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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >Identification and proteomic profiling of exosomes in human urine.
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Identification and proteomic profiling of exosomes in human urine.

机译：人尿中外泌体的鉴定和蛋白质组学分析。

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Urine provides an alternative to blood plasma as a potential source of disease biomarkers. One urinary biomarker already exploited in clinical studies is aquaporin-2. However, it remains a mystery how aquaporin-2 (an integral membrane protein) and other apical transporters are delivered to the urine. Here we address the hypothesis that these proteins reach the urine through the secretion of exosomes [membrane vesicles that originate as internal vesicles of multivesicular bodies (MVBs)]. Low-density urinary membrane vesicles from normal human subjects were isolated by differential centrifugation. ImmunoGold electron microscopy using antibodies directed to cytoplasmic or anticytoplasmic epitopes revealed that the vesicles are oriented "cytoplasmic-side inward," consistent with the unique orientation of exosomes. The vesicles were small (<100 nm), consistent with studies of MVBs and exosomes from other tissues. Proteomic analysis of urinary vesicles through nanospray liquid chromatography-tandem mass spectrometry identified numerous protein components of MVBs and of the endosomal pathway in general. Full liquid chromatography-tandem MS analysis revealed 295 proteins, including multiple protein products of genes already known to be responsible for renal and systemic diseases, including autosomal dominant polycystic kidney disease, Gitelman syndrome, Bartter syndrome, autosomal recessive syndrome of osteopetrosis with renal tubular acidosis, and familial renal hypomagnesemia. The results indicate that exosome isolation may provide an efficient first step in biomarker discovery in urine.
机译：尿液可替代血浆，作为疾病生物标志物的潜在来源。在临床研究中已经利用的一种尿液生物标志物是水通道蛋白2。然而，如何将aquaporin-2（一种完整的膜蛋白）和其他根尖转运蛋白输送到尿液仍是一个谜。在这里，我们解决以下假设：这些蛋白质通过外泌体[起源于多囊泡体内囊泡（MVBs）的膜囊泡]的分泌到达尿液。通过差速离心分离来自正常人的低密度尿膜囊泡。使用针对细胞质或抗细胞质表位的抗体进行的ImmunoGold电子显微镜检查显示，囊泡的方向是“胞质侧向内”，与外泌体的独特方向一致。囊泡很小（<100 nm），与对来自其他组织的MVB和外泌体的研究一致。通过纳米喷雾液相色谱-串联质谱对尿囊进行蛋白质组学分析，可以确定MVB和内体途径的许多蛋白质成分。全液相色谱-串联质谱分析揭示了295种蛋白质，包括已知与肾脏和全身性疾病有关的多种蛋白质产物，包括常染色体显性遗传多囊肾，吉特曼综合征，巴特综合征，骨化病伴肾小管性酸中毒的常染色体隐性遗传综合征和家族性肾低镁血症。结果表明，外泌体分离可为尿液中生物标志物的发现提供有效的第一步。

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