Purification and Identification of Membrane Proteins from Urinary Extracellular Vesicles using Triton X-114 Phase Partitioning
Abstract
Urinary extracellular vesicles (uEVs) have emerged as a valuable source of biomarkers, offering insights into biochemical changes associated with kidney and urogenital diseases. Notably, uEVs are rich in membrane proteins involved in essential cellular processes such as adhesion, transport, and signaling. As a result, these membrane proteins represent a distinct and biologically significant class.
In this study, we optimized the Triton X-114 detergent partitioning protocol specifically for membrane protein isolation from uEVs while minimizing interference from Tamm-Horsfall protein, the most abundant urinary protein. This is the first report dedicated to enriching and characterizing integral transmembrane proteins in human urinary vesicles. Initially, uEVs were isolated using a hydrostatic filtration dialysis system, and their enrichment was confirmed via transmission electron microscopy. Subsequently, Triton X-114 phase partitioning was applied, yielding an insoluble pellet fraction along with aqueous (AP) and detergent (DP) phase fractions, which were analyzed using LC-MS/MS.
Both in-gel and off-gel protein digestion methods revealed an increased number of uEV membrane proteins. Compared to previous studies without phase separation, we identified 199 distinct proteins in the DP fraction. Transmembrane domain (TMD) predictions indicated a higher prevalence of TMD-containing proteins in DP than in other fractions. Hydrophobicity analysis further supported this finding, as the GRAVY score for DP was significantly higher than for other fractions. Additionally, proteins in DP exhibited a greater number of lipid anchors, reinforcing their membrane-associated nature. KEGG pathway analysis showed that DP proteins are involved in endocytosis and signaling, aligning with the expected functions of membrane proteins. Western blotting results further confirmed the enrichment of membrane proteins in DP rather than AP.
In conclusion, our study validates the use of Triton X-114 phase partitioning for the targeted isolation of membrane proteins from uEVs, effectively reducing sample complexity. This method enhances the detection of potential biomarkers and druggable targets, facilitating future research in kidney and urogenital disease diagnostics and therapeutics.