发展的方法论biotinylate TGN-resident蛋白质。(A)的示意图HRP-TGN46内生TGN-resident蛋白质的生物素酰化标签。在biotin-phenol和过氧化氢的存在,合催化形成membrane-impermeable biotin-phenoxyl附近内源性蛋白质共价标签。(B)的透射电子显微照片HRP-TGN46-expressing海拉细胞孵化与民建联和过氧化氢为10分钟。(比例尺,500海里;缩放尺度酒吧,100 nm)。呃,内质网;GA,高尔基体;米,线粒体;N,核;不,核膜。 (C) STED microscopy of HRP-TGN46–expressing HeLa cells following biotinylation, stained with fluorescent streptavidin and immunofluorescence labeling of Golgin-97. (Scale bars, 5 µm; Insets, 2 µm.) (D) Streptavidin affinity isolation of biotinylated proteins from total cell lysate of HRP-TGN46–expressing cells incubated with H2O2 in the presence or absence of BP, and their corresponding known subcellular localization. (E) Scatterplot of all HRP-TGN46–labeled proteins, with known mannose-6-phosphate–tagged proteins overlaid in red, and example TGN retrograde cargoes highlighted by blue labels; n = 5 independent repeats. (F–G) Graphical representation of overrepresentation of gene ontology cellular compartments (F) and biological processes (G) within the HRP-TGN46–proteome according to their calculated P value. Overrepresented gene ontology terms are colored green, and underrepresented terms are colored red. The numbers of identified proteins belonging to each category are provided within parentheses. Credit:美国国家科学院院刊》上(2022)。DOI: 10.1073 / pnas.2201980119