使用串联亲和纯化结合质谱技术在哺乳动物细胞中建立蛋白质-蛋白质相互作用网络的方案。

Key Laboratory of Structural Biology of Zhejiang Province, School of Life Sciences, Westlake University, Hangzhou, Zhejiang 310024, China; Westlake Laboratory of Life Sciences and Biomedicine, Hangzhou, Zhejiang 310024, China; Institute of Biology, Westlake Institute for Advanced Study, Hangzhou, Zhejiang 310024, China.

Department of Experimental Radiation Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA.

STAR Protoc. 2022 Jul 19;3(3):101569. doi: 10.1016/j.xpro.2022.101569. eCollection 2022 Sep 16.

Identification of protein interactors is fundamental to understanding their functions. Here, we describe a modified protocol for tandem affinity purification coupled with mass spectrometry (TAP/MS), which includes two-step purification. We detail the S-, 2×FLAG-, and Streptavidin-Binding Peptide (SBP)- tandem tags (SFB-tag) system for protein purification. This protocol can be used to identify protein interactors and establish a high-confidence protein-protein interaction network based on computational models. This is particularly useful for identifying interacting proteins for subsequent functional studies. For complete details on the use and execution of this protocol, please refer to Bian et al. (2021).

鉴定蛋白质相互作用因子对于理解其功能至关重要。在这里，我们描述了一种改良的串联亲和纯化与质谱联用（TAP/MS）的方案，该方案包括两步纯化。我们详细介绍了 S-、2×FLAG-和 Streptavidin-Binding Peptide（SBP）-串联标签（SFB-tag）系统的蛋白质纯化。该方案可用于鉴定蛋白质相互作用因子，并根据计算模型建立高可信度的蛋白质-蛋白质相互作用网络。这对于鉴定后续功能研究的相互作用蛋白特别有用。如需了解该方案的使用和执行的完整详细信息，请参考 Bian 等人（2021 年）。