Investigation of the N-Glycoproteome in the Urinary Exosomes: Technical Challenges

Abstract

Urinary Exosomes (UE) are membranous nanometer-sized vesicles that can originate from endothelial cells, podocytes or tubular epithelial cells. Their molecular composition depends upon the type, and even status, of the producer cell. Their presence in urine makes them readily accessible, representing a liquid biopsy, non-invasive modality capable to provide diagnostic and prognostic information about kidney health state. Additionally, the UE proteome includes many species that are N-glycomodified. N-glycosylation is one of the most complex and frequent protein post-translational modifications, playing diverse biological roles. Additionally, it is reported that disease-specific glycosylation profiles of circulating microvesicles were detected in autosomal dominant polycystic kidney disease and ovarian carcinoma. This study is set in this context. Our aim is to provide the best MS-sample preparation protocol to study the UE glycosylated protein content, focusing our efforts on the enrichment of the glycosylated species and simultaneously depleting the most abundant contaminant protein (Uromodulin).