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.Electronic supplementary materialThe online version of this article (10.1007/s00018-018-2773-4) contains supplementary material, which is available to authorized users.

Adipose‐Derived Biogenic Nanoparticles for Suppression of Inflammation

“…Lipo‐NPs are envisioned for future autologous applications, but could also have potential allogeneic uses considering that BiNPs are frequently exchanged between individuals in the clinical setting. For example, plasma transfusions seldom cause adverse reactions,79 although this biological fluid contains a large amount of BiNPs from various cells 80. Moreover, it is possible that Lipo‐NPs could be lyophilized to aid in storage for clinical applications.…”

Optimizing Size Exclusion Chromatography for Extracellular Vesicle Enrichment and Proteomic Analysis from Clinically Relevant Samples

“…To evaluate whether a specific MDA‐MB‐231 EV signal could be detected in each of the spiked samples, we identified seven putative MDA‐MB‐231 EV proteins which are not reported in the healthy plasma EV proteome dataset by Karimi et al . but are reported in the MDA‐MB‐231 EV proteome published by Hurwitz et al .…”

“…To evaluate whether a specific MDA-MB-231 EV signal could be detected in each of the spiked samples, we identified seven putative MDA-MB-231 EV proteins which are not reported in the healthy plasma EV proteome dataset by Karimi et al [17] but are reported in the MDA-MB-231 EV proteome published by Hurwitz et al [28] and functionally implicated in MDA-MB-231 cell activity. [29][30][31][32][33][34][35] The LC-MS/MS data (total intensity, % coverage, # unique peptides) for 6-phosphogluconate dehydrogenase (PGD), ADP-ribosylation factor 1 (ARF1), eukaryotic elongation factor 2 (EEF2), Rab GDP dissociation inhibitor beta (GDI2), heterogeneous nuclear ribonuclear protein K (HNRNPK), syntenin-1 (SDCBP) and vimentin (VIM) are summarized in Table 1.…”

“…There is, however, yet to be a consensus on the methodological details of SEC. For example, various column types have been used, including Sepharose CL‐2B, Sepharose CL‐4B, Sephacryl S‐400, and pre‐packed commercially available columns . This methodological variability makes it difficult to ascertain the true utility of this method for preparing EVs suitable for proteomic analysis.…”

Proteomic Signature of Mesenchymal Stromal Cell‐Derived Small Extracellular Vesicles

“…Proteomic profiles of MSC‐EV isolated by SEC are likely to differ from those of MSC‐EV isolated by ultracentrifugation, HPLC, tangential flow filtration, or precipitation, as SEC is likely to better remove overabundant soluble proteins compared to other techniques, resulting in relatively more pure EV preparations. Thus, comparative studies on SEC‐isolated MSC‐EV, with a strong focus on EV purity and potential contaminations will be required to verify our conclusions on MSC‐EV cargo and their potential functional implications …”