title: Size-exclusion chromatography as a stand-alone methodology identifies novel markers in mass spectrometry analyses of plasma-derived vesicles from healthy individuals.
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.
authors.affiliation: ISGlobal, Barcelona Centre for International Health Research (CRESIB), Hospital Clínic - Universitat de Barcelona, Barcelona, Spain.
authors.forename: Armando
authors.initials: A
authors.lastname: de Menezes-Neto
authors.affiliation: Proteomic Unit from Scientific and Technological Centers, University of Barcelona (CCIT-UB), Barcelona, Spain.
authors.forename: María José Fidalgo
authors.initials: MJ
authors.lastname: Sáez
authors.affiliation: IVECAT Group, Germans Trias i Pujol Research Institute (IGTP), Badalona, Spain.
authors.forename: Inés
authors.initials: I
authors.lastname: Lozano-Ramos
authors.affiliation: ISGlobal, Barcelona Centre for International Health Research (CRESIB), Hospital Clínic - Universitat de Barcelona, Barcelona, Spain.
authors.forename: Joan
authors.initials: J
authors.lastname: Segui-Barber
authors.affiliation: ISGlobal, Barcelona Centre for International Health Research (CRESIB), Hospital Clínic - Universitat de Barcelona, Barcelona, Spain.
authors.forename: Lorena
authors.initials: L
authors.lastname: Martin-Jaular
authors.affiliation: Proteomic Unit from Scientific and Technological Centers, University of Barcelona (CCIT-UB), Barcelona, Spain.
authors.forename: Josep M Estanyol
authors.initials: JM
authors.lastname: Ullate
authors.affiliation: ISGlobal, Barcelona Centre for International Health Research (CRESIB), Hospital Clínic - Universitat de Barcelona, Barcelona, Spain.
authors.forename: Carmen
authors.initials: C
authors.lastname: Fernandez-Becerra
authors.affiliation: IVECAT Group, Germans Trias i Pujol Research Institute (IGTP), Badalona, Spain.|Nephrology Service, Germans Trias i Pujol University Hospital, Badalona, Spain.
authors.forename: Francesc E
authors.initials: FE
authors.lastname: Borrás
authors.affiliation: ISGlobal, Barcelona Centre for International Health Research (CRESIB), Hospital Clínic - Universitat de Barcelona, Barcelona, Spain.|Institució Catalana de Recerca i Estudis Avançats (ICREA), Barcelona, Spain; hernandoa.delportillo@cresib.cat.
authors.forename: Hernando A
authors.initials: HA
authors.lastname: Del Portillo
keywords: CD5L; LGALS3BP; comparative analysis; exosomes; low infrastructure settings; mass spectrometry; plasma-derived exosomes