State Key Laboratory of Pharmaceutical Biotechnology and Collaborative Innovation Center of Chemistry for Life Sciences, Department of Biochemistry, Nanjing University , Nanjing 210093, P. R. China.
Center for Molecular Recognition and Biosensing, School of Life Sciences, Shanghai University , Shanghai 200444, P. R. China.
Anal Chem. 2017 Oct 17;89(20):10776-10782. doi: 10.1021/acs.analchem.7b02025. Epub 2017 Oct 5.
Quantitation of plasma membrane proteins (PMPs) is fundamental and frequently performed daily in the lab. However, challenged by the inherent/interacting heterostructures and complex surroundings of the PMPs in lipid membrane, quantitative techniques for PMP often require complex treatments (e.g., labeling, isolation, purification, and determination), and the sensitivity is usually not satisfactory. To address this problem, we have proposed a novel method that enables quantitation of PMPs with extremely high sensitivity, in an easier-to-manipulate and more streamlined way. This method is based on the design of an in situ rolling cycling replication-templated amplification strategy (isRTA). In fact, two rounds of DNA cascade isothermal amplifications have been conducted. The first round of amplification can provide templates for the second round of amplification; thus, significant enhancement of quantitative signals can be achieved. In this way, PMPs are quantified with ultrahigh sensitivity; as few as 25 copies of PMPs can be detected per cell. Moreover, the advantages of isRTA have been demonstrated by simultaneous identification of several PMP biomarkers (MUC1, EpCAM, and HER2) that are expressed over a wide distribution range on breast cancer cells. The precise typing of breast cancer cell subsets is thus possible because of the "quantitative-to-qualitative" strategy. Therefore, the unprecedented sensitivity and high usability of the isRTA method may present significant prospects for delving into membrane proteins and their related biofunctions in many research fields.
定量分析质膜蛋白（PMPs）是实验室中一项基础且日常进行的工作。然而，由于 PMPs 在脂质膜中具有固有/相互作用的异质结构和复杂环境，定量分析 PMP 的技术通常需要复杂的处理（例如标记、分离、纯化和测定），且灵敏度通常不能令人满意。为了解决这个问题，我们提出了一种新的方法，可以更轻松、更精简地实现对 PMP 的超高灵敏度定量分析。该方法基于原位滚环复制模板扩增策略（isRTA）的设计。实际上，已经进行了两轮 DNA 级联等温扩增。第一轮扩增可以为第二轮扩增提供模板；因此，可以实现定量信号的显著增强。通过这种方式，可以实现 PMP 的超高灵敏度定量分析；每个细胞中可以检测到多达 25 个拷贝的 PMP。此外，通过同时鉴定几种在乳腺癌细胞上广泛分布的 PMP 标志物（MUC1、EpCAM 和 HER2），证明了 isRTA 的优势。因此，由于“定量到定性”的策略，有可能对乳腺癌细胞亚群进行精确分类。因此，isRTA 方法具有前所未有的灵敏度和高可用性，可能为深入研究许多研究领域中的膜蛋白及其相关生物功能提供重要前景。
