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Extracellular vesicles are rapidly purified from human plasma by PRotein Organic Solvent PRecipitation (PROSPR).

Gallart-Palau X, Serra A, Wong AS, Sandin S, Lai MK, Chen CP, Kon OL, Sze SK - Sci Rep (2015)

Bottom Line: Here, we report a novel and inexpensive method of rapidly isolating EVs from small volumes of human blood plasma by PRotein Organic Solvent PRecipitation (PROSPR).PROSPR encompasses a rapid three-step protocol to remove soluble proteins from plasma via precipitation in cold acetone, leaving the lipid-encapsulated EVs behind in suspension.We foresee that PROSPR-based purification of EVs will significantly accelerate the discovery of new disease biomarkers and the characterization of EVs with potential for clinical applications.

View Article: PubMed Central - PubMed

Affiliation: School of Biological Sciences, Nanyang Technological University, Singapore, 637551.

ABSTRACT
Extracellular vesicles (EVs) such as exosomes and microvesicles mediate intercellular communication and regulate a diverse range of crucial biological processes. Host cells that are damaged, infected or transformed release biomarker-containing EVs into the peripheral circulation, where they can be readily accessed for use in diagnostic or prognostic testing. However, current methods of EV isolation from blood plasma are complex and often require relatively large sample volumes, hence are inefficient for widespread use in clinical settings. Here, we report a novel and inexpensive method of rapidly isolating EVs from small volumes of human blood plasma by PRotein Organic Solvent PRecipitation (PROSPR). PROSPR encompasses a rapid three-step protocol to remove soluble proteins from plasma via precipitation in cold acetone, leaving the lipid-encapsulated EVs behind in suspension. This generates higher purity EVs that can then be obtained from filtration or classical ultracentrifugation methods. We foresee that PROSPR-based purification of EVs will significantly accelerate the discovery of new disease biomarkers and the characterization of EVs with potential for clinical applications.

No MeSH data available.


Related in: MedlinePlus

PROSPR generates higher purity plasma EVs as opposed to conventional ultra-cushion methods.LC-MS/MS label-free quantitation revealed that PROSPR separation yields high purity microvesicle fractions containing lower levels of contaminating plasma proteins when compared with samples obtained by ultra-cushion (a). The list of EV proteins identified by LC-MS/MS in ultra-cushion and PROSPR isolated fractions were compared with the list of human EV proteome contained in vesiclepedia20 as shown by the venn diagrams (b). Percentage of match between ultra-cushion and PROSPR was analyzed; PROSPR revealed higher percentage of match 90.7% compared to ultra-cushion 78.0% with vesiclepedia EV proteins. Cross-contamination in ultra-cushion and PROSPR isolated EV between proteins and high density lipoproteins was analyzed (c) the lipoproteins A-II and C-III were found at higher levels in PROSPR EV fractions whereas the lipoproteins A-I, E, A-IV, M and D were found at higher levels in ultra-cushion EV fractions. EV protein datasets were subjected to functional enrichment analyses using the FunRich tool21. Higher percentage of genes was identified in PROSPR fractions compared to ultra-cushion regarding site of expression (d) and analyzed microvesicle components (e).
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f5: PROSPR generates higher purity plasma EVs as opposed to conventional ultra-cushion methods.LC-MS/MS label-free quantitation revealed that PROSPR separation yields high purity microvesicle fractions containing lower levels of contaminating plasma proteins when compared with samples obtained by ultra-cushion (a). The list of EV proteins identified by LC-MS/MS in ultra-cushion and PROSPR isolated fractions were compared with the list of human EV proteome contained in vesiclepedia20 as shown by the venn diagrams (b). Percentage of match between ultra-cushion and PROSPR was analyzed; PROSPR revealed higher percentage of match 90.7% compared to ultra-cushion 78.0% with vesiclepedia EV proteins. Cross-contamination in ultra-cushion and PROSPR isolated EV between proteins and high density lipoproteins was analyzed (c) the lipoproteins A-II and C-III were found at higher levels in PROSPR EV fractions whereas the lipoproteins A-I, E, A-IV, M and D were found at higher levels in ultra-cushion EV fractions. EV protein datasets were subjected to functional enrichment analyses using the FunRich tool21. Higher percentage of genes was identified in PROSPR fractions compared to ultra-cushion regarding site of expression (d) and analyzed microvesicle components (e).

Mentions: High concentrations of unwanted plasma proteins in ultra-centrifuged samples are likely to impede the identification of EV-associated proteins and mRNAs with potential for use as disease biomarkers19. We therefore proceeded to use LC-MS/MS label-free proteomics to evaluate the relative levels of contaminating proteins present in the EV fractions obtained by ultra-cushion and PROSPR. When compared with the EV fraction obtained by ultra-cushion, we observed that the PROSPR-separated EV fraction exhibited dramatically reduced levels of serum albumin (ALB) and lower quantities of other plasma high abundant proteins (Fig. 5a). Indeed, albumin is the most abundant protein in human plasma, and the PROSPR-separated EV fractions contained less than 1% of the albumin levels present in the ultra-cushion purified fractions. When subsequently analyzed using the high-throughput Orbitrap mass spectrometers Elite and QExactive (Thermo Scientific Inc., Bremen, Germany), we detected a total of 1539 proteins in the PROSPR-isolated fraction, and a total of 610 proteins in the ultra-cushion EVs fraction (Supplemental Data Set 1). We then compared the percentage of match of these respective obtained datasets with Vesiclepedia, an extensive database containg proteins previously identified in isolated EVs20. The PROSPR-isolated fractions exhibited 90.7% of match with vesiclepedia and the ultra-cushion isolated fractions exhibited 78.0% of match (Fig. 5b). A total of 1396 EV proteins were identified in common between PROSPR and vesiclepedia and a total of 476 EV proteins were identified in common between ultra-cushion and vesiclepedia.


Extracellular vesicles are rapidly purified from human plasma by PRotein Organic Solvent PRecipitation (PROSPR).

Gallart-Palau X, Serra A, Wong AS, Sandin S, Lai MK, Chen CP, Kon OL, Sze SK - Sci Rep (2015)

PROSPR generates higher purity plasma EVs as opposed to conventional ultra-cushion methods.LC-MS/MS label-free quantitation revealed that PROSPR separation yields high purity microvesicle fractions containing lower levels of contaminating plasma proteins when compared with samples obtained by ultra-cushion (a). The list of EV proteins identified by LC-MS/MS in ultra-cushion and PROSPR isolated fractions were compared with the list of human EV proteome contained in vesiclepedia20 as shown by the venn diagrams (b). Percentage of match between ultra-cushion and PROSPR was analyzed; PROSPR revealed higher percentage of match 90.7% compared to ultra-cushion 78.0% with vesiclepedia EV proteins. Cross-contamination in ultra-cushion and PROSPR isolated EV between proteins and high density lipoproteins was analyzed (c) the lipoproteins A-II and C-III were found at higher levels in PROSPR EV fractions whereas the lipoproteins A-I, E, A-IV, M and D were found at higher levels in ultra-cushion EV fractions. EV protein datasets were subjected to functional enrichment analyses using the FunRich tool21. Higher percentage of genes was identified in PROSPR fractions compared to ultra-cushion regarding site of expression (d) and analyzed microvesicle components (e).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4588595&req=5

f5: PROSPR generates higher purity plasma EVs as opposed to conventional ultra-cushion methods.LC-MS/MS label-free quantitation revealed that PROSPR separation yields high purity microvesicle fractions containing lower levels of contaminating plasma proteins when compared with samples obtained by ultra-cushion (a). The list of EV proteins identified by LC-MS/MS in ultra-cushion and PROSPR isolated fractions were compared with the list of human EV proteome contained in vesiclepedia20 as shown by the venn diagrams (b). Percentage of match between ultra-cushion and PROSPR was analyzed; PROSPR revealed higher percentage of match 90.7% compared to ultra-cushion 78.0% with vesiclepedia EV proteins. Cross-contamination in ultra-cushion and PROSPR isolated EV between proteins and high density lipoproteins was analyzed (c) the lipoproteins A-II and C-III were found at higher levels in PROSPR EV fractions whereas the lipoproteins A-I, E, A-IV, M and D were found at higher levels in ultra-cushion EV fractions. EV protein datasets were subjected to functional enrichment analyses using the FunRich tool21. Higher percentage of genes was identified in PROSPR fractions compared to ultra-cushion regarding site of expression (d) and analyzed microvesicle components (e).
Mentions: High concentrations of unwanted plasma proteins in ultra-centrifuged samples are likely to impede the identification of EV-associated proteins and mRNAs with potential for use as disease biomarkers19. We therefore proceeded to use LC-MS/MS label-free proteomics to evaluate the relative levels of contaminating proteins present in the EV fractions obtained by ultra-cushion and PROSPR. When compared with the EV fraction obtained by ultra-cushion, we observed that the PROSPR-separated EV fraction exhibited dramatically reduced levels of serum albumin (ALB) and lower quantities of other plasma high abundant proteins (Fig. 5a). Indeed, albumin is the most abundant protein in human plasma, and the PROSPR-separated EV fractions contained less than 1% of the albumin levels present in the ultra-cushion purified fractions. When subsequently analyzed using the high-throughput Orbitrap mass spectrometers Elite and QExactive (Thermo Scientific Inc., Bremen, Germany), we detected a total of 1539 proteins in the PROSPR-isolated fraction, and a total of 610 proteins in the ultra-cushion EVs fraction (Supplemental Data Set 1). We then compared the percentage of match of these respective obtained datasets with Vesiclepedia, an extensive database containg proteins previously identified in isolated EVs20. The PROSPR-isolated fractions exhibited 90.7% of match with vesiclepedia and the ultra-cushion isolated fractions exhibited 78.0% of match (Fig. 5b). A total of 1396 EV proteins were identified in common between PROSPR and vesiclepedia and a total of 476 EV proteins were identified in common between ultra-cushion and vesiclepedia.

Bottom Line: Here, we report a novel and inexpensive method of rapidly isolating EVs from small volumes of human blood plasma by PRotein Organic Solvent PRecipitation (PROSPR).PROSPR encompasses a rapid three-step protocol to remove soluble proteins from plasma via precipitation in cold acetone, leaving the lipid-encapsulated EVs behind in suspension.We foresee that PROSPR-based purification of EVs will significantly accelerate the discovery of new disease biomarkers and the characterization of EVs with potential for clinical applications.

View Article: PubMed Central - PubMed

Affiliation: School of Biological Sciences, Nanyang Technological University, Singapore, 637551.

ABSTRACT
Extracellular vesicles (EVs) such as exosomes and microvesicles mediate intercellular communication and regulate a diverse range of crucial biological processes. Host cells that are damaged, infected or transformed release biomarker-containing EVs into the peripheral circulation, where they can be readily accessed for use in diagnostic or prognostic testing. However, current methods of EV isolation from blood plasma are complex and often require relatively large sample volumes, hence are inefficient for widespread use in clinical settings. Here, we report a novel and inexpensive method of rapidly isolating EVs from small volumes of human blood plasma by PRotein Organic Solvent PRecipitation (PROSPR). PROSPR encompasses a rapid three-step protocol to remove soluble proteins from plasma via precipitation in cold acetone, leaving the lipid-encapsulated EVs behind in suspension. This generates higher purity EVs that can then be obtained from filtration or classical ultracentrifugation methods. We foresee that PROSPR-based purification of EVs will significantly accelerate the discovery of new disease biomarkers and the characterization of EVs with potential for clinical applications.

No MeSH data available.


Related in: MedlinePlus