Large-Scale Proteomics and Phosphoproteomics of Urinary Exosomes
Large-Scale Proteomics and Phosphoproteomics of Urinary Exosomes
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DOI：
10.1681/asn.2008040406
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发表时间：
2009-02-01
影响因子：
13.6
通讯作者：
Knepper, Mark A.
中科院分区：
文献类型：
--
作者：
Gonzales, Patricia A.;Pisitkun, Trairak;Knepper, Mark A.
正常人尿液含有大量的外泌体，它们是 40 至 100 nm 的囊泡，起源于面向尿腔的每种肾上皮细胞类型的多囊泡体内的内囊泡。在这里，我们使用 LC-MS/MS 来分析人尿外泌体的蛋白质组。总体而言，该分析明确鉴定了 1132 种蛋白质，其中 177 种存在于人类疾病相关基因的在线孟德尔遗传数据库中，这表明外泌体分析是发现尿液生物标志物的潜在方法。我们使用中性丢失扫描将蛋白质组分析扩展到磷酸蛋白质组分析，这产生了多个新的磷酸化位点，包括噻嗪类敏感的 Na-Cl 协同转运蛋白 NCC 中的丝氨酸 811。为了证明外泌体分析在识别遗传性肾病方面的潜在用途，我们对临床诊断为 1 型 Bartter 综合征的患者的尿液样本中的外泌体进行了免疫印迹，结果显示不存在钠钾氯化物协同转运蛋白 2 NKCC2。蛋白质组数据可在 http://dir.nhlbi.nih.gov/papers/lkem/exosome/ 上公开获取。
Normal human urine contains large numbers of exosomes, which are 40- to 100-nm vesicles that originate as the internal vesicles in multivesicular bodies from every renal epithelial cell type facing the urinary space. Here, we used LC-MS/MS to profile the proteome of human urinary exosomes. Overall, the analysis identified 1132 proteins unambiguously, including 177 that are represented on the Online Mendelian Inheritance in Man database of disease-related genes, suggesting that exosome analysis is a potential approach to discover urinary biomarkers. We extended the proteomic analysis to phospho-proteomic profiling using neutral loss scanning, and this yielded multiple novel phosphorylation sites, including serine-811 in the thiazide-sensitive Na-Cl co-transporter, NCC. To demonstrate the potential use of exosome analysis to identify a genetic renal disease, we carried out immunoblotting of exosomes from urine samples of patients with a clinical diagnosis of Bartter syndrome type 1, showing an absence of the sodium-potassium-chloride co-transporter 2, NKCC2. The proteomic data are publicly accessible at http://dir.nhlbi.nih.gov/papers/lkem/exosome/.