Simple urinary sample preparation for proteomic analysis
Simple urinary sample preparation for proteomic analysis
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DOI：
10.1021/pr060305y
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发表时间：
2006-10-06
影响因子：
4.4
通讯作者：
Packer, Nicolle H.
中科院分区：
文献类型：
--
作者：
Khan, Alamgir;Packer, Nicolle H.
自从人类基因组序列完成以来，现在的注意力集中在建立体液（如血浆和尿液）的参考图谱，以检测疾病的诊断标记物。尽管取得了一些进展，但在开发尿液蛋白质组学分析的最佳样品制备方法方面仍然存在挑战。我们开发了一种简单有效的尿液制备方法，用于二维凝胶电泳，该方法涉及蛋白质的沉淀和同时脱盐。与其他有机溶剂沉淀法相比，乙腈沉淀法产生的二维凝胶分离具有最高的分辨率和最多的蛋白质斑点。该方法用于观察6周内尿蛋白质组的变化，并建立健康受试者的参考图。采用肽质量指纹图谱技术，从健康男性尿液中鉴定出159个基因的339个蛋白。将1天内3次和4天的尿蛋白质组图谱进行比较，发现蛋白质在图谱上的数量和空间位置有所不同。该方法也被证明适用于卵巢癌患者尿液中较高浓度的蛋白质。我们已经开发了一种简单而可靠的方法来制备尿液用于二维凝胶，这将鼓励进一步使用尿液。
Since the completion of the human genome sequence, attention has now focused on establishing reference maps of body fluids such as plasma and urine for detecting diagnostic markers of disease. Although some progress has been made, challenges still remain in the development of an optimal sample preparation method for proteomic analysis of urine. We have developed a simple and efficient urine preparation method for two-dimensional (2-D) gel electrophoresis which involves precipitation of proteins with simultaneous desalting. Acetonitrile precipitation produced 2-D gel separations with the highest resolution and the greatest number of protein spots compared to precipitation by other organic solvents. The method was applied to observe changes in the urinary proteome over a 6 week period and to establish a reference map of a healthy subject. A total of 339 proteins from 159 genes was identified from healthy male urine by peptide mass fingerprinting. The profiles of the urinary proteome at three times in 1 day and on four different days were compared and were found to vary in number and spatial location of the proteins on the map. The method was also shown to be applicable to the higher concentrations of protein found in the urine of an ovarian cancer subject. We have developed a facile and robust method for preparing urine for 2-D gels that will encourage further use of urine.