Combinatorial Peptide Libraries Facilitate Development of Multiple Reaction Monitoring Assays for Low-Abundance Proteins
Combinatorial Peptide Libraries Facilitate Development of Multiple Reaction Monitoring Assays for Low-Abundance Proteins
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DOI：
10.1021/pr900729g
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发表时间：
2010-03-01
影响因子：
4.4
通讯作者：
Diamandis, Eleftherios P.
中科院分区：
文献类型：
--
作者：
Drabovich, Andrei P.;Diamandis, Eleftherios P.
生物体液中存在的低丰度蛋白质通常被认为是新疾病生物标志物的有吸引力的来源。由于大量高丰度蛋白质在蛋白质组规模的发现实验中很难观察到此类蛋白质，因此开发低丰度蛋白质的定量多反应监测（MRM）测定是一项具有挑战性的任务。在这里，我们提出了一种策略，有助于开发大量未纯化的低丰度蛋白质的 MRM 检测方法。我们的发现策略基于通过一珠一化合物组合肽库 (CPL) 降低生物液体中蛋白质浓度的动态范围。我们的 2D-LC-MS/MS 方法使我们能够鉴定卵巢癌腹水中总共 484 种独特的蛋白质，其中 216 种蛋白质被指定为低丰度蛋白质。有趣的是，其中 74 种蛋白质以前从未在腹水中被描述过。使用 CPL 处理可以鉴定出大量低丰度蛋白质的独特肽，并为 MRM 测定的开发提供重要的经验碎片信息。最后，我们证实 MRM 检测适用于普通腹水消化物中的 30 种低丰度蛋白质。使用多重 MRM 方法，测定了一组卵巢癌腹水中五种蛋白质（激肽释放酶 6、金属蛋白酶抑制剂 1、巨噬细胞迁移抑制因子、卵泡抑素相关蛋白和间皮素）的相对含量。针对大量蛋白质的多重 MRM 测定可用于开发具有高灵敏度和选择性的综合生物标志物组，并研究复杂的蛋白质网络。
Low-abundance proteins present in biological fluids are often considered an attractive source of new disease biomarkers. Since such proteins are poorly observed in proteome-scale discovery experiments due to an overwhelming mass of high-abundance proteins, the development of quantitative multiple reaction monitoring (MRM) assays for low-abundance proteins is a challenging task. Here, we present a strategy that facilitates the development of MRM assays for large numbers of unpurified low-abundance proteins. Our discovery strategy is based on the reduction of the dynamic range of protein concentrations in biological fluids by means of one-bead one-compound combinatorial peptide libraries (CPL). Our 2D-LC-MS/MS approach allowed us to identify a total of 484 unique proteins in ovarian cancer ascites, and 216 proteins were assigned as low-abundance ones. Interestingly, 74 of those proteins have never been previously described in ascites fluid. Treatment with CPL allowed identification of a significantly higher number of unique peptides for low-abundance proteins and provided important empirical fragmentation information for development of MRM assays. Finally, we confirmed that MRM assays worked for 30 low-abundance proteins in the unfractionated ascites digest. Using a multiplexed MRM method, relative amounts of five proteins (kallikrein 6, metalloproteinase inhibitor 1, macrophage migration inhibitory factor, follistatin-related protein, and mesothelin) were determined in a set of ovarian cancer ascites. Multiplexed MRM assays targeting large numbers of proteins can be used to develop comprehensive panels of biomarkers with high sensitivity and selectivity, and to study complex protein networks.