使用亲和纯化质谱法绘制差异蛋白质-蛋白质相互作用网络
Mapping Differential Protein-Protein Interaction Networks using Affinity Purification Mass Spectrometry.
作者信息
Kaushal Prashant, Ummadi Manisha R, Jang Gwendolyn M, Delgado Yennifer, Makanani Sara K, Blanc Sophie F, Winters Decan M, Xu Jiewei, Polacco Benjamin, Zhou Yuan, Stevenson Erica, Eckhardt Manon, Zuliani-Alvarez Lorena, Kaake Robyn, Swaney Danielle L, Krogan Nevan, Bouhaddou Mehdi
机构信息
Department of Microbiology, Immunology, and Molecular Genetics, University of California, Los Angeles, Los Angeles, CA, USA.
Institute for Quantitative and Computational Biosciences, University of California, Los Angeles, Los Angeles, CA, USA.
出版信息
ArXiv. 2024 May 15:arXiv:2405.09699v1.
Proteins congregate into complexes to perform fundamental cellular functions. Phenotypic outcomes, in health and disease, are often mechanistically driven by the remodeling of protein complexes by protein-coding mutations or cellular signaling changes in response to molecular cues. Here, we present an affinity purification-mass spectrometry (APMS) proteomics protocol to quantify and visualize global changes in protein-protein interaction (PPI) networks between pairwise conditions. We describe steps for expressing affinity-tagged "bait" proteins in mammalian cells, identifying purified protein complexes, quantifying differential PPIs, and visualizing differential PPI networks. Specifically, this protocol details steps for designing affinity-tagged "bait" gene constructs, transfection, affinity purification, mass spectrometry sample preparation, data acquisition, database search, data quality control, PPI confidence scoring, cross-run normalization, statistical data analysis, and differential PPI visualization. Our protocol discusses caveats and limitations with applicability across cell types and biological areas. For complete details on the use and execution of this protocol, please refer to Bouhaddou et al. 2023.
蛋白质聚集形成复合物以执行基本的细胞功能。在健康和疾病状态下，表型结果通常由蛋白质编码突变或细胞对分子信号的反应所导致的细胞信号变化对蛋白质复合物进行重塑而机械驱动。在这里，我们提出了一种亲和纯化-质谱（APMS）蛋白质组学方案，用于量化和可视化成对条件之间蛋白质-蛋白质相互作用（PPI）网络的全局变化。我们描述了在哺乳动物细胞中表达亲和标记的“诱饵”蛋白、鉴定纯化的蛋白质复合物、量化差异PPI以及可视化差异PPI网络的步骤。具体而言，该方案详细介绍了设计亲和标记的“诱饵”基因构建体、转染、亲和纯化、质谱样品制备、数据采集、数据库搜索、数据质量控制、PPI置信度评分、跨运行归一化、统计数据分析以及差异PPI可视化的步骤。我们的方案讨论了注意事项和局限性，并适用于各种细胞类型和生物学领域。有关本方案使用和执行的完整详细信息，请参考Bouhaddou等人2023年的文献。
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