Optimizing a Proteomics Platform for Urine Biomarker Discovery
Optimizing a Proteomics Platform for Urine Biomarker Discovery
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DOI：
10.1074/mcp.m110.000992
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发表时间：
2010-10-01
影响因子：
7
通讯作者：
Libermann, Towia A.
中科院分区：
文献类型：
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作者：
Afkarian, Maryam;Bhasin, Manoj;Libermann, Towia A.
尿液中的生物标志物发现方法受到蛋白质组学方案的可重复性和标准化不足的影响。在这项研究中，我们描述了一个优化的定量蛋白质组学策略，尿液生物标志物的发现，这是适用于新鲜或长期冷冻样本。我们使用健康对照的尿液标准化iTRAQ（用于相对和绝对定量的同量异位素标签），用于蛋白酶抑制剂诱导的变异、起始蛋白和iTRAQ标记量、蛋白提取方法以及白蛋白和免疫球蛋白G（IgG）的消耗。我们观察到以下情况：（a）蛋白酶抑制剂的缺乏不影响高置信度蛋白质的数量或身份。(b)每个样品使用少于20 μ g的蛋白质导致鉴定的蛋白质数量显著下降。(c)使用少至四分之一单位的iTRAQ标记不会影响所鉴定蛋白质的数量或身份。(d)通过甲醇沉淀的蛋白质提取导致最高的蛋白质产率和最可重复的光谱。(e)白蛋白和IgG的消耗没有增加所鉴定的蛋白质的数量或加深蛋白质组覆盖。将此优化方案应用于患有或不患有肾病的糖尿病皮马印第安人的四对长冷冻尿液样本，我们观察到的模式表明iTRAQ光谱分离病例和对照。我们还确定了几个先前报道的候选生物标志物，这些生物标志物显示出差异表达的趋势，尽管在这个小样本集中没有达到统计学显著性。Molecular & Cellular Proteomics 9：2195-2204，2010.
Biomarker discovery approaches in urine have been hindered by concerns for reproducibility and inadequate standardization of proteomics protocols. In this study, we describe an optimized quantitative proteomics strategy for urine biomarker discovery, which is applicable to fresh or long frozen samples. We used urine from healthy controls to standardize iTRAQ (isobaric tags for relative and absolute quantitation) for variation induced by protease inhibitors, starting protein and iTRAQ label quantities, protein extraction methods, and depletion of albumin and immunoglobulin G (IgG). We observed the following: (a) Absence of protease inhibitors did not affect the number or identity of the high confidence proteins. (b) Use of less than 20 mu g of protein per sample led to a significant drop in the number of identified proteins. (c) Use of as little as a quarter unit of an iTRAQ label did not affect the number or identity of the identified proteins. (d) Protein extraction by methanol precipitation led to the highest protein yields and the most reproducible spectra. (e) Depletion of albumin and IgG did not increase the number of identified proteins or deepen the proteome coverage. Applying this optimized protocol to four pairs of long frozen urine samples from diabetic Pima Indians with or without nephropathy, we observed patterns suggesting segregation of cases and controls by iTRAQ spectra. We also identified several previously reported candidate biomarkers that showed trends toward differential expression, albeit not reaching statistical significance in this small sample set. Molecular & Cellular Proteomics 9:2195-2204, 2010.