Exosomes are membranous nanovesicles of endocytic origin that carry and transfer regulatory bioactive molecules and mediate intercellular communication between cells and tissues. Although seminal exosomes have been identified in human seminal plasma, their exact composition and possible physiologic function remain unknown. The objective of this study was to perform a comprehensive proteomics analysis of exosomes derived from human seminal plasma. Seminal exosomes were isolated and purified from 12 healthy donors using a 30%
sucrose cushion-based exosome-isolation protocol, followed by characterization by western blot, transmission electron microscopy, and nanoparticle tracking analysis before performing extensive liquid chromatography tandem mass spectrometry proteomics analysis. The identified
proteins were analyzed by bioinformatics analysis, and seminal exosomes-associated
proteins were selectively validated by western blot. A total of 1474
proteins were identified in all seminal exosomes samples, with Gene Ontology analysis demonstrating that these identified seminal exosomes-associated
proteins were mostly linked to 'exosomes,' 'cytoplasm,' and 'cytosol.' Bioinformatics analysis indicated that these
proteins were mainly involved in biologic processes, including metabolism, energy pathways,
protein metabolism, cell growth and maintenance, and transport. Of these identified
proteins, PHGDH, LGALS3BP, SEMG1, ACTB, GAPDH, and the exosomal-marker
protein ALIX were validated by western blot. This study provided a more comprehensive description of the seminal exosomes
proteome and could also be a resource for further screening of
biomarkers and comparative proteomics studies, including those associated with
male infertility and
prostate cancer.