Host-Guest Protein Assembly for Affinity Purification of Methyllysine Proteomes
Host-Guest Protein Assembly for Affinity Purification of Methyllysine Proteomes
复制标题
用于甲基赖氨酸蛋白质组亲和纯化的主客体蛋白质组装
DOI：
10.1021/acs.analchem.0c01643
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发表时间：
2020-07-07
影响因子：
7.4
通讯作者：
Wang, Ruibing
中科院分区：
文献类型：
--
作者：
Li, Linting;Liu, Min;Wang, Ruibing
蛋白质-蛋白质相互作用驱动生物大分子的自组装，从而在细胞水平上实现重要的生理功能。超分子化学家已经开发出人工主客体相互作用，与天然蛋白质-蛋白质相互作用相似但又不同且正交。例如，葫芦脲是合成受体，可以以高亲和力特异性识别具有 N 末端芳香残基的蛋白质，但这种相互作用可以通过金刚烷胺等小分子的竞争来逆转。在此，我们通过结合基于葫芦[7]尿素的主客体相互作用和共价蛋白-肽反应，开发了一种位点特异性、定向的蛋白质展示方法。甲基赖氨酸结合蛋白 HP1 β 染色结构域 (CD) 通过宿主-客体相互作用被固定，并用作从癌细胞捕获甲基赖氨酸蛋白质组的“诱饵”。捕获的“鱼”——含有甲基赖氨酸的蛋白质——可以通过金刚烷胺的竞争性置换以非变性和无痕的方式释放。通过随后的质谱分析，这种亲和纯化方法从癌细胞中 255 个 HP1 β CD 结合蛋白的 66 个甲基化蛋白中的 101 个已识别位点中发现了 73 个新的甲基赖氨酸位点。因此，这项工作提出了一种与质谱联用的甲基赖氨酸蛋白亲和纯化中人工主客体蛋白组装的新策略。
Protein-protein interactions drive self-assembly of biomacromolecules and thus enable important physiological functions at a cellular level. Supramolecular chemists have developed artificial host-guest interactions that are similar with, yet distinct from and orthogonal to, the natural protein-protein interactions. For instance, cucurbit[n]urils are synthetic receptors that can specifically recognize proteins with N-terminal aromatic residues with high affinities, yet this interaction can be reversed by the competition of small molecules such as amantadine. Herein, we develop a site-specific, oriented protein-display method by combining the host-guest interaction based on cucurbit[7]uril and a covalent protein-peptide reaction. A methyllysine-binding protein HP1 beta chromodomain (CD) is immobilized via host-guest interactions and used as the "bait" to capture methyllysine proteomes from cancer cells. The captured "fish"-methyllysine-containing proteins-can be released via competitive displacement by amantadine in a nondenaturing and traceless manner. This affinity purification method found 73 novel methyllysine sites from 101 identified sites among 66 methylated proteins from 255 HP1 beta CD-binding proteins in cancer cells via subsequent mass spectrometric analysis. This work thereby presents a new strategy of artificial host-guest protein assembly in affinity purification of methyllysine proteins in coupling to mass spectrometry.