Size‐exclusion chromatography as a stand‐alone methodology identifies novel markers in mass spectrometry analyses of plasma‐derived vesicles from healthy individuals

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.