Probing Protein–Protein Interactions with Label-Free Mass Spectrometry Quantification in Combination with Affinity Purification by Spin-Tip Affinity Columns
Guizhen Liu, Tao Fu, Ying Han, Shichen Hu, Xuepei Zhang, Meng‐Meng Zheng, Piliang Hao, Lifeng Pan, Jingwu Kang
Abstract
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.