Size-exclusion chromatography identifies novel markers in plasma-derived vesicles

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, researchers at the Universitat de Barcelona 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, they also observed a paucity of “classical exosome markers.” Independent of the isolation method, however, the researchers 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, these 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.

exosome rna

Exosome markers in plasma-derived exosomal preparations. (a) Mass spectrometry results from Exo-Spin and SEC samples (Preparation 1) were compared to previous studies that have isolated exosomes by other methodologies. The total number of identified proteins, of abundant plasma proteins and of exosome markers, is shown. (b) A panel of 34 exosome markers are compared between high-throughput proteomic data sets of plasma-derived exosomes (n = 13) and non-plasma-derived exosomes retrieved from ExoCarta (n = 67). Statistical significance was tested for with Mann–Whitney–Wicoxon test for non-parametric distributions at a level of significance of 5% (p-value < 0.05). SEC: size-exclusion chromatography, FPLC: fast protein liquid chromatography, SCG: continuous sucrose gradient, UC: ultracentrifugation, Filt: filtration at 0.22 or 0.1 µm, SucC: sucrose cushion, DG: density gradient, Ab-I: antibody incubation, MCS: magnetic column separation, ¥according to data extracted from ExoCarta for previous publications, §list of common plasma proteins.

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. (2015) Size-exclusion chromatography as a stand-alone methodology identifies novel markers in mass spectrometry analyses of plasma-derived vesicles from healthy individuals. J Extracell Vesicles 4:27378. [article]

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