Purification and Identification of Membrane Proteins from Urinary Extracellular Vesicles using Triton X-114 Phase Partitioning
Purification and Identification of Membrane Proteins from Urinary Extracellular Vesicles using Triton X-114 Phase Partitioning
复制标题
使用 Triton X-114 相分配纯化和鉴定尿细胞外囊泡的膜蛋白
DOI：
10.1021/acs.jproteome.7b00386
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发表时间：
2018-01-01
影响因子：
4.4
通讯作者：
Holthofer, Harry
中科院分区：
文献类型：
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作者：
Hu, Shuiwang;Musante, Luca;Holthofer, Harry
尿液细胞外囊泡（uEVs）已成为一种很有前景的生物标志物来源，能准确反映肾脏及泌尿生殖系统疾病中的生化变化。其特点是，uEVs富含与多种细胞功能（如黏附、转运和信号传导）相关的膜蛋白。因此，uEVs的膜蛋白应是一类具有独特生物学特性、令人关注的蛋白质。在本研究中，我们利用uEVs优化了针对膜蛋白的Triton X - 114去污剂相分离方案，并在消除尿液中含量最丰富的干扰蛋白——Tamm Horsfall蛋白的影响后，对膜蛋白进行后续表征。这是首篇旨在富集和表征人尿液囊泡中完整跨膜蛋白的报告。首先，使用“静水压过滤透析”装置富集uEVs，然后通过透射电子显微镜对富集的uEVs及其裂解物进行验证。采用Triton X - 114相分离后，我们得到了不溶性沉淀组分、水相（AP）和去污剂相（DP）组分，并通过液相色谱 - 串联质谱（LC - MS/MS）对其进行分析。采用胶内和胶外蛋白质消化方法，以揭示更多uEVs的膜蛋白。与我们早期发表的未进行相分离所鉴定出的蛋白质进行比较后，在DP中检测到199种不同的蛋白质。对这些蛋白质组分的跨膜结构域（TMDs）预测表明，DP中的TMDs比其他组更多。疏水性分析显示，DP的亲水性平均系数（GRAVY）得分远高于其他组分。此外，对具有脂质锚定的蛋白质分析表明，DP中的蛋白质比其他组分具有更多的脂质锚定。另外，京都基因与基因组百科全书（KEGG）通路分析显示，检测到的DP蛋白质参与内吞作用和信号传导，这与膜蛋白预期的生物学功能一致。最后，蛋白质免疫印迹（Western blotting）结果证实，膜蛋白条带出现在DP组分而非AP中。总之，我们的研究验证了在uEVs上使用Triton X - 114相分离方案可靶向分离膜蛋白并降低样本复杂性。该方法成功助力于检测uEVs中的潜在生物标志物和药物作用靶点。
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.