Probing Protein–Protein Interactions with Label-Free Mass Spectrometry Quantification in Combination with Affinity Purification by Spin-Tip Affinity Columns
| Autor: | Jingwu Kang, Lifeng Pan, Shichen Hu, Ying Han, Tao Fu, Guizhen Liu, Xuepei Zhang, Piliang Hao, Mengmeng Zheng |
|---|---|
| Rok vydání: | 2020 |
| Předmět: |
Nitrilotriacetic Acid
Proteomics Recombinant Fusion Proteins Quantitative proteomics Ion chromatography 010402 general chemistry Tandem mass spectrometry 01 natural sciences Interactome Chromatography Affinity Analytical Chemistry Protein–protein interaction chemistry.chemical_compound Affinity chromatography Tandem Mass Spectrometry Humans Histidine Protein Interaction Maps Chromatography High Pressure Liquid Chromatography 010401 analytical chemistry Nitrilotriacetic acid Signal transducing adaptor protein 0104 chemical sciences chemistry Peptides Microtubule-Associated Proteins Oligopeptides Protein Binding |
| Zdroj: | Analytical Chemistry. 92:3913-3922 |
| ISSN: | 1520-6882 0003-2700 |
| Popis: | We describe an affinity purification-mass spectrometry (AP-MS) method for probing the 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 pipet (spin-tip columns). The recombinant His6-tagged protein (bait protein) was reversibly immobilized on the affinity column through the chelating group 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 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 seven literature-reported FYCO1-associating proteins were identified. Among them CCZ1 and MON1A were further biochemically validated, and the direct interaction between the FYCO1 GOLD domain with CCZ1 was confirmed by co-immunoprecipitation experiments. |
| Databáze: | OpenAIRE |
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