse brain tissue. a Venn diagram matched to mouse exosome proteins compiled in Exocarta [19] and Funrich [24] corresponding to the 42 % of total identified proteins in mouse PROSPR-CNS-EV fractions. Mouse microvesicle proteins compiled in Vesiclepedia [21] and EVpedia [22] corresponding to the 33 % of all identified proteins in mouse PROSPR-CNS-EV fractions. Finally, the 49.5 % of the EV proteins found in mouse PROSPR-CNS-EVs were novel identifications not previously found in enriched/isolated EVs. b Funrich [24] site of expression analysis of unique EV proteins from mouse PROSPR-CNS-EVs. c Funrich cellular origin categories of unique EV proteins from mouse PROSPR-CNS-EVs
Fig. 5
Fig. 5
Workflow of the methodology applied to enrich EVs from human CNS tissues. Dissected brain tissue is homogenized in two cycles of medium and higher speed as depicted here. *PROSPR workflow was adapted from our previous report [16]. Obtained PROSPR-CNS-EVs were characterized by lipidomics and proteomics as illustrated in this diagram
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