Dataset Information

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.

SUBMITTER: Karimi N

PROVIDER: S-EPMC6021463 | biostudies-literature | 2018 Aug

REPOSITORIES: biostudies-literature

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

Karimi Nasibeh N Cvjetkovic Aleksander A Jang Su Chul SC Crescitelli Rossella R Hosseinpour Feizi Mohammad Ali MA Nieuwland Rienk R Lötvall Jan J Lässer Cecilia C

Cellular and molecular life sciences : CMLS 20180213 15

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 we ...[more]

PMID: 29441425

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Project description:Bacteria release nano-sized extracellular vesicles (EVs) into the extracellular milieu. Bacterial EVs contain molecular cargo originating from the parent bacterium and have important roles in bacterial survival and pathogenesis. Using 8-plex iTRAQ approaches, we profiled the EV proteome of two Escherichia coli strains, uropathogenic (UPEC) 536 and probiotic Nissle 1917. For these strains, we compared the proteome of crude input EVs prepared by ultracentrifugation alone with EVs purified by either density gradient centrifugation (DGC) or size exclusion chromatography (SEC). We further compared the proteome of EVs from bacterial cultures that were grown in iron-restricted (R) and iron-supplemented (RF) conditions. Overall, outer membrane components were highly enriched, and bacterial inner membrane components were significantly depleted in both UPEC and Nissle EVs, in keeping with an outer membrane origin. In addition, we found enrichment of ribosome-related Gene Ontology terms in UPEC EVs and proteins involved in glycolytic processes and ligase activity in Nissle EVs. We have identified that three proteins (RbsB of UPEC in R; YoeA of UPEC in RF; BamA of Nissle in R) were consistently enriched in the DGC- and SEC-purified EV samples in comparison to their crude input EV, whereas conversely the 60 kDa chaperonin GroEL was enriched in the crude input EVs for both UPEC and Nissle in R condition. Such proteins may have utility as technical markers for assessing the purity of E. coli EV preparations. Several proteins were changed in their abundance depending on the iron availability in the media. Data are available via ProteomeXchange with identifier PXD011345. In summary, we have undertaken a comprehensive characterization of the protein content of E. coli EVs and found evidence of specific EV cargos for physiological activity and conserved protein cargo that may find utility as markers in the future. Abbreviation: DGC: density gradient centrifugation; DTT: 1,4-dithiothreitol; EV: extracellular vesicles; FDR: false discovery rate; GO: Gene Ontology; R: iron-restricted; RF: iron-supplemented; iTRAQ: isobaric tags for relative and absolute quantitation; OMV: outer membrane vesicle; SWATH-MS: sequential window acquisition of all theoretical mass spectra; SEC: size exclusion chromatography.

Dataset Information

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

Publications

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

Similar Datasets

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