Leibniz-Forschungsinstitut für Molekulare Pharmakologie, Robert-Rössle-Straße 10, 13125 Berlin, Germany.
Institut für Chemie, Humboldt-Universität zu Berlin, Brook-Taylor-Straße 2, 12489 Berlin, Germany.
STAR Protoc. 2021 Jan 14;2(1):100277. doi: 10.1016/j.xpro.2020.100277. eCollection 2021 Mar 19.
This protocol describes an affinity enrichment approach from mammalian cell extracts to identify protein binding partners of inositol hexakisphosphate (InsP) and 5-diphosphoinositol pentakisphosphate (5PP-InsP), two important eukaryotic metabolites. The interactomes are annotated using mass spectrometry-based proteomics, and comparison against a control resin can uncover hundreds of protein targets. Quantitative analysis of InsP- versus 5PP-InsP-binding proteins highlights specific protein-ligand interactions. The approach is applicable to different cells and organisms and will contribute to a mechanistic understanding of inositol poly- and pyrophosphate signaling. For complete details on the use and execution of this protocol, please refer to Furkert et al. (2020).
本方案描述了一种从哺乳动物细胞提取物中鉴定肌醇六磷酸（InsP）和 5-二磷酸肌醇五磷酸（5PP-InsP）的蛋白质结合伴侣的亲和富集方法，这两种物质是两种重要的真核代谢物。互作组使用基于质谱的蛋白质组学进行注释，与对照树脂的比较可以发现数百个蛋白质靶标。InsP 与 5PP-InsP 结合蛋白的定量分析突出了特定的蛋白质-配体相互作用。该方法适用于不同的细胞和生物体，将有助于深入了解肌醇多磷酸和焦磷酸信号。有关本方案使用和执行的完整详细信息，请参阅 Furkert 等人（2020 年）。
