Proteomic analysis of normal human urinary proteins isolated by acetone precipitation or ultracentrifugation
Proteomic analysis of normal human urinary proteins isolated by acetone precipitation or ultracentrifugation
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DOI：
10.1111/j.1523-1755.2002.kid565.x
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发表时间：
2002-10-01
影响因子：
19.6
通讯作者：
Klein, JB
中科院分区：
文献类型：
--
作者：
Thongboonkerd, V;Mcleish, KR;Klein, JB
背景。蛋白质组学技术最近可用于大规模蛋白质分析。这些技术在尿蛋白鉴定中的应用还不明确。采用两种差分分离技术，构建了正常人尿液蛋白质组图谱，作为参考蛋白数据库。采用丙酮沉淀法或超离心法从正常人尿液中分离蛋白质，采用二维聚丙烯酰胺凝胶电泳（2D-PAGE）分离蛋白质，采用基质辅助激光解吸电离飞行时间（MALDI-TOF）质谱法和多肽质量指纹图谱对蛋白质进行鉴定。共鉴定出47种独特蛋白的67种蛋白形式，包括转运蛋白、粘附分子、补体、伴侣蛋白、受体、酶、蛇形蛋白、细胞信号蛋白和基质蛋白。丙酮沉淀出更多的酸性和亲水蛋白，而超离心分离出更多的碱性、疏水和膜蛋白。生物信息学分析预测糖基化是同一蛋白多种形式的最常见解释。结合两种差异分离技术，放大了人类尿液中蛋白质的鉴定。尿蛋白的蛋白质组学分析是研究肾脏生理和病理生理以及确定肾脏疾病生物标志物的一种很有前途的工具。
Background. Proteomic techniques have recently become available for large-scale protein analysis. The utility of these techniques in identification of urinary proteins is poorly defined. We constructed a proteome map of normal human urine as a reference protein database by using two differential fractionated techniques to isolate the proteins.Methods. Proteins were isolated from urine obtained from normal human volunteers by acetone precipitation or ultracentrifugation, separated by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and identified by matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) mass spectrometry followed by peptide mass fingerprinting.Results. A total of 67 protein forms of 47 unique proteins were identified, including transporters, adhesion molecules, complement, chaperones, receptors, enzymes, serpins, cell signaling proteins and matrix proteins. Acetone precipitated more acidic and hydrophilic proteins, whereas ultracentrifugation fractionated more basic, hydrophobic, and membrane proteins. Bioinformatic analysis predicted glycosylation to be the most common explanation for multiple forms of the same protein.Conclusions. Combining two differential isolation techniques magnified protein identification from human urine. Proteomic analysis of urinary proteins is a promising tool to study renal physiology and pathophysiology and to determine biomarkers of renal disease.