Abstract：
Quantitative and qualitative analyses of cell protein composition using liquid chromatography/tandem mass spectrometry are now standard techniques in biological and clinical research. However, the quantitative analysis of protein-protein interactions (PPIs) in cells is also important since these interactions are the bases of many processes, such as the cell cycle and signaling pathways. This paper describes the application of Skyline software for the identification and quantification of the biotinylated form of the biotin acceptor peptide (BAP) tag, which is a marker of in vivo PPIs. The tag was used in the Proximity Utilizing Biotinylation (PUB) method, which is based on the co-expression of BAP-X and BirA-Y in mammalian cells, where X or Y are interacting proteins of interest. A high level of biotinylation was detected in the model experiments where X and Y were pluripotency transcription factors Sox2 and Oct4, or heterochromatin protein HP1γ. MRM data processed by Skyline were normalized and recalculated. Ratios of biotinylation levels in experiment versus controls were 86 ± 6 (3 h biotinylation time) and 71 ± 5 (9 h biotinylation time) for BAP-Sox2 + BirA-Oct4 and 32 ± 3 (4 h biotinylation time) for BAP-HP1γ + BirA-HP1γ experiments. Skyline can also be applied for the analysis and identification of PPIs from shotgun proteomics data downloaded from publicly available datasets and repositories.
摘要：
使用液相色谱/串联质谱法对细胞蛋白质组成进行定量和定性分析现在是生物学和临床研究中的标准技术。然而,细胞中蛋白质-蛋白质相互作用（PPI）的定量分析也很重要，因为这些相互作用是许多过程的基础，如细胞周期和信号通路。本文介绍了Skyline软件在生物素化形式的生物素受体肽（BAP）标签的鉴定和定量中的应用，它是体内PPI的标志物。该标签用于利用生物素化（PUB）方法的邻近，基于BAP-X和BirA-Y在哺乳动物细胞中的共表达，其中X或Y是感兴趣的相互作用蛋白。在模型实验中检测到高水平的生物素化，其中X和Y是多能性转录因子Sox2和Oct4或异染色质蛋白HP1γ。对Skyline处理的MRM数据进行归一化和重新计算。对于BAP-Sox2BirA-Oct4，实验与对照的生物素化水平比率为86±6（3h生物素化时间）和71±5（9h生物素化时间），对于BAP-HP1γBirA-HP1γ实验为32±3（4h生物素化时间）。Skyline还可以应用于从公开可用的数据集和存储库下载的shot弹枪蛋白质组学数据中的PPI的分析和识别。
