Six Biomarkers Expressed Stably in Urinary Exosomes During All Life Stages - As the Reference Markers in Urinary Quantification

Background: Urinary extracellular exosomes (uEVs) have been identified as a novel, stable and no-invasive source of biomarkers. However, the potential clinical value of uEVs is limited by the lack of standard quantitative proteomics data. It is necessary to uncover ubiquitous and stable proteins of uEVs as the reference markers in urinary quantification. Samples and methods The samples from 210 healthy individuals (3~90 years old), were divided into seven different stages of life. The uEVs samples were identified by LC-MS/MS and data-independent acquisition (DIA) methods. Eight stably expressed uEVs proteins were obtained by bioinformatics analysis. Moreover, 42 samples were used to validate by Western blot, ELISA, and immunofluorescence. Results: A total of 3,002 proteins and 1,393 co-expression uEVs proteins were identified by LC-MS/MS. The bioinformatics analysis showed 1,393 co-expression proteins mostly enriched in endocytosis. Eight proteins were stably expressed throughout the seven age stages ( p <0.05). Furthermore, RAB8A, RAB8B, Semaphorin-5A, Plexin-B2, JAMA, and STUB1 were validated by Western blot. Above all, RAB8A and RAB8B are the most stably expressed proteins in different age stages. Conclusion: RAB8A, RAB8B, Semaphorin-5A, Plexin-B2, JAMA, and STUB1 were expressed stably proteins throughout the age stages. These six proteins might be the standard reference markers in the analysis of urine exosomal proteomics. RAB8A and RAB8B have been validated are the putative reference markers.