The GFP Interactome: Implications for Assessing GFP-tagged Protein Interactions Using Immunoaffinity Purification-Mass Spectrometry Analysis

摘要

An IAP-MS method using in vivo chemical crosslinking for characterizing in planta protein interactions in Arabidopsis was established for LRR RLK-GFP expression lines by creating and using a GFP expression line as a control [2]. Based on extensive IAP-MS analysis of LRR RLK-GFP proteins, a GFP interactome has been defined which contains 79 proteins that have been observed in 90% of all GFP controls. Relative label-free quantification using the GFP control accounts for protein interactors that bind to the epitope tag of LRR RLK-GFP proteins, thus enhancing the assignments of bona fide LRR RLK interacting partners. The implications of our findings and approach can be extended to other affinity protein-tags of sufficient size, such as MBP or mCherry, in order to more confidently identify protein interactors using IAP-MS analysis.