Profiling Protein Interactions with Label-Free Quantification Proteomics in Combination with Affinity Purification by micro- Affinity Column
Profiling Protein Interactions with Label-Free Quantification Proteomics in Combination with Affinity Purification by micro- Affinity Column
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结合微亲和柱的亲和纯化，利用无标记定量蛋白质组学分析蛋白质相互作用
DOI：
10.1021/acs.analchem.9b05355
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发表时间：
2020
期刊：
影响因子：
--
通讯作者：
Jingwu Kang
中科院分区：
文献类型：
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作者：
Guizhen Liu;Tao Fu;Han Ying;Shichen Hu;Xuepei Zhang;Mengmeng Zheng;Hao Piliang;Lifeng Pan;Jingwu Kang
We describe an affinity purification-mass spectrometry (AP-MS) method for probing interactome of a special targeting protein. The AP was implemented with monolithic micro immobilized metal ion affinity chromatography columns (m-IMAC) which were prepared by photoinitiated polymerization in the tip of a pipette (Spin-tip columns). The recombinant His6-tagged protein (bait protein) was reversibly immobilized on the affinity column through chelating group the nitrilotriacetic acid (NTA)-Ni2+. The bait protein and its interacting partners can be easily eluted from the affinity matrix. The pulled-down cellular proteins were then analyzed with the label-free quantitative proteomics. We used this method for probing the interactome concerning the GOLD (Golgi dynamics) domain of the autophagy associated adaptor protein FYCO1. Totally 96 proteins including 7 literature reported FYCO1-associating proteins were identified. Among them CCZ1 and MON1A were further biochemically validated, and the direct interaction between FYCO1 GOLD domain with CCZ1 were confirmed by co-immunoprecipitation experiments.
