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.