色谱法鉴定适体-蛋白质靶标的研究进展,Journal of Proteome Research

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色谱法鉴定适体-蛋白质靶标的研究进展
Journal of Proteome Research ( IF 3.8 ) Pub Date : 2018-05-04 , DOI: 10.1021/acs.jproteome.8b00122
Anna Drabik ₁ , Joanna Ner-Kluza ₁ , Przemyslaw Mielczarek ₁ , Laia Civit ₂ , Günter Mayer _{2,

3} , Jerzy Silberring ₁

Affiliation

自从开发通过指数富集的配体系统进化（SELEX）的方法以来，适体已广泛用于各种研究中，包括探索新的诊断工具和发现新的治疗方法。通常，与抗体相比，适体以高亲和力和特异性结合蛋白质的能力使寻找潜在的癌症生物标志物成为可能，并有助于我们了解致癌机理。这些研究的盲点通常是难以选择性地提取与适体相连的靶标。有许多研究描述了SELEX细胞，它们是在动物模型中针对活体癌细胞乃至整个肿瘤的适体的首选。然而，当大量蛋白质被鉴定为潜在靶标时，通常会发生两难境地。在本文中，我们提出了一种新的分析方法，旨在选择性地靶向与适体结合的蛋白质。在研究过程中，我们专注于以对前列腺癌细胞高度特异性为特征的适体分子靶标的明确鉴定。我们比较了使用电泳和色谱方法“析出”适体蛋白靶标并随后进行质谱鉴定的四种测定方法。我们已经建立了一种基于荧光标记的寡核苷酸的新方法，该寡核苷酸通常用于流式细胞术实验或作为光学适体传感器，可以通过质谱检测特定的适体-蛋白质相互作用。使用atto488标记的适体来追踪特定的适体-靶标复合物的形成为研究假定的蛋白质对应物提供了可能，而无需应用富集技术。值得注意的是，atto488标记的适体-蛋白质复合物疏水特性的变化促进了它们的分离，方法是反相色谱结合荧光检测，然后进行基于质谱的蛋白质鉴定。几种方法学方法的比较结果证实，该方法具有很高的灵敏度，可广泛用于研究适体与蛋白质的相互作用，与下拉法相比具有优越的性能。

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Advances in the Study of Aptamer–Protein Target Identification Using the Chromatographic Approach

Ever since the development of the process known as the systematic evolution of ligands by exponential enrichment (SELEX), aptamers have been widely used in a variety of studies, including the exploration of new diagnostic tools and the discovery of new treatment methods. Aptamers’ ability to bind to proteins with high affinity and specificity, often compared to that of antibodies, enables the search for potential cancer biomarkers and helps us understand the mechanisms of carcinogenesis. The blind spot of those investigations is usually the difficulty in the selective extraction of targets attached to the aptamer. There are many studies describing the cell SELEX for the prime choice of aptamers toward living cancer cells or even whole tumors in the animal models. However, a dilemma arises when a large number of proteins are being identified as potential targets, which is often the case. In this article, we present a new analytical approach designed to selectively target proteins bound to aptamers. During studies, we have focused on the unambiguous identification of the molecular targets of aptamers characterized by high specificity to the prostate cancer cells. We have compared four assay approaches using electrophoretic and chromatographic methods for “fishing out” aptamer protein targets followed by mass spectrometry identification. We have established a new methodology, based on the fluorescent-tagged oligonucleotides commonly used for flow-cytometry experiments or as optic aptasensors, that allowed the detection of specific aptamer–protein interactions by mass spectrometry. The use of atto488-labeled aptamers for the tracking of the formation of specific aptamer–target complexes provides the possibility of studying putative protein counterparts without needing to apply enrichment techniques. Significantly, changes in the hydrophobic properties of atto488-labeled aptamer–protein complexes facilitate their separation by reverse-phase chromatography combined with fluorescence detection followed by mass-spectrometry-based protein identification. These comparative results of several methodological approaches confirmed the universal applicability of this method to studying aptamer–protein interactions with high sensitivity, showing superior properties compared with pull-down techniques.

更新日期：2018-05-05

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