Selective Proteomic Proximity Labeling Assay Using Tyramide (SPPLAT): A Quantitative Method for the Proteomic Analysis of Localized Membrane-Bound Protein Clusters.
Selective Proteomic Proximity Labeling Assay Using Tyramide (SPPLAT): A Quantitative Method for the Proteomic Analysis of Localized Membrane-Bound Protein Clusters.
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DOI：
10.1002/0471140864.ps1927s80
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发表时间：
2015-04-01
影响因子：
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通讯作者：
Jackson, Antony Philip
中科院分区：
文献类型：
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作者：
Rees, Johanna Susan;Li, Xue-Wen;Jackson, Antony Philip
这份手稿描述了一种新的和一般的方法来识别蛋白质定位到空间受限的膜微环境。辣根过氧化物酶（HRP）通过与一抗或二抗、抗原或底物、药物或毒素共价连接而与靶蛋白接触。然后加入生物素化酪胺基试剂。在HRP和过氧化氢的存在下，该试剂被转化为自由基，该自由基在共价标记距离目标几十到几百纳米内的蛋白质之前仅扩散很短的距离。生物素化的蛋白质然后可以通过标准亲和色谱法分离，并通过液相色谱法（LC）和质谱法（MS）鉴定。该测定可以通过使用细胞培养物中氨基酸的稳定同位素标记（SILAC）或肽水平的同量异位素标记来定量进行。
This manuscript describes a new and general method to identify proteins localized into spatially restricted membrane microenvironments. Horseradish peroxidase (HRP) is brought into contact with a target protein by being covalently linked to a primary or secondary antibody, an antigen or substrate, a drug, or a toxin. A biotinylated tyramide-based reagent is then added. In the presence of HRP and hydrogen peroxide, the reagent is converted into a free radical that only diffuses a short distance before covalently labeling proteins within a few tens to hundreds of nanometers from the target. The biotinylated proteins can then be isolated by standard affinity chromatography and identified by liquid chromatography (LC) and mass spectrometry (MS). The assay can be made quantitative by using stable isotope labeling with amino acids in cell culture (SILAC) or isobaric tagging at the peptide level.