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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

Proteomic analysis of samples from donor 1. Mass spectrometry was performed on 4 preparations from the same plasma sample: (i) chromatographic fractions 7+8, (ii) chromatographic fractions 9+10, (iii) chromatographic fraction 11 and (iv) Exo-Spin™. (a) Venn diagram showing the overlap of proteins detected by nanoLC–MS/MS. (b) The relative abundance (NSAF) of the exosome markers detected in the data set of fractions 7+8 (in red) and of exosome markers identified by single peptides (orange) were compared to the relative abundance of the remaining proteins for the same sample. (c) The overall relative abundance of all proteins in each sample plotted in descending order. NSAF=normalized spectrum abundance factor.
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Figure 0003: Proteomic analysis of samples from donor 1. Mass spectrometry was performed on 4 preparations from the same plasma sample: (i) chromatographic fractions 7+8, (ii) chromatographic fractions 9+10, (iii) chromatographic fraction 11 and (iv) Exo-Spin™. (a) Venn diagram showing the overlap of proteins detected by nanoLC–MS/MS. (b) The relative abundance (NSAF) of the exosome markers detected in the data set of fractions 7+8 (in red) and of exosome markers identified by single peptides (orange) were compared to the relative abundance of the remaining proteins for the same sample. (c) The overall relative abundance of all proteins in each sample plotted in descending order. NSAF=normalized spectrum abundance factor.

Mentions: To further evaluate the impact of chromatography on the separation of vesicles from free plasma proteins, we have followed the progression of the fractionation by MS through the analysis of consecutive fractions from donor “1.” Based on flow cytometry and NTA results, we have prepared 3 samples: (a) the mix (1:1) of fractions 7 and 8, (b) fractions 9 and 10 and (c) fraction 11. The set of identified proteins were compared among the 3 preparations and also to the Exo-Spin™ preparation from the same donor (Fig. 3a). In total, 269 proteins were detected from donor “1,” and a set of 24 proteins were found in all 4 preparations, most of these shared proteins are known plasma components such as immunoglobulins, complement factors, apolipoproteins, fibrinogen and albumin. Among the SEC-derived preparations, “fractions 7+8” was the one featuring the highest number of unique hits and also the one sharing the least number of proteins with “Exo-Spin.” Of interest, in “fractions 7+8,” and only in this sample, the exosome markers actin (UniProt: ACTB), moesin (UniProt: MSN) and 2 proteins from the 14-3-3 family (UniProt: YWHAB and YWHAZ) were detected (Supplementary Tables 3–5). Moreover, if MS protein identification parameters were lowered to encompass proteins identified by only one high confidence peptide, 3 additional exosome markers: annexin A2 (UniProt: ANXA2), Ras-related protein Rap-1b (UniProt: RAP1B) and glyceraldehyde-3-phosphate dehydrogenase (UniProt: GAPDH) were also detected (Supplementary Table 6and Supplementary Fig. 2). Of importance, even under these more permissive MS parameters, no exosomal markers were detected in the remaining samples. To address this question, we have calculated the relative abundance of proteins within each sample as a normalized function of their spectra counts. In “fractions 7+8,” the relative abundances of the exosome markers support the notion that these proteins are in low abundance (Fig. 3b). Interestingly, when the overall relative abundance of the entire protein sets was compared among the 4 samples it was evident that “fractions 7+8” had the most balanced distribution of proteins, in sharp contrast to “Exo-Spin” (Fig. 3c). The interpretation is that, in “fractions 7+8,” optimal conditions were met for the MS-based detection of proteins classically associated with exosomes. At this point, vesicles were already eluting while plasma proteins were mostly trapped in the column, and even though plasma proteins were still detected, their detection levels were not disproportionately high. On the other hand, in the consecutive fractions “9+10” and “11” and in the Exo-Spin™ preparation, a few proteins, in particular immunoglobulins, were over-represented and possibly interfered in the detection of less abundant proteins.


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)

Proteomic analysis of samples from donor 1. Mass spectrometry was performed on 4 preparations from the same plasma sample: (i) chromatographic fractions 7+8, (ii) chromatographic fractions 9+10, (iii) chromatographic fraction 11 and (iv) Exo-Spin™. (a) Venn diagram showing the overlap of proteins detected by nanoLC–MS/MS. (b) The relative abundance (NSAF) of the exosome markers detected in the data set of fractions 7+8 (in red) and of exosome markers identified by single peptides (orange) were compared to the relative abundance of the remaining proteins for the same sample. (c) The overall relative abundance of all proteins in each sample plotted in descending order. NSAF=normalized spectrum abundance factor.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 0003: Proteomic analysis of samples from donor 1. Mass spectrometry was performed on 4 preparations from the same plasma sample: (i) chromatographic fractions 7+8, (ii) chromatographic fractions 9+10, (iii) chromatographic fraction 11 and (iv) Exo-Spin™. (a) Venn diagram showing the overlap of proteins detected by nanoLC–MS/MS. (b) The relative abundance (NSAF) of the exosome markers detected in the data set of fractions 7+8 (in red) and of exosome markers identified by single peptides (orange) were compared to the relative abundance of the remaining proteins for the same sample. (c) The overall relative abundance of all proteins in each sample plotted in descending order. NSAF=normalized spectrum abundance factor.
Mentions: To further evaluate the impact of chromatography on the separation of vesicles from free plasma proteins, we have followed the progression of the fractionation by MS through the analysis of consecutive fractions from donor “1.” Based on flow cytometry and NTA results, we have prepared 3 samples: (a) the mix (1:1) of fractions 7 and 8, (b) fractions 9 and 10 and (c) fraction 11. The set of identified proteins were compared among the 3 preparations and also to the Exo-Spin™ preparation from the same donor (Fig. 3a). In total, 269 proteins were detected from donor “1,” and a set of 24 proteins were found in all 4 preparations, most of these shared proteins are known plasma components such as immunoglobulins, complement factors, apolipoproteins, fibrinogen and albumin. Among the SEC-derived preparations, “fractions 7+8” was the one featuring the highest number of unique hits and also the one sharing the least number of proteins with “Exo-Spin.” Of interest, in “fractions 7+8,” and only in this sample, the exosome markers actin (UniProt: ACTB), moesin (UniProt: MSN) and 2 proteins from the 14-3-3 family (UniProt: YWHAB and YWHAZ) were detected (Supplementary Tables 3–5). Moreover, if MS protein identification parameters were lowered to encompass proteins identified by only one high confidence peptide, 3 additional exosome markers: annexin A2 (UniProt: ANXA2), Ras-related protein Rap-1b (UniProt: RAP1B) and glyceraldehyde-3-phosphate dehydrogenase (UniProt: GAPDH) were also detected (Supplementary Table 6and Supplementary Fig. 2). Of importance, even under these more permissive MS parameters, no exosomal markers were detected in the remaining samples. To address this question, we have calculated the relative abundance of proteins within each sample as a normalized function of their spectra counts. In “fractions 7+8,” the relative abundances of the exosome markers support the notion that these proteins are in low abundance (Fig. 3b). Interestingly, when the overall relative abundance of the entire protein sets was compared among the 4 samples it was evident that “fractions 7+8” had the most balanced distribution of proteins, in sharp contrast to “Exo-Spin” (Fig. 3c). The interpretation is that, in “fractions 7+8,” optimal conditions were met for the MS-based detection of proteins classically associated with exosomes. At this point, vesicles were already eluting while plasma proteins were mostly trapped in the column, and even though plasma proteins were still detected, their detection levels were not disproportionately high. On the other hand, in the consecutive fractions “9+10” and “11” and in the Exo-Spin™ preparation, a few proteins, in particular immunoglobulins, were over-represented and possibly interfered in the detection of less abundant proteins.

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