Robust, quantitative analysis of proteins using peptide immunoreagents, in vitro translation, and an ultrasensitive acoustic resonant sensor.
Robust, quantitative analysis of proteins using peptide immunoreagents, in vitro translation, and an ultrasensitive acoustic resonant sensor.
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DOI：
10.1021/ac500084d
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发表时间：
2014-05-20
影响因子：
7.4
通讯作者：
Roberts, Richard W.
中科院分区：
文献类型：
--
作者：
Jalali-Yazdi, Farzad;Corbin, Jasmine M.;Takahashi, Terry T.;Roberts, Richard W.
蛋白质组学和基因组学数据的一个主要好处是有可能开发出数千种新的细胞、组织和临床样本的诊断和分析测试。单克隆抗体技术、噬菌体展示和mRNA展示是可以用于产生针对蛋白质组的每个成员的亲和配体的方法。越来越多的挑战不是配体生成，而是通过这些方法生成的许多配体的分析和亲和力排序。在这里，我们开发了一种定量的方法来分析蛋白质相互作用，使用体外翻译的配体。在该测定中，体外翻译的配体通过同时结合固定在磁珠上的靶标和商业声学传感装置中的传感器表面来产生信号。然后，我们将每个配体的结合与其相对翻译效率归一化，以便对不同的配体进行排序。我们证明了针对癌症标志物Bcl-xL的肽的方法。我们的方法具有4至10倍的灵敏度，使用100倍的蛋白质和5倍的抗体，每个样品，直接与ELISA相比。此外，所有分析都可以在生理离子强度的复杂混合物中进行。最后，我们证明了使用肽作为在复杂基质中起作用的亲合性试剂的能力，这在诊断应用中是需要的。
A major benefit of proteomic and genomic data is the potential for developing thousands of novel diagnostic and analytical tests of cells, tissues, and clinical samples. Monoclonal antibody technologies, phage display and mRNA display, are methods that could be used to generate affinity ligands against each member of the proteome. Increasingly, the challenge is not ligand generation, rather the analysis and affinity rank-ordering of the many ligands generated by these methods. Here, we developed a quantitative method to analyze protein interactions using in vitro translated ligands. In this assay, in vitro translated ligands generate a signal by simultaneously binding to a target immobilized on a magnetic bead and to a sensor surface in a commercial acoustic sensing device. We then normalize the binding of each ligand with its relative translation efficiency in order to rank-order the different ligands. We demonstrate the method with peptides directed against the cancer marker Bcl-xL. Our method has 4- to 10-fold higher sensitivity, using 100-fold less protein and 5-fold less antibody per sample, as compared directly with ELISA. Additionally, all analysis can be conducted in complex mixtures at physiological ionic strength. Lastly, we demonstrate the ability to use peptides as ultrahigh affinity reagents that function in complex matrices, as would be needed in diagnostic applications.
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