Moon Pyong-Gon, Hwang Hyun-Ho, Boo Yong Chool, Kwon Joseph, Cho Je-Yoel, Baek Moon-Chang
Department of Molecular Medicine, School of Medicine, Kyungpook National University, Deagu, Republic of Korea.
Electrophoresis. 2008 Nov;29(21):4324-31. doi: 10.1002/elps.200800070.
Many different types of urine proteome studies have been done, but urine glycoprotein studies are insufficient. Therefore, we studied the glycoproteins from rat urine, which could be used to identify biomarkers in an animal model. First, urinary proteins were prepared by using the dialysis and lyophilizing methods from rat urine. Glycoproteins enriched with lectin affinity purification, concanavalin A, jacalin and wheat germ agglutinin from the urinary proteins were separated by means of reverse-phase fast protein LC (FPLC) or 1-D PAGE. Each FPLC fraction and 1-D PAGE gel band were trypsin-digested and analyzed by means of nanoLC-MS/MS. LC-MS/MS analyses were carried out by using linear ion trap MS. A total of 318 rat urinary glycoproteins were identified from the FPLC fractions and gel bands; approximately 90% of identified proteins were confirmed as glycoproteins in Swiss-Prot. Many glycoproteins, known as biomarkers, including C-reactive protein, uromodulin, amyloid beta A4 protein, alpha-1-inhibitor 3, vitamin D-binding protein, kallikrein 3 and fetuin-A were identified in this study. By studying urinary glycoproteins collected from rat, these results may help to assist in identifying urinary biomarkers regarding various types of disease models.
已经开展了许多不同类型的尿蛋白质组研究，但尿糖蛋白研究尚不充分。因此，我们对大鼠尿液中的糖蛋白进行了研究，其可用于在动物模型中鉴定生物标志物。首先，采用透析和冻干方法从大鼠尿液中制备尿蛋白。通过反相快速蛋白质液相色谱（FPLC）或一维聚丙烯酰胺凝胶电泳（1-D PAGE），从尿蛋白中分离出用凝集素亲和纯化法富集的糖蛋白，所用凝集素为伴刀豆球蛋白A、红豆凝集素和麦胚凝集素。对每个FPLC级分和一维聚丙烯酰胺凝胶电泳条带进行胰蛋白酶消化，并通过纳升液相色谱-串联质谱（nanoLC-MS/MS）进行分析。采用线性离子阱质谱进行液相色谱-串联质谱分析。从FPLC级分和凝胶条带中总共鉴定出318种大鼠尿糖蛋白；在瑞士蛋白质数据库（Swiss-Prot）中，约90%的已鉴定蛋白质被确认为糖蛋白。本研究鉴定出了许多作为生物标志物的糖蛋白，包括C反应蛋白、尿调节蛋白、β淀粉样蛋白A4、α-1抗胰蛋白酶3、维生素D结合蛋白、激肽释放酶3和胎球蛋白A。通过研究从大鼠收集的尿糖蛋白，这些结果可能有助于识别与各种疾病模型相关的尿生物标志物。
