Host–Guest Protein Assembly for Affinity Purification of Methyllysine Proteomes
Linting Li, Min Liu, Ludan Yue, Rui Wang, Rui Wang, Ning Zhang, Yujie Liang, Liang Zhang, Lixin Cheng, Jiang Xia, Ruibing Wang, Ruibing Wang
Abstract
Protein–protein interactions drive self-assembly of biomacromolecules and thus enable important physiological functions at a cellular level. Supramolecular chemists have developed artificial host–guest interactions that are similar with, yet distinct from and orthogonal to, the natural protein–protein interactions. For instance, cucurbit[n]urils are synthetic receptors that can specifically recognize proteins with N-terminal aromatic residues with high affinities, yet this interaction can be reversed by the competition of small molecules such as amantadine. Herein, we develop a site-specific, oriented protein-display method by combining the host–guest interaction based on cucurbit[7]uril and a covalent protein–peptide reaction. A methyllysine-binding protein HP1β chromodomain (CD) is immobilized via host–guest interactions and used as the “bait” to capture methyllysine proteomes from cancer cells. The captured “fish”—methyllysine-containing proteins—can be released via competitive displacement by amantadine in a nondenaturing and traceless manner. This affinity purification method found 73 novel methyllysine sites from 101 identified sites among 66 methylated proteins from 255 HP1β CD-binding proteins in cancer cells via subsequent mass spectrometric analysis. This work thereby presents a new strategy of artificial host–guest protein assembly in affinity purification of methyllysine proteins in coupling to mass spectrometry.