ABSTRACT: Urinary extracellular vesicles (uEVs) denote minuscule membranous entities generated by cells lining the urinary tract and adjoining tissues(1). The examination of uEVs as an exploratory instrument has garnered substantial focus owing to their prospective utility as non-invasive biomarkers for diagnosing and monitoring kidney disorders, like hypertension, diabetes, immune-related ailments, and malignancies(2). The protein payload of uEVs reflects both the protein composition of tissues(3) as well as physiologic changes within the kidney(4). As a result of technological advancements in mass spectrometry, such as the innovation of the Orbitrap ion trap mass analyzer, the identification of proteins has become more meticulous, expeditious, and capable of detecting subtle variations in protein quantities(5). Thus far urinary EV proteomic and phosphoproteomic analyses have identified more than 4000 unique proteins(6). High speed ultracentrifugation is the most widely used method for the isolation of EVs. Differential ultracentrifugation is commonly used to capture the whole population of vesicles at the size range 40nm–200nm(7). Recently, investigations into the low-speed pellet resulting from centrifugation have shown its potential as a valuable source of EVs as well(8–10). Ultracentrifugation can remove the vast majority of contaminant protein, Tamm-Horsfall protein (THP; also known as uromodulin), which tends to co sediment with EV’s(11–13). THP polymerization gives rise to multiple isoforms, including high molecular weight networks(14,15) that precipitate at low speeds, forming urinary casts, along with short oligomers that co-segregate with EVs, acting as impurities(16,17) . This situation can complicate downstream analysis by either masking low-abundance proteins during mass spectrometry(18) or interfering with glycan analysis due to THP's extensive glycosylation(17). To overcome residual contamination, THP depolymerization by DTT (dithiothreitol) treatment(12) or uEV isolation by sucrose gradient centrifugation(19) have been routinely used, but both approaches have disadvantages(20). The use of DTT doesn't entirely eliminate THP from uEVs (21), potentially affecting the folding and biological functionality of specific proteins in uEV preparations(21) . Incorporating chemical agents like iodixanol to counter viscous contaminants complicates proteomic analysis due to mass spectrometry sensitivity. On the other hand, separation via sucrose gradients or cushions (20,22) is time-intensive, rendering uEV preparations derived from these methods less suitable for functional investigations(23). The utilization of filters to remove THP has been extensively explored for uEV isolation. However, combining filtration with other techniques like differential ultracentrifugation as an enrichment step to enhance EV yield is labor-intensive and impractical for large-scale studies(24–26). Density gradient could enhance EV fraction purity by eliminating contaminants such as THP, but this method leads to EV loss and isn't feasible in clinical settings(27). The absence of standardized protocols for EV isolation techniques has prompted the development of innovative methods to optimize EV extraction from diverse biological fluids(28). Currently, the optimal method for EV isolation is chosen based on factors like the type and quantity of bodily fluid being analyzed, availability of specialized equipment, intended therapeutic application, administration route, and desired vesicle fraction. As a result, the literature abounds with numerous protocols for EV isolation and strategies to counter THP interference(29). Previously, we demonstrated consistent retrieval of uEVs through centrifugation at 20,000g(8) following unfolding of THP filaments by reducing agent (TCEP). In this study we explore simplified methods for high throughput EV preparation for large scale analysis and refine conditions to unravel THP filaments without the need for reducing agents or filters, all in pursuit of identifying biomarkers. We report a comparative mass spectrometry analysis of high centrifugation vs. low centrifugation uEV pellets following THP removal and compare the results to the combination of both high and low centrifugation uEV pellets. This easy high throughput uEVs enrichment method covers the majority of the uEVs proteome and requires only small microcentrifuge rather than ultracentrifugation .
henrik molina 
