Rees Johanna Susan, Li Xue-Wen, Perrett Sarah, Lilley Kathryn Susan, Jackson Antony Philip
Department of Biochemistry, University of Cambridge, Cambridge, United Kingdom.
Cambridge Centre for Proteomics, University of Cambridge, Cambridge, United Kingdom.
Curr Protoc Protein Sci. 2015 Apr 1;80:19.27.1-19.27.18. doi: 10.1002/0471140864.ps1927s80.
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.
本手稿描述了一种鉴定定位于空间受限膜微环境中的蛋白质的全新通用方法。通过将辣根过氧化物酶（HRP）与一抗或二抗、抗原或底物、药物或毒素共价连接，使其与靶蛋白接触。然后加入一种基于生物素化酪胺的试剂。在HRP和过氧化氢存在的情况下，该试剂会转化为自由基，该自由基在共价标记距靶标几十到几百纳米范围内的蛋白质之前，只会扩散很短的距离。然后可以通过标准亲和色谱法分离生物素化的蛋白质，并通过液相色谱（LC）和质谱（MS）进行鉴定。通过在细胞培养中使用氨基酸稳定同位素标记（SILAC）或在肽水平进行等压标记，该测定可以实现定量。
