Enhanced Detection of Low Abundance Human Plasma Proteins Using a Tandem IgY12-SuperMix Immunoaffinity Separation Strategy
Enhanced Detection of Low Abundance Human Plasma Proteins Using a Tandem IgY12-SuperMix Immunoaffinity Separation Strategy
复制标题
DOI：
10.1074/mcp.m800008-mcp200
复制
发表时间：
2008-10-01
影响因子：
7
通讯作者：
Smith, Richard D.
中科院分区：
文献类型：
--
作者：
Qian, Wei-Jun;Kaleta, David T.;Smith, Richard D.
人类体液蛋白质组的巨大动态范围对当前基于MS的蛋白质组学技术提出了重大挑战，因为它使得特别难以检测人类生物流体（例如血浆）中的低丰度蛋白质，这是成功的生物标志物发现工作的重要方面。在这里，我们提出了一种新的串联IgY 12-SuperMix免疫亲和分离系统，用于增强检测人血浆中的低丰度蛋白。串联IgY 12-SuperMix系统从血浆中的低丰度蛋白质中分离出类似于60种丰度蛋白质，从而使SuperMix流通级分中的低丰度血浆蛋白质显著富集。在样品处理回收率和基于光谱计数数据的LC-MS/MS鉴别结果方面，观察到串联分离的重现性较高。通过将三种不同水平的六种非人标准蛋白加标至血浆中，证明了应用串联分离后定量测量差异蛋白丰度的能力。通过一维和二维LC-MS/MS分析的SuperMix流通样品和IgY 12流通样品之间的并排比较显示，由于SuperMix分离，蛋白质组覆盖率增加了60-80%，这表明低丰度蛋白质的检测显著增强。通过将串联分离策略与二维LC-MS/MS相结合，在单次分析中（每种蛋白质至少两种肽）确定了总共695种血浆蛋白质，包括42种报告正常浓度为100 pg/ml至100 ng/ml的蛋白质。两种选定的蛋白质，巨噬细胞集落刺激因子1和基质金属蛋白酶-8的浓度，通过ELISA分别独立验证为202 pg/ml和12.4 ng/ml。结合效率的评估显示，45种中等丰度蛋白质被SuperMix柱有效捕获，保留率> 90%。综上所述，这些结果说明了这种串联IgY 12-SuperMix策略对于涉及人类生物流体的蛋白质组学应用的潜在广泛实用性，其中有效地解决标本的动态范围挑战是必要的。Molecular & Cellular Proteomics 7：1963-1973，2008.
The enormous dynamic range of human bodily fluid proteomes poses a significant challenge for current MS-based proteomics technologies as it makes it especially difficult to detect low abundance proteins in human biofluids such as blood plasma, which is an essential aspect for successful biomarker discovery efforts. Here we present a novel tandem IgY12-SuperMix immunoaffinity separation system for enhanced detection of low abundance proteins in human plasma. The tandem IgY12-SuperMix system separates similar to 60 abundant proteins from the low abundance proteins in plasma, allowing for significant enrichment of low abundance plasma proteins in the SuperMix flow-through fraction. High reproducibility of the tandem separations was observed in terms of both sample processing recovery and LC-MS/MS identification results based on spectral count data. The ability to quantitatively measure differential protein abundances following application of the tandem separations was demonstrated by spiking six non-human standard proteins at three different levels into plasma. A side-by-side comparison between the SuperMix flow-through and IgY12 flow-through samples analyzed by both one- and two-dimensional LC-MS/MS revealed a 60-80% increase in proteome coverage as a result of the SuperMix separations, suggesting significantly enhanced detection of low abundance proteins. A total of 695 plasma proteins were confidently identified in a single analysis ( with a minimum of two peptides per protein) by coupling the tandem separation strategy with two-dimensional LC-MS/MS, including 42 proteins with reported normal concentrations of similar to 100 pg/ml to 100 ng/ml. The concentrations of two selected proteins, macrophage colony-stimulating factor 1 and matrix metalloproteinase-8, were independently validated by ELISA as 202 pg/ml and 12.4 ng/ml, respectively. Evaluation of binding efficiency revealed that 45 medium abundance proteins were efficiently captured by the SuperMix column with > 90% retention. Taken together, these results illustrate the potential broad utilities of this tandem IgY12-SuperMix strategy for proteomics applications involving human biofluids where effectively addressing the dynamic range challenge of the specimen is imperative. Molecular & Cellular Proteomics 7: 1963-1973, 2008.