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

• Published in: Journal of extracellular vesicles Vol. 4; no. 1; pp. 27378 - n/a
• Main Authors: de Menezes-Neto, Armando, Sáez, María José Fidalgo, Lozano-Ramos, Inés, Segui-Barber, Joan, Martin-Jaular, Lorena, Ullate, Josep M. Estanyol, Fernandez-Becerra, Carmen, Borrás, Francesc E., del Portillo, Hernando A.
• Format: Journal Article
• Language: English
• Published: Sweden Taylor & Francis 01.01.2015; John Wiley & Sons, Inc; Co-Action Publishing; Wiley
• Subjects: Antibodies; Antigens; Blood platelets; CD5 antigen; CD5L; Cell adhesion & migration; Chromatography; Cloning; Comparative analysis; Exosomes; Flow cytometry; Funding; Hospitals; LGALS3BP; low infrastructure settings; Mass spectrometry; Mass spectroscopy; Medical research; Nanoparticles; Original; Plasma; Plasma proteins; plasma-derived exosomes; Protein composition; Protein purification; Proteins; Proteomics; Scientific imaging; Solvents; Studies; Ultracentrifugation; Vesicles; Spain; United Kingdom > UK; United States > US; LGALS3BP; plasma-derived exosomes; comparative analysis; exosomes; CD5L; mass spectrometry; low infrastructure settings
• ISSN: 2001-3078; 2001-3078
• DOI: 10.3402/jev.v4.27378

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.