Guangdong Provincial Key Laboratory of Proteomics, State Key Laboratory of Organ Failure Research, Southern Medical University , Guangzhou, China.
Institute of Nephrology and Urology, The Third Affiliated Hospital, Southern Medical University , Guangzhou, China.
J Proteome Res. 2018 Jan 5;17(1):86-96. doi: 10.1021/acs.jproteome.7b00386. Epub 2017 Nov 20.
Urinary extracellular vesicles (uEVs) have become a promising source for biomarkers accurately reflecting biochemical changes in kidney and urogenital diseases. Characteristically, uEVs are rich in membrane proteins associated with several cellular functions like adhesion, transport, and signaling. Hence, membrane proteins of uEVs should represent an exciting protein class with unique biological properties. In this study, we utilized uEVs to optimize the Triton X-114 detergent partitioning protocol targeted for membrane proteins and proceeded to their subsequent characterization while eliminating effects of Tamm-Horsfall protein, the most abundant interfering protein in urine. This is the first report aiming to enrich and characterize the integral transmembrane proteins present in human urinary vesicles. First, uEVs were enriched using a "hydrostatic filtration dialysis'' appliance, and then the enriched uEVs and lysates were verified by transmission electron microscopy. After using Triton X-114 phase partitioning, we generated an insoluble pellet fraction and aqueous phase (AP) and detergent phase (DP) fractions and analyzed them with LC-MS/MS. Both in- and off-gel protein digestion methods were used to reveal an increased number of membrane proteins of uEVs. After comparing with the identified proteins without phase separation as in our earlier publication, 199 different proteins were detected in DP. Prediction of transmembrane domains (TMDs) from these protein fractions showed that DP had more TMDs than other groups. The analyses of hydrophobicity revealed that the GRAVY score of DP was much higher than those of the other fractions. Furthermore, the analysis of proteins with lipid anchor revealed that DP proteins had more lipid anchors than other fractions. Additionally, KEGG pathway analysis showed that the DP proteins detected participate in endocytosis and signaling, which is consistent with the expected biological functions of membrane proteins. Finally, results of Western blotting confirmed that the membrane protein bands are found in the DP fraction instead of AP. In conclusion, our study validates the use of Triton X-114 phase partitioning protocol on uEVs for a targeted isolation of membrane proteins and to reduce sample complexity. This method successfully facilitates detection of potential biomarkers and druggable targets in uEVs.
尿液细胞外囊泡（uEVs）已成为反映肾脏和泌尿生殖系统疾病生化变化的有前途的生物标志物来源。其特征在于，uEVs 富含与几种细胞功能相关的膜蛋白，如粘附、运输和信号转导。因此，uEVs 的膜蛋白应该代表一个具有独特生物学特性的令人兴奋的蛋白质类别。在这项研究中，我们利用 uEVs 来优化针对膜蛋白的 Triton X-114 去污剂分配方案，并在消除尿液中最丰富的干扰蛋白 Tamm-Horsfall 蛋白的影响后对其进行后续表征。这是旨在富集和表征人尿囊泡中存在的完整跨膜蛋白的第一份报告。首先，使用“静水过滤透析”装置富集 uEVs，然后通过透射电子显微镜验证富集的 uEVs 和裂解物。在使用 Triton X-114 相分离后，我们生成了不溶性沉淀级分和水相 (AP) 和去污剂相 (DP) 级分，并通过 LC-MS/MS 对其进行分析。使用胶内和胶外蛋白消化方法来揭示 uEVs 的膜蛋白数量增加。与我们早期出版物中未进行相分离的鉴定蛋白进行比较后，在 DP 中检测到 199 种不同的蛋白。从这些蛋白级分预测跨膜结构域 (TMD) 表明，DP 比其他组具有更多的 TMD。疏水性分析表明，DP 的 GRAVY 评分明显高于其他级分。此外，带有脂质锚的蛋白分析表明，DP 蛋白比其他级分具有更多的脂质锚。此外，KEGG 途径分析表明，检测到的 DP 蛋白参与内吞作用和信号转导，这与膜蛋白的预期生物学功能一致。最后，Western 印迹分析结果证实膜蛋白条带存在于 DP 级分中，而不是 AP 级分中。总之，我们的研究验证了使用 Triton X-114 相分离方案在 uEVs 上靶向分离膜蛋白并减少样品复杂性的方法。该方法成功地促进了 uEVs 中潜在生物标志物和可药物靶标的检测。
