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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

Plasma-derived exosomes isolated by Exo-Spin™ from the plasma of 3 healthy donors. (a) Electrophoretic profile of proteins on silver-stained polyacrylamide gels. The following amount of proteins were loaded onto the gels: 1.05 µg (1); 1.14 µg (2) and 4.6 µg (3). (b) NTA was performed on a NanoSight LM10 (software version 2.3) after dilution of samples in PBS. (c) Venn diagram showing the overlap of proteins detected by LC–MS/MS in each preparation (red: “Preparation from donor 1”; green: “Preparation from donor 2”; blue: “Preparation from donor 3”).
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Figure 0001: Plasma-derived exosomes isolated by Exo-Spin™ from the plasma of 3 healthy donors. (a) Electrophoretic profile of proteins on silver-stained polyacrylamide gels. The following amount of proteins were loaded onto the gels: 1.05 µg (1); 1.14 µg (2) and 4.6 µg (3). (b) NTA was performed on a NanoSight LM10 (software version 2.3) after dilution of samples in PBS. (c) Venn diagram showing the overlap of proteins detected by LC–MS/MS in each preparation (red: “Preparation from donor 1”; green: “Preparation from donor 2”; blue: “Preparation from donor 3”).

Mentions: Electrophoresis of the protein content of the vesicles isolated by Exo-Spin™ revealed that the 3 preparations shared a comparable complex mixture of proteins (Fig. 1a). Moreover, NTA revealed that preparations from donors “1” and “2” were highly homogeneous in terms of particle size (97 and 89 nm) and concentration (1.54×108 and 1.49×108 particles/µL), whereas the preparation from donor “3” contained bigger modal particle size (127 nm) and a lower concentration (0.98×108 particles/µL; Fig. 1b). Vesicle isolation from donor “3” was performed from frozen plasma (6 days), and perhaps it could account for the observed variability in this sample. In spite of these apparent differences in size, MS of Exo-Spin™ isolated vesicles revealed a substantial overlapping of identified proteins among all 3 preparations (Supplementary Table 3). Thus, from the total 152 detected proteins, 77 were common to all 3 preparations and 112 were found in at least 2 out of 3 samples (Fig. 1c). Strikingly, no exosome markers were detected in the Exo-Spin™ preparations, whereas several of the most abundant plasma proteins such as serum albumin, immunoglobulins, fibrinogen, plasminogen and apolipoproteins were. These results suggest that, although vesicles isolated by Exo-Spin™ were within the expected size range of exosomes according to NTA data, the carryover of abundant plasma proteins might have masked the detection of the less abundant proteins, including those that are classically associated to exosomes.


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)

Plasma-derived exosomes isolated by Exo-Spin™ from the plasma of 3 healthy donors. (a) Electrophoretic profile of proteins on silver-stained polyacrylamide gels. The following amount of proteins were loaded onto the gels: 1.05 µg (1); 1.14 µg (2) and 4.6 µg (3). (b) NTA was performed on a NanoSight LM10 (software version 2.3) after dilution of samples in PBS. (c) Venn diagram showing the overlap of proteins detected by LC–MS/MS in each preparation (red: “Preparation from donor 1”; green: “Preparation from donor 2”; blue: “Preparation from donor 3”).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 0001: Plasma-derived exosomes isolated by Exo-Spin™ from the plasma of 3 healthy donors. (a) Electrophoretic profile of proteins on silver-stained polyacrylamide gels. The following amount of proteins were loaded onto the gels: 1.05 µg (1); 1.14 µg (2) and 4.6 µg (3). (b) NTA was performed on a NanoSight LM10 (software version 2.3) after dilution of samples in PBS. (c) Venn diagram showing the overlap of proteins detected by LC–MS/MS in each preparation (red: “Preparation from donor 1”; green: “Preparation from donor 2”; blue: “Preparation from donor 3”).
Mentions: Electrophoresis of the protein content of the vesicles isolated by Exo-Spin™ revealed that the 3 preparations shared a comparable complex mixture of proteins (Fig. 1a). Moreover, NTA revealed that preparations from donors “1” and “2” were highly homogeneous in terms of particle size (97 and 89 nm) and concentration (1.54×108 and 1.49×108 particles/µL), whereas the preparation from donor “3” contained bigger modal particle size (127 nm) and a lower concentration (0.98×108 particles/µL; Fig. 1b). Vesicle isolation from donor “3” was performed from frozen plasma (6 days), and perhaps it could account for the observed variability in this sample. In spite of these apparent differences in size, MS of Exo-Spin™ isolated vesicles revealed a substantial overlapping of identified proteins among all 3 preparations (Supplementary Table 3). Thus, from the total 152 detected proteins, 77 were common to all 3 preparations and 112 were found in at least 2 out of 3 samples (Fig. 1c). Strikingly, no exosome markers were detected in the Exo-Spin™ preparations, whereas several of the most abundant plasma proteins such as serum albumin, immunoglobulins, fibrinogen, plasminogen and apolipoproteins were. These results suggest that, although vesicles isolated by Exo-Spin™ were within the expected size range of exosomes according to NTA data, the carryover of abundant plasma proteins might have masked the detection of the less abundant proteins, including those that are classically associated to exosomes.

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