Optimizing protein recovery for urinary proteomics, a tool to monitor renal transplantation
Optimizing protein recovery for urinary proteomics, a tool to monitor renal transplantation
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DOI：
10.1111/j.1399-0012.2008.00833.x
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发表时间：
2008-09-01
影响因子：
2.1
通讯作者：
Sarwal, Minnie
中科院分区：
文献类型：
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作者：
Sigdel, Tara K.;Lau, Ken;Sarwal, Minnie
尽管尿液对于发现全身性和肾脏疾病的生物标志物具有吸引力，但尿液中高丰度血浆蛋白的混杂效应以及缺乏尿蛋白回收方法的优化是尿蛋白质组学的瓶颈。进行了三种方法并比较了蛋白质产率百分比、产率一致性、分析的容易性和成本：（i）有机溶剂沉淀，（ii）透析/冻干，和（iii）离心过滤。将尿液样品置于免疫亲和柱上以去除高丰度蛋白质。进行差异凝胶电泳以评估用于检测低丰度蛋白质的耗尽策略的使用。使用来自健康志愿者（n = 10）和具有蛋白尿的肾移植受者（n = 11）的尿液。离心过滤对于分析容易性和产量一致性表现最好。从透析/冻干获得最高的百分比产率，但是费力并且残留的盐干扰随后的凝胶电泳。有机溶剂沉淀是廉价的，但遭受不同的产率一致性。在高丰度蛋白质耗尽后，注意到一些低丰度和先前未检测到的蛋白质的斑点强度增加。总之，我们比较了不同蛋白质回收方法的优缺点，并揭示了耗尽策略后蛋白质检测的动态范围的增加，这对于生物标志物的发现可能是至关重要的，特别是在处理来自临床试验的人类研究样本时。
Despite attractiveness of urine for biomarker discovery for systemic and renal diseases, the confounding effect of the high abundance plasma proteins in urine, and a lack of optimization of urine protein recovery methods are bottlenecks for urine proteomics. Three methods were performed and compared for percentage protein yield, yield consistency, ease and cost of analysis: (i) organic solvent precipitation, (ii) dialysis/lyophilization, and (iii) centrifugal filtration. Urine samples were subjected to an immunoaffinity column to deplete high abundance proteins. Difference gel electrophoresis was performed to assess use of depletion strategy for detection of low abundance proteins. Urine from healthy volunteers (n = 10) and kidney transplant recipients with proteinuria (n = 11) were used. Centrifugal filtration performed best for analysis ease and yield consistency. Highest percentage yield was obtained from dialysis/lyophilization but was laborious and residual salt interfered with subsequent gel electrophoresis. Organic solvent precipitation was inexpensive, but suffered from varying yield consistency. Increased spot intensity for some low abundance and previously undetected proteins were noted after depletion of high abundance proteins. In conclusion, we compare the pros and cons of different protein recovery methods and reveal an increase in the dynamic range of protein detection after depletional strategy that could be critical for biomarker discovery, particularly with reference to processing human study samples from clinical trials.