Multiomics analysis to evaluate the enrichment of extracellular vesicles from human plasma

Figures

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  Fig. 1. Schematic overview of the plasma EVs isolation workflow. Blood samples were collected in K2E EDTA tubes and spun at 1,500 g for 20 min to separate plasma. Plasma was spun at 2,500 g for 10 min twice to deplete platelets from the plasma. Platelet depleted plasma (PDP) was collected and stored at −80°C until use. For EVs isolation, 6 ml of PDP was layered on top of an OptiPrep™ cushion and subjected to 178,000 g avg, 4°C, for 128 min in a SW40 rotor. Aliquots of LD and HD bands were collected for downstream immunoblotting and omics analyses. The remaining HD fractions were pooled before SEC column separation (Sepharose® CL-6B (24 nm), IZON qEVoriginal™ Legacy 35 nm, Sepharose® CL-4B (42 nm), and IZON qEVoriginal™ Legacy 70 nm). EV = extracellular vesicles, LD = low density, HD = high density.

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  Fig. 2. Protein quantitation and immunoblot characterization of plasma EVs isolated by density gradient ultracentrifugation (DGUC) and size-exclusion chromatography (SEC). A: Protein yield in each particle fraction from different SEC columns. B: Immunoblotting of PDP, LD, and HD samples (2.3 μg) and SEC fractions (equal volume). Samples were electrophoresed for 60 min at 160 V. Total proteins were stained and imaged, followed by immunoblotting with EV markers, flotillin-1 and syntenin, and ApoB100 (LDL marker), and ApoA-I (HDL marker). EV = extracellular vesicle, PDP = platelet depleted plasma, LD = low density, HD = high density.

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  Fig. 3. Size-exclusion chromatography (SEC) columns vary in their ability to enrich for EVs and deplete non-EV particles and abundant plasma proteins following density gradient ultracentrifugation (DGUC). A: Number of protein identifications in each sample. The EVs enriched fractions are highlighted by the red boxes and the numbers in red indicate the number of proteins identified in these fractions. B: The normalized LFQ intensity comparison of selected EV markers. The EVs enriched fractions contained higher level of EV markers (ANXA2, ANXA5, CD63, CD9, flotillin-1, flotillin-2, syntenin, and TSG101). C: The normalized LFQ intensity comparison of contaminating proteins. The EVs enriched fractions contained substantially low intensity of contaminant proteins (ALB, APOA1, APOA2, APOB, complement C3, and prothrombin). PDP = platelet depleted plasma, in red; LD band = low-density band, in orange; HD band = high-density band, in yellow; 6B = Sepharose™ CL-6B, in purple; 35 nm = qEVoriginal™ 35 nm, in green; 4B = Sepharose™ CL-4B, in pink; 70 nm = qEVoriginal™ 70 nm in blue, LFQ = label-free quantification.

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  Fig. 4. Comparison of lipid class in F7-9 EV fractions from qEVoriginal™ 70 nm SEC column against PDP and HD. Comparison is shown at (A) PC, (B) PE, (C) PS, (D) Cer, (E) SM, (F) TG, (G) CE and (H). Chol lipid class level. Data represent log2-transformed normalized abundance fold change (NAFC) ± standard deviation. One sample t test on the log2 NAFC values followed by multiple testing Benjamin–Hochberg FDR correction was used to determine statistical significance, with adjusted ∗P < 0.05; ∗∗P < 0.01, and ∗∗∗P < 0.001. PDP = platelet depleted plasma, EV = extracellular vesicle; PC = phosphocholine, PE = glycerophosphoethanoamine, PS = glycerophosphoserine, Cer = ceramide, SM = sphingomyelin, TG = triglyceride, CE = cholesteryl ester, and Chol = cholesterol.

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  Fig. 5. Immunoblot and proteomic characterization of highly enriched plasma EVs. A: Normalized PDP, HD and EVs samples (2.3 μg) were electrophoresed for 60 min at 160 V. Total proteins were stained, followed by immunoblotting with ApoB100 (LDL marker), ApoA-I (HDL marker), and syntenin and flotillin-1 (general EVs markers). B: The number of proteins identified in each sample (PDP, HD, and EVs). The red frame and number indicate total proteins identified. C: Venn diagram of identified proteins in plasma EVs. D: The normalized LFQ intensity comparison of representative EVs markers. E: The normalized LFQ intensity comparison of plasma proteins and lipoproteins. PDP = platelet depleted plasma, HD band = high-density band, EV = extracellular vesicle, sub = volunteer subject, LFQ = label-free quantification.

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  Fig. 6. Enrichment analysis of the proteome of highly enriched plasma EVs. A: Gene ontology enrichment analyses including top 10 biological process, cellular component, and molecular function pathways. B: Top 10 enriched KEGG pathways. C: Top 10 enriched Reactome pathways. D: Venn diagram showing numbers of proteins identified in plasma EVs compared with Human Plasma Proteome (Deutsch et al., 2021). Histograms show identification and enrichment of (E) neurodegeneration/CNS associated proteins, (F) mitochondrial proteins, (G) endosomal-autophagic-lysosomal (EAL) pathway proteins, and (H) lipid metabolism proteins in EVs. The databases were from Human Protein Atlas databases and KEGG pathways. PDP = platelet depleted plasma, HD band = high-density band, EV = extracellular vesicle, sub = volunteer subject, CNS = central nervous system, KEGG = Kyoto Encyclopedia of Genes and Genomes.

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  Fig. 7. Comparison of lipid in EVs against PDP and HD. A: Comparison at lipid category level. Four lipid categories include glycerophospholipids (GPs), sphingolipids (SPs), glycerolipids (GLs), and sterol (ST) lipids. Comparison at (B) GP lipid class level, (C) SP lipid class level, (D) GL lipid class level and (E.) ST lipid class level. Data represent log2-transformed normalized abundance fold change (NAFC) ± standard deviation. One sample t test on the log2 NAFC values followed by multiple testing Benjamin–Hochberg FDR correction was used to determine statistical significance, with adjusted ∗P < 0.05; ∗∗P < 0.01. F: Volcano plot showing comparison between PDP and EVs at lipid sum composition level. Multiple t test followed by Benjamin–Hochberg FDR correction was used to determine statistical significance. Significantly differential lipids with adjusted P value < 0.05 are highlighted in red. PDP = platelet depleted plasma, HD band = high-density band, EV = extracellular vesicle, PC = phosphocholine, PE = glycerophosphoethanoamine, PS = glycerophosphoserine, PI = glycerophosphoinositol, PA = glycerophosphoric acid, PG = glycerophosphoglycerol, Sph_base = sphingoid base, Cer = ceramide,…