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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

CD5L and LGALS3BP have similar elution profiles to the exosome marker CD81. Plasma from “Donor 1” was submitted to size-exclusion chromatography and fractions 6–12 were analysed by NTA, flow cytometry and transmission electron microscopy (TEM). (a) NTA was performed on a NanoSight LM10 (software version 3.0). For flow cytometry, samples were coupled to 4 µm beads and incubated with primary antibodies against CD81 (1:10), CD5L (1:100) or LGALS3BP (1:1,000). The secondary antibody was conjugated to Alexa 488 was used at a 1:1,000 dilution. MFI: mean fluorescence intensity. (b) Fraction 6 from size-exclusion chromatography was submitted to cryo-EM and (c) immunostained with anti-CD5L antibodies conjugated to gold spheres of 20 nm.
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Figure 0006: CD5L and LGALS3BP have similar elution profiles to the exosome marker CD81. Plasma from “Donor 1” was submitted to size-exclusion chromatography and fractions 6–12 were analysed by NTA, flow cytometry and transmission electron microscopy (TEM). (a) NTA was performed on a NanoSight LM10 (software version 3.0). For flow cytometry, samples were coupled to 4 µm beads and incubated with primary antibodies against CD81 (1:10), CD5L (1:100) or LGALS3BP (1:1,000). The secondary antibody was conjugated to Alexa 488 was used at a 1:1,000 dilution. MFI: mean fluorescence intensity. (b) Fraction 6 from size-exclusion chromatography was submitted to cryo-EM and (c) immunostained with anti-CD5L antibodies conjugated to gold spheres of 20 nm.

Mentions: Despite the many limitations that prevent drawing a clear comparison among these data sets, it was noteworthy that there was a small set of proteins that were more frequently identified and listed in most of the preparations irrespective of the isolation method (Fig. 5, red box). As expected, most of the proteins in this subset are recognizably plasma contaminants such as albumin, fibrinogen, apolipoproteins and complement factors. Yet, 2 of these proteins, CD5 antigen-like (CD5L) and galectin-3 binding protein (LGALS3BP), were consistently detected and had already been associated to exosomes and EVs in previous studies (6, 27). To demonstrate the presence of these proteins in our samples, we obtained the FACS profiles of CD5L and LGALS3BP throughout the chromatographic separation of plasma and compared them to the profile of CD81 (Fig. 6a and Supplementary Fig. 2: Characterization of markers present on exosomes coated to latex beads by FACs analysis). As shown in Fig. 6a, the expression profiles for CD5L and LGALS3BP were almost identical to that of CD81. Vesicles on fraction 6 were then submitted to transmission electron microscopy and their size as well as their morphology was confirmed (Fig. 6b). Moreover, immunoelectron microscopy indicates that CD5L is found in the surface of these vesicles (Fig. 6c). These results strongly suggest that CD5L and LGALS3BP are also associated to exosomes. Considering that CD81 has not been detected by MS in any of the plasma-derived exosome preparations, CD5L and LGALS3BP could be considered as surrogate markers for future studies involving MS-based proteomic analysis of vesicles derived from plasma.


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)

CD5L and LGALS3BP have similar elution profiles to the exosome marker CD81. Plasma from “Donor 1” was submitted to size-exclusion chromatography and fractions 6–12 were analysed by NTA, flow cytometry and transmission electron microscopy (TEM). (a) NTA was performed on a NanoSight LM10 (software version 3.0). For flow cytometry, samples were coupled to 4 µm beads and incubated with primary antibodies against CD81 (1:10), CD5L (1:100) or LGALS3BP (1:1,000). The secondary antibody was conjugated to Alexa 488 was used at a 1:1,000 dilution. MFI: mean fluorescence intensity. (b) Fraction 6 from size-exclusion chromatography was submitted to cryo-EM and (c) immunostained with anti-CD5L antibodies conjugated to gold spheres of 20 nm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 0006: CD5L and LGALS3BP have similar elution profiles to the exosome marker CD81. Plasma from “Donor 1” was submitted to size-exclusion chromatography and fractions 6–12 were analysed by NTA, flow cytometry and transmission electron microscopy (TEM). (a) NTA was performed on a NanoSight LM10 (software version 3.0). For flow cytometry, samples were coupled to 4 µm beads and incubated with primary antibodies against CD81 (1:10), CD5L (1:100) or LGALS3BP (1:1,000). The secondary antibody was conjugated to Alexa 488 was used at a 1:1,000 dilution. MFI: mean fluorescence intensity. (b) Fraction 6 from size-exclusion chromatography was submitted to cryo-EM and (c) immunostained with anti-CD5L antibodies conjugated to gold spheres of 20 nm.
Mentions: Despite the many limitations that prevent drawing a clear comparison among these data sets, it was noteworthy that there was a small set of proteins that were more frequently identified and listed in most of the preparations irrespective of the isolation method (Fig. 5, red box). As expected, most of the proteins in this subset are recognizably plasma contaminants such as albumin, fibrinogen, apolipoproteins and complement factors. Yet, 2 of these proteins, CD5 antigen-like (CD5L) and galectin-3 binding protein (LGALS3BP), were consistently detected and had already been associated to exosomes and EVs in previous studies (6, 27). To demonstrate the presence of these proteins in our samples, we obtained the FACS profiles of CD5L and LGALS3BP throughout the chromatographic separation of plasma and compared them to the profile of CD81 (Fig. 6a and Supplementary Fig. 2: Characterization of markers present on exosomes coated to latex beads by FACs analysis). As shown in Fig. 6a, the expression profiles for CD5L and LGALS3BP were almost identical to that of CD81. Vesicles on fraction 6 were then submitted to transmission electron microscopy and their size as well as their morphology was confirmed (Fig. 6b). Moreover, immunoelectron microscopy indicates that CD5L is found in the surface of these vesicles (Fig. 6c). These results strongly suggest that CD5L and LGALS3BP are also associated to exosomes. Considering that CD81 has not been detected by MS in any of the plasma-derived exosome preparations, CD5L and LGALS3BP could be considered as surrogate markers for future studies involving MS-based proteomic analysis of vesicles derived from plasma.

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