Detailed analysis of the plasma extracellular vesicle proteome after separation from lipoproteins

Abstract

The isolation of extracellular vesicles (EVs) from blood is of great importance to understand the biological role of circulating EVs and to develop EVs as biomarkers of disease. Due to the concurrent presence of lipoprotein particles, however, blood is one of the most difficult body fluids to isolate EVs from. The aim of this study was to develop a robust method to isolate and characterise EVs from blood with minimal contamination by plasma proteins and lipoprotein particles. Plasma and serum were collected from healthy subjects, and EVs were isolated by size-exclusion chromatography (SEC), with most particles being present in fractions 8-12, while the bulk of the plasma proteins was present in fractions 11-28. Vesicle markers peaked in fractions 7-11; however, the same fractions also contained lipoprotein particles. The purity of EVs was improved by combining a density cushion with SEC to further separate lipoprotein particles from the vesicles, which reduced the contamination of lipoprotein particles by 100-fold. Using this novel isolation procedure, a total of 1187 proteins were identified in plasma EVs by mass spectrometry, of which several proteins are known as EV-associated proteins but have hitherto not been identified in the previous proteomic studies of plasma EVs. This study shows that SEC alone is unable to completely separate plasma EVs from lipoprotein particles. However, combining SEC with a density cushion significantly improved the separation of EVs from lipoproteins and allowed for a detailed analysis of the proteome of plasma EVs, thus making blood a viable source for EV biomarker discovery.

Keywords: Density cushion; Exosomes; Extracellular vesicles; Lipoproteins; Mass spectrometry; Plasma; Proteomics; Serum; Size-exclusion chromatography.

Fig. 1

Schematic overview of the experimental workflow. Blood was collected in the morning from overnight-fasting healthy subjects, and plasma and serum were isolated. Several approaches were used to isolate EVs from plasma and serum and to separate them from lipoprotein particles and plasma proteins. HDL high-density lipoprotein, IDC iodixanol density cushion, RT room temperature, SEC size exclusion chromatography, UCF ultracentrifugation

Fig. 2

Evaluation of EVs isolated with size-exclusion chromatography (SEC). One millilitre of plasma or serum was loaded onto 10 mL Sepharose CL-2B columns, and up to 30 fractions of 500 µL were collected from each column. a Concentrations of particles and proteins in the SEC fractions were determined with nanoparticle tracking analysis (NTA; ZetaView^®, blue) and BCA (green), respectively. Data are presented as the percentage of the total amount of particles or proteins in fractions 5–16. N = 4–6, and the results are presented as the average ± SEM. b ELISA was used to determine the expression of CD9, CD63, and CD81 on the vesicles in the SEC fractions. Data are presented as the percentage of the total expression for each protein  in fractions 5–16. N = 3–5, and the results are presented as the average ± SEM. c Presence of the vesicle marker flotillin-1 and the HDL marker Apo-A1 was determined in fractions 7–12 (40 µL/fraction) with Western blot. d, e Fifteen microliters (1–20 µg protein) from fractions 8–11 were loaded onto grids, negative stained, and evaluated with electron microscopy. Examples of EV-like structures (cup-shaped) are indicated by black arrows, and examples of lipoprotein particle-like structures (white structures) are indicated by white arrows. e Enlargements from fraction 8–10 from the plasma sample showed in d. Scale bars are 200 nm in d and 100 nm in e

Fig. 3

Schematic overview of the size and density of lipoproteins and EVs. Several of the lipoproteins such as high-density lipoproteins (HDL), low-density lipoproteins (LDL), intermediate-density lipoproteins (IDL), very low-density lipoproteins (VLDL), and chylomicrons overlap with extracellular vesicles (EVs) in terms of size or density. With an iodixanol density cushion of 10%/30%/50%, the 10% layer will create a density cutoff at approximate 1.06 g/cm³ (indicated by the orange dashed line). With Sepharose CL-2B SEC columns, the size cutoff is approximately 75 nm (indicated by the blue dashed line). The picture is modified from [11]

Fig. 4

Evaluation of EVs isolated with the combination of density cushion and size-exclusion chromatography (IDC + SEC). Six millilitres of plasma were loaded on top of a density cushion (50%/30%/10% iodixanol), and visible bands after ultracentrifugation were further loaded onto 10 mL Sepharose CL-2B columns, and up to 30 fractions of 500 µL each were collected. a After centrifuging of the plasma sample on top of the cushion, two bands were visible. One band contained material floating above 1.025 g/cm³ (low-density band), and the second band contained material floating at approximately 1.06–1.16 g/cm³ (high-density band). b Low-density and high-density bands were loaded onto individual SEC columns, and fractions were collected. The concentrations of particles and proteins in the SEC fractions were determined by nanoparticle tracking analysis (NTA; ZetaView^®, blue) and BCA (green), respectively. Data are presented as the percentage of the total amount of particles or proteins in fractions 5–16. N = 3, and the results are presented as the average ± SEM. c Total number of particles was determined by ZetaView^® in fractions 5–16 from the high-density and low-density bands isolated from the same plasma samples, and the fold change was calculated. N = 3, and the results are presented as the average ± SEM. LD low-density, HD high-density. d Presence of the vesicle markers flotillin-1 and TSG-101 as well as the HDL marker Apo-A1 was determined by Western blot of fractions 7–14 (40 µL/fraction) isolated from both the high-density and low-density band. e Fifteen microliters (1–6 µg protein) from fractions 8–10 from the high-density and low-density bands were loaded onto grids, negative stained, and evaluated with electron microscopy. Scale bars are 200 nm

Fig. 5

Evaluation of EVs isolated with the combination of ultracentrifugation, density cushion, and size-exclusion chromatography (UCF + IDC + SEC). To be able to increase the starting volume of plasma, two centrifugation steps were added. The pellets from 16,500×g and 118,000×g spins were re-suspended in PBS, mixed, loaded on top of a density cushion (50%/30%/10% iodixanol), and centrifuged. The band between 30 and 10% was subsequently loaded onto a 10 mL Sepharose CL-2B column, and up to 30 fractions of 500 µL each were collected. a Concentrations of particles and proteins in the SEC fractions were determined with nanoparticle tracking analysis (NTA; ZetaView^®, blue) and BCA (green), respectively. Data are presented as the percentage of the total amount of particles or proteins in fractions 5–16. N = 1. b Presence of the vesicle marker flotillin-1 and the HDL marker Apo-A1 was determined with Western blot in fractions 7–14 (40 µL/fraction). c Five micrograms of protein (11–19 µL) from SEC fractions 7–10 were loaded onto grids, negative stained, and evaluated with electron microscopy. Scale bars are 500 nm. d LC–MS/MS was performed on the EVs isolated from fractions 8 and 9 and pooled. In total, 1187 proteins were identified and were analysed with DAVID Bioinformatics Resources 6.8 (https://david.ncifcrf.gov/). The ten most associated cellular compartments (based on p value) are listed in the graph. e The  1187 identified proteins were compared to previously published proteomes of plasma EVs [–31, 33]

Fig. 6

RNA isolation from EV-enriched and lipoprotein-enriched fractions. a Six millilitres of plasma were loaded onto a 50%/30%/10% iodixanol density cushion, and the low-density and high-density bands were isolated. RNA was isolated with a miRCURY RNA Isolation Kit—Cell and Plant (Exiqon) directly from 300 µL of the high-density and low-density bands and analysed with a Bioanalyzer^® (Agilent). N = 4–6, and the results are presented as the average ± SEM. **p value < 0.01. LD low-density, HD high-density. b Fifty-eight millilitres of plasma were ultracentrifuged, and the pellets from a 16,500×g and 118,000×g spin were re-suspended in PBS, mixed, loaded on top of a density cushion (50%/30%/10% iodixanol) and centrifuged. The band between 30 and 10% was subsequently loaded onto a 10 mL Sepharose CL-2B column and 30 fractions of 500 µL each were collected in pools of 6 fractions (3 mL in total/pool). The sample pools were ultracentrifuged, and RNA was isolated and analysed as in a