Target Protein Identification on Photocatalyst-Functionalized Magnetic Affinity Beads
Target Protein Identification on Photocatalyst-Functionalized Magnetic Affinity Beads
复制标题
光触媒功能化磁力亲和珠上的目标蛋白鉴定
DOI：
10.1002/cpps.108
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发表时间：
2020
影响因子：
--
通讯作者：
中村浩之
中科院分区：
文献类型：
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作者：
對馬理彦;佐藤伸一;中根啓太;中村浩之
尽管已经开发了各种亲和层析和光亲和标记方法用于生物活性分子的靶蛋白鉴定，但使用这些技术通常难以检测以弱瞬时亲和力结合配体的蛋白质。我们开发了使用钌光催化剂的单电子转移介导的酪氨酸标记。使用 1-甲基-4-芳基-乌拉唑 (MAUra) 的邻近标记可高效、高选择性地标记靠近光催化剂的蛋白质。在亲和珠上进行这种标记反应可以标记以弱瞬时亲和力结合配体的蛋白质。在本文中，描述了使用钌光催化剂功能化磁性亲和珠上的光催化剂邻近标记来识别目标蛋白的新方案。 © 2020 Wiley periodicals LLC. 基本方案 1：钌光催化剂的合成基本方案 2：叠氮化物或脱硫生物素标记试剂的合成基本方案 3：光催化剂和配体功能化亲和珠的制备基本方案 4：细胞裂解液中的目标蛋白标记基本方案 5：使用 MAUra-DTB 富集标记蛋白LC-MS/MS 分析基本方案 6：荧光标记蛋白的 2D-DIGE 分析
Although various affinity chromatography and photoaffinity labeling methods have been developed for target protein identification of bioactive molecules, it is often difficult to detect proteins that bind the ligand with weak transient affinity using these techniques. We have developed single electron transfer–mediated tyrosine labeling using ruthenium photocatalysts. Proximity labeling using 1‐methyl‐4‐aryl‐urazole (MAUra) labels proteins in close proximity to the photocatalyst with high efficiency and selectivity. Performing this labeling reaction on affinity beads makes it possible to label proteins that bind the ligand with weak transient affinity. In this article, novel protocols are described for target protein identification using photocatalyst proximity labeling on ruthenium photocatalyst‐functionalized magnetic affinity beads. © 2020 Wiley Periodicals LLC.Basic Protocol 1: Synthesis of ruthenium photocatalystBasic Protocol 2: Synthesis of azide‐ or desthiobiotin‐conjugated labeling reagentsBasic Protocol 3: Preparation of photocatalyst and ligand‐functionalized affinity beadsBasic Protocol 4: Target protein labeling in cell lysateBasic Protocol 5: Enrichment of labeled proteins with MAUra‐DTB for LC‐MS/MS analysisBasic Protocol 6: 2D‐DIGE analysis of fluorescence‐labeled proteins