Research Center for Analytical Sciences, College of Sciences, Northeastern University , Shenyang 110819, PR China.
State Key Laboratory of Proteomics, Beijing Proteome Research Center, National Center for Protein Sciences (Beijing), Beijing Institute of Lifeomics , Beijing 102206, PR China.
Anal Chem. 2018 Feb 6;90(3):2186-2192. doi: 10.1021/acs.analchem.7b04531. Epub 2018 Jan 16.
Western-blot (WB) is a powerful analytical technique for protein identification in complex biological samples and has been widely used in biological studies for decades. Detection specificity and sensitivity of WB largely relies on quality of the antibodies and performance of the conjugated HRP. However, the application of WB analysis for the detection of protein post-translational modifications (PTMs) is hampered by the low abundance of protein PTMs and by the limited availability of antibodies that specifically differentiate various kinds of PTMs from their protein substrates. Therefore, new recognition mechanisms and signal amplification strategies for WB analysis of protein PTMs is in high demand. In this work, we prepared a soluble polymer that detects various azide-tagged PTM proteins in WB analysis using triarylphosphine and HRP modified thermoresponsive polymer. Specific and efficient detection of azide-tagged PTM protein is achieved via the bioorthogonal reaction between azide and triarylphosphine. More importantly, the chemiluminiscent signal in the WB analysis is largely amplified by the temperature induced self-assembly of numerous thermoresponsive polymer chains carrying multiple HRPs. As a result, approximately 100 times more sensitive detection than commercial antibodies is achieved by this method using standard PTM proteins. Though, this new reagent does not directly detect native PTMs in cell, tissue or blood samples, it still has important application potential in protein PTM studies, considering the wide availability of azide-tagging techniques to a variety of PTMs.
Western blot（WB）是一种强大的分析技术，用于鉴定复杂生物样品中的蛋白质，几十年来已广泛应用于生物学研究。WB 的检测特异性和灵敏度在很大程度上依赖于抗体的质量和结合的 HRP 的性能。然而，WB 分析在检测蛋白质翻译后修饰（PTMs）方面的应用受到蛋白质 PTMs 丰度低和特异性区分各种 PTMs 与其蛋白质底物的抗体有限的限制。因此，对 PTMs 的 WB 分析需要新的识别机制和信号放大策略。在这项工作中，我们使用三芳基膦和 HRP 修饰的温敏聚合物制备了一种可检测 WB 分析中各种叠氮标记 PTM 蛋白的可溶性聚合物。通过叠氮化物和三芳基膦之间的生物正交反应实现了对叠氮标记 PTM 蛋白的特异性和高效检测。更重要的是，通过携带多个 HRP 的大量温敏聚合物链的温度诱导自组装，WB 分析中的化学发光信号得到了极大的放大。因此，与商业抗体相比，该方法使用标准 PTM 蛋白可实现约 100 倍的更灵敏检测。虽然这种新试剂不能直接检测细胞、组织或血液样本中的天然 PTMs，但考虑到各种 PTMs 广泛应用的叠氮标记技术，它在蛋白质 PTM 研究中仍具有重要的应用潜力。
