Identification of protein interaction partners in mammalian cells using SILAC-immunoprecipitation quantitative proteomics.
Identification of protein interaction partners in mammalian cells using SILAC-immunoprecipitation quantitative proteomics.
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DOI：
10.3791/51656
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发表时间：
2014-07-06
期刊：
影响因子：
--
通讯作者：
Goodfellow I
中科院分区：
文献类型：
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作者：
Emmott E;Goodfellow I
定量蛋白质组学与免疫亲和纯化、SILAC免疫沉淀相结合，为发现新的蛋白质：蛋白质相互作用提供了强有力的手段。通过准确地相对量化对照和测试样品中的蛋白质丰度，可以很容易地将真正的相互作用与实验污染物区分开来。低亲和力的相互作用可以通过使用不那么严格的缓冲条件来保存，并保持容易识别。该方案讨论了用稳定同位素标记的氨基酸标记组织培养细胞，转染和免疫沉淀亲和标记的感兴趣蛋白，然后准备提交给质谱仪。该方案然后讨论如何分析和解释从质谱仪返回的数据，以便识别与感兴趣的蛋白质相互作用的细胞伙伴。作为一个例子，该技术被应用于鉴定与真核翻译起始因子eIF4AI和eIF4AII结合的蛋白质。
Quantitative proteomics combined with immuno-affinity purification, SILAC immunoprecipitation, represent a powerful means for the discovery of novel protein:protein interactions. By allowing the accurate relative quantification of protein abundance in both control and test samples, true interactions may be easily distinguished from experimental contaminants. Low affinity interactions can be preserved through the use of less-stringent buffer conditions and remain readily identifiable. This protocol discusses the labeling of tissue culture cells with stable isotope labeled amino acids, transfection and immunoprecipitation of an affinity tagged protein of interest, followed by the preparation for submission to a mass spectrometry facility. This protocol then discusses how to analyze and interpret the data returned from the mass spectrometer in order to identify cellular partners interacting with a protein of interest. As an example this technique is applied to identify proteins binding to the eukaryotic translation initiation factors: eIF4AI and eIF4AII.