A proteomics approach to the cell-surface interactome using the enzyme-mediated activation of radical sources reaction
A proteomics approach to the cell-surface interactome using the enzyme-mediated activation of radical sources reaction
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DOI：
10.1002/pmic.201100551
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发表时间：
2012-01-01
期刊：
影响因子：
3.4
通讯作者：
Honke, Koichi
中科院分区：
文献类型：
--
作者：
Jiang, Songlin;Kotani, Norihiro;Honke, Koichi
我们之前报道了一种简单的方法来分析活细胞中细胞表面分子的相互作用。这种方法被称为酶介导的自由基源激活（EMARS），其特点是由辣根过氧化物酶（HRP）形成标记试剂的自由基。在此，我们提出了一种结合EMARS反应和基于质谱的蛋白质组学技术来研究细胞表面分子相互作用组的方法。在目前的研究中，我们采用了一种新的标记试剂，荧光素偶联芳基酰胺。用荧光图像分析仪在电泳凝胶中直接检测EMARS反应产生的荧光素标记蛋白。用抗荧光素抗体固定化树脂对这些产物进行了免疫亲和层析纯化和浓缩。纯化的荧光素标记蛋白随后进行基于质谱的蛋白质组学分析。利用酶标霍乱毒素亚基B（识别脂质筏标记物神经节苷脂GM1）进行分析，发现30种膜和分泌蛋白是与GM1共聚的细胞表面分子的候选蛋白。所提出的方法将为研究细胞表面各种生物事件中的功能分子相互作用提供线索。
We previously reported a simple method to analyze the interaction of cell-surface molecules in living cells. This method termed enzyme-mediated activation of radical sources (EMARS) is featured by radical formation of the labeling reagent by horseradish peroxidase (HRP). Herein, we propose an approach to the cell-surface molecular interactome by using combination of this EMARS reaction and MS-based proteomics techniques. In the current study, we employed a novel labeling reagent, fluorescein-conjugated arylazide. The fluorescein-tagged proteins resulting from the EMARS reaction were directly detected in the electrophoresis gels with a fluorescence image analyzer. These products were also purified and concentrated by immunoaffinity chromatography with anti-fluorescein antibody-immobilized resins. The purified fluorescein-tagged proteins were subsequently subjected to an MS-based proteomics analysis. Analysis using HRP-conjugated cholera toxin subunit B, which recognizes a lipid raft marker, ganglioside GM1, revealed 30 membrane and secreted proteins that were candidates for the cell-surface molecules coclustering with GM1. The proposed approach will provide a clue to study functional molecular interactions in a variety of biological events on the cell surface.