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Size-exclusion chromatography as a stand-alone methodology identifies novel markers in mass spectrometry analyses of plasma-derived vesicles from healthy individuals.

de Menezes-Neto A, Sáez MJ, Lozano-Ramos I, Segui-Barber J, Martin-Jaular L, Ullate JM, Fernandez-Becerra C, Borrás FE, Del Portillo HA - J Extracell Vesicles (2015)

Bottom Line: In this study, we have addressed both challenges by carrying-out mass spectrometry (MS) analyses of plasma-derived vesicles, in the size range of exosomes, from healthy donors obtained by 2 alternative methodologies: size-exclusion chromatography (SEC) on sepharose columns and Exo-Spin™.Noticeably, after a cross-comparative analysis of all published studies using MS to characterize plasma-derived exosomes from healthy individuals, we also observed a paucity of "classical exosome markers." Independent of the isolation method, however, we consistently identified 2 proteins, CD5 antigen-like (CD5L) and galectin-3-binding protein (LGALS3BP), whose presence was validated by a bead-exosome FACS assay.Altogether, our results support the use of SEC as a stand-alone methodology to obtain preparations of extracellular vesicles, in the size range of exosomes, from plasma and suggest the use of CD5L and LGALS3BP as more suitable markers of plasma-derived vesicles in MS.

View Article: PubMed Central - PubMed

Affiliation: ISGlobal, Barcelona Centre for International Health Research (CRESIB), Hospital Clínic - Universitat de Barcelona, Barcelona, Spain.

ABSTRACT
Plasma-derived vesicles hold a promising potential for use in biomedical applications. Two major challenges, however, hinder their implementation into translational tools: (a) the incomplete characterization of the protein composition of plasma-derived vesicles, in the size range of exosomes, as mass spectrometric analysis of plasma sub-components is recognizably troublesome and (b) the limited reach of vesicle-based studies in settings where the infrastructural demand of ultracentrifugation, the most widely used isolation/purification methodology, is not available. In this study, we have addressed both challenges by carrying-out mass spectrometry (MS) analyses of plasma-derived vesicles, in the size range of exosomes, from healthy donors obtained by 2 alternative methodologies: size-exclusion chromatography (SEC) on sepharose columns and Exo-Spin™. No exosome markers, as opposed to the most abundant plasma proteins, were detected by Exo-Spin™. In contrast, exosomal markers were present in the early fractions of SEC where the most abundant plasma proteins have been largely excluded. Noticeably, after a cross-comparative analysis of all published studies using MS to characterize plasma-derived exosomes from healthy individuals, we also observed a paucity of "classical exosome markers." Independent of the isolation method, however, we consistently identified 2 proteins, CD5 antigen-like (CD5L) and galectin-3-binding protein (LGALS3BP), whose presence was validated by a bead-exosome FACS assay. Altogether, our results support the use of SEC as a stand-alone methodology to obtain preparations of extracellular vesicles, in the size range of exosomes, from plasma and suggest the use of CD5L and LGALS3BP as more suitable markers of plasma-derived vesicles in MS.

No MeSH data available.


Related in: MedlinePlus

Isolation of plasma-derived exosomes by size-exclusion chromatography. An aliquot (1 mL) of undiluted plasma from Donor 1 was passed through a sepharose (CL-2B) column, and 30 fractions of 0.5 mL each were collected. (a) SDS–PAGE stained with silver and protein concentration values of fractions 6–14 were measured by Bradford assay (fractions 6, 7 and 8 were below the lower limit of detection) and fractions 6–9 were analysed by flow cytometry, after coupling of vesicles to 4 µm latex beads, for the presence of antigens CD9 (1:10) and CD81 (1:10). The secondary anti-mouse antibody was conjugated to FITC and was used at a 1:100 dilution. MFI: mean fluorescence intensity. (b) Fractions 7–11 were submitted to NTA on a NanoSight LM10 (software version 3.0).
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Figure 0002: Isolation of plasma-derived exosomes by size-exclusion chromatography. An aliquot (1 mL) of undiluted plasma from Donor 1 was passed through a sepharose (CL-2B) column, and 30 fractions of 0.5 mL each were collected. (a) SDS–PAGE stained with silver and protein concentration values of fractions 6–14 were measured by Bradford assay (fractions 6, 7 and 8 were below the lower limit of detection) and fractions 6–9 were analysed by flow cytometry, after coupling of vesicles to 4 µm latex beads, for the presence of antigens CD9 (1:10) and CD81 (1:10). The secondary anti-mouse antibody was conjugated to FITC and was used at a 1:100 dilution. MFI: mean fluorescence intensity. (b) Fractions 7–11 were submitted to NTA on a NanoSight LM10 (software version 3.0).

Mentions: Aiming to exclude abundant plasma proteins, the 3 preparations were processed for vesicle isolation by SEC. Upon chromatographic fractionation, vesicles should elute in early fractions, prior to the bulk of plasma proteins (11). We thus performed electrophoresis, protein quantification, NTA analysis and flow cytometry for early fractions. As illustrated with data from donor “1,” the complexity and total amount of protein steadily increased as the separation progressed (Fig. 2a). Moreover, a bead-exosome flow cytometry assay (8) revealed that the highest MFIs for CD9 or CD81, 2 tetraspanins considered “classical” exosomal markers, were observed in fraction 8 (Fig. 2a). In addition, as detected by NTA, particles within the expected size range of exosomes began to elute in higher concentration from fraction 8 onwards (Fig. 2b). Remarkably, neither NTA nor flow cytometry results held any correlation to the protein content of the corresponding fractions. Thus, while virtually no protein was detected until fraction 8, it is clear that from fraction 11 onwards there was a disproportional accumulation of proteins, mainly in the mass range of 55–70 kDa, which encloses the molecular weight of serum albumin (66 kDa), the most abundant protein of plasma. Results from donors “2” and “3” were similar (Supplementary Fig. 1). Altogether, these results demonstrate that chromatographic isolation efficiently separates vesicles from non-related abundant plasma proteins that would otherwise contaminate the preparations.


Size-exclusion chromatography as a stand-alone methodology identifies novel markers in mass spectrometry analyses of plasma-derived vesicles from healthy individuals.

de Menezes-Neto A, Sáez MJ, Lozano-Ramos I, Segui-Barber J, Martin-Jaular L, Ullate JM, Fernandez-Becerra C, Borrás FE, Del Portillo HA - J Extracell Vesicles (2015)

Isolation of plasma-derived exosomes by size-exclusion chromatography. An aliquot (1 mL) of undiluted plasma from Donor 1 was passed through a sepharose (CL-2B) column, and 30 fractions of 0.5 mL each were collected. (a) SDS–PAGE stained with silver and protein concentration values of fractions 6–14 were measured by Bradford assay (fractions 6, 7 and 8 were below the lower limit of detection) and fractions 6–9 were analysed by flow cytometry, after coupling of vesicles to 4 µm latex beads, for the presence of antigens CD9 (1:10) and CD81 (1:10). The secondary anti-mouse antibody was conjugated to FITC and was used at a 1:100 dilution. MFI: mean fluorescence intensity. (b) Fractions 7–11 were submitted to NTA on a NanoSight LM10 (software version 3.0).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4495624&req=5

Figure 0002: Isolation of plasma-derived exosomes by size-exclusion chromatography. An aliquot (1 mL) of undiluted plasma from Donor 1 was passed through a sepharose (CL-2B) column, and 30 fractions of 0.5 mL each were collected. (a) SDS–PAGE stained with silver and protein concentration values of fractions 6–14 were measured by Bradford assay (fractions 6, 7 and 8 were below the lower limit of detection) and fractions 6–9 were analysed by flow cytometry, after coupling of vesicles to 4 µm latex beads, for the presence of antigens CD9 (1:10) and CD81 (1:10). The secondary anti-mouse antibody was conjugated to FITC and was used at a 1:100 dilution. MFI: mean fluorescence intensity. (b) Fractions 7–11 were submitted to NTA on a NanoSight LM10 (software version 3.0).
Mentions: Aiming to exclude abundant plasma proteins, the 3 preparations were processed for vesicle isolation by SEC. Upon chromatographic fractionation, vesicles should elute in early fractions, prior to the bulk of plasma proteins (11). We thus performed electrophoresis, protein quantification, NTA analysis and flow cytometry for early fractions. As illustrated with data from donor “1,” the complexity and total amount of protein steadily increased as the separation progressed (Fig. 2a). Moreover, a bead-exosome flow cytometry assay (8) revealed that the highest MFIs for CD9 or CD81, 2 tetraspanins considered “classical” exosomal markers, were observed in fraction 8 (Fig. 2a). In addition, as detected by NTA, particles within the expected size range of exosomes began to elute in higher concentration from fraction 8 onwards (Fig. 2b). Remarkably, neither NTA nor flow cytometry results held any correlation to the protein content of the corresponding fractions. Thus, while virtually no protein was detected until fraction 8, it is clear that from fraction 11 onwards there was a disproportional accumulation of proteins, mainly in the mass range of 55–70 kDa, which encloses the molecular weight of serum albumin (66 kDa), the most abundant protein of plasma. Results from donors “2” and “3” were similar (Supplementary Fig. 1). Altogether, these results demonstrate that chromatographic isolation efficiently separates vesicles from non-related abundant plasma proteins that would otherwise contaminate the preparations.

Bottom Line: In this study, we have addressed both challenges by carrying-out mass spectrometry (MS) analyses of plasma-derived vesicles, in the size range of exosomes, from healthy donors obtained by 2 alternative methodologies: size-exclusion chromatography (SEC) on sepharose columns and Exo-Spin™.Noticeably, after a cross-comparative analysis of all published studies using MS to characterize plasma-derived exosomes from healthy individuals, we also observed a paucity of "classical exosome markers." Independent of the isolation method, however, we consistently identified 2 proteins, CD5 antigen-like (CD5L) and galectin-3-binding protein (LGALS3BP), whose presence was validated by a bead-exosome FACS assay.Altogether, our results support the use of SEC as a stand-alone methodology to obtain preparations of extracellular vesicles, in the size range of exosomes, from plasma and suggest the use of CD5L and LGALS3BP as more suitable markers of plasma-derived vesicles in MS.

View Article: PubMed Central - PubMed

Affiliation: ISGlobal, Barcelona Centre for International Health Research (CRESIB), Hospital Clínic - Universitat de Barcelona, Barcelona, Spain.

ABSTRACT
Plasma-derived vesicles hold a promising potential for use in biomedical applications. Two major challenges, however, hinder their implementation into translational tools: (a) the incomplete characterization of the protein composition of plasma-derived vesicles, in the size range of exosomes, as mass spectrometric analysis of plasma sub-components is recognizably troublesome and (b) the limited reach of vesicle-based studies in settings where the infrastructural demand of ultracentrifugation, the most widely used isolation/purification methodology, is not available. In this study, we have addressed both challenges by carrying-out mass spectrometry (MS) analyses of plasma-derived vesicles, in the size range of exosomes, from healthy donors obtained by 2 alternative methodologies: size-exclusion chromatography (SEC) on sepharose columns and Exo-Spin™. No exosome markers, as opposed to the most abundant plasma proteins, were detected by Exo-Spin™. In contrast, exosomal markers were present in the early fractions of SEC where the most abundant plasma proteins have been largely excluded. Noticeably, after a cross-comparative analysis of all published studies using MS to characterize plasma-derived exosomes from healthy individuals, we also observed a paucity of "classical exosome markers." Independent of the isolation method, however, we consistently identified 2 proteins, CD5 antigen-like (CD5L) and galectin-3-binding protein (LGALS3BP), whose presence was validated by a bead-exosome FACS assay. Altogether, our results support the use of SEC as a stand-alone methodology to obtain preparations of extracellular vesicles, in the size range of exosomes, from plasma and suggest the use of CD5L and LGALS3BP as more suitable markers of plasma-derived vesicles in MS.

No MeSH data available.


Related in: MedlinePlus