Direct Profiling the Post-Translational Modification Codes of a Single Protein Immobilized on a Surface Using Cu-free Click Chemistry,ACS Central Science

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Direct Profiling the Post-Translational Modification Codes of a Single Protein Immobilized on a Surface Using Cu-free Click Chemistry
ACS Central Science ( IF 12.7 ) Pub Date : 2018-04-18 00:00:00 , DOI: 10.1021/acscentsci.8b00114
Kyung Lock Kim ₁ , Kyeng Min Park _{1,

2} , James Murray ₁ , Kimoon Kim ₁ , Sung Ho Ryu

Affiliation

Combinatorial post-translational modifications (PTMs), which can serve as dynamic “molecular barcodes”, have been proposed to regulate distinct protein functions. However, studies of combinatorial PTMs on single protein molecules have been hindered by a lack of suitable analytical methods. Here, we describe erasable single-molecule blotting (eSiMBlot) for combinatorial PTM profiling. This assay is performed in a highly multiplexed manner and leverages the benefits of covalent protein immobilization, cyclic probing with different antibodies, and single molecule fluorescence imaging. Especially, facile and efficient covalent immobilization on a surface using Cu-free click chemistry permits multiple rounds (>10) of antibody erasing/reprobing without loss of antigenicity. Moreover, cumulative detection of coregistered multiple data sets for immobilized single-epitope molecules, such as HA peptide, can be used to increase the antibody detection rate. Finally, eSiMBlot enables direct visualization and quantitative profiling of combinatorial PTM codes at the single-molecule level, as we demonstrate by revealing the novel phospho-codes of ligand-induced epidermal growth factor receptor. Thus, eSiMBlot provides an unprecedentedly simple, rapid, and versatile platform for analyzing the vast number of combinatorial PTMs in biological pathways.

中文翻译：

使用无铜点击化学方法直接分析固定在表面上的单个蛋白质的翻译后修饰码

已经提出可以充当动态“分子条形码”的组合翻译后修饰（PTM），以调节独特的蛋白质功能。但是，由于缺乏合适的分析方法，对单一蛋白质分子上的组合PTM的研究受到了阻碍。在这里，我们描述了用于组合式PTM分析的可擦写单分子印迹（eSiMBlot）。该测定以高度复用的方式进行，并利用了共价蛋白固定，使用不同抗体进行的循环探测以及单分子荧光成像的好处。尤其是，使用无铜点击化学物质将表面轻松有效地共价固定在表面上，可进行多轮（> 10）的抗体擦除/重新捕获，而不会失去抗原性。而且，可以使用固定注册的单表位分子（例如HA肽）的共同注册的多个数据集的累积检测来提高抗体的检测率。最后，正如我们通过揭示配体诱导的表皮生长因子受体的新型磷酸化密码所证明的那样，eSiMBlot能够在单分子水平上直接显示组合的PTM代码并进行定量分析。因此，eSiMBlot提供了前所未有的简单，快速和通用的平台，用于分析生物途径中大量的组合PTM。正如我们通过揭示配体诱导的表皮生长因子受体的新型磷酸化密码所证明的那样。因此，eSiMBlot提供了前所未有的简单，快速和通用的平台，用于分析生物途径中大量的组合PTM。正如我们通过揭示配体诱导的表皮生长因子受体的新型磷酸化密码所证明的那样。因此，eSiMBlot提供了前所未有的简单，快速和通用的平台，用于分析生物途径中大量的组合PTM。

更新日期：2018-04-18

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