Separation of phosphorylated peptides utilizing dual pH- and temperature-responsive chromatography

The phosphorylation of a peptide is considered to be one of the most important post-translational modification reactions that can alter protein function in mammalian cells. To separate and purify, we developed a dual temperature- and pH-responsive chromatography based on terpolymer composed of N-isopropylacrylamide, N,N'-dimethylaminopropylacrylamide and butylmethacrylate. The property of the surface of the terpolymer-grafted stationary phase altered from hydrophilic to hydrophobic, and from changed to non-charged by changes in the temperature and the pH, respectively. In addition, it was possible to appear and hide ion-exchange groups on the polymer chain surface by temperature changes. These phenomena resulted from changes in the charge and the hydrophobicity of the pH- and temperature-responsive polymer on the stationary surface by controlling the temperature. In the developed environmental-responsive chromatographic system, the ionizable dimethylamino group of N,N'-dimethylaminopropylacrylamide in terpolymer played a key role for the separation. We applied the developed chromatographic system to the separation of phosphorylated compounds, such as phospho-tyrosine, phosphopeptide and oligonucleotides. At a low column temperature, the electrostatic interaction plays a predominant role for retain anionic phosphorylated compounds, because of the strong interaction between the cationic dimethylamino group in the stationary phase and the anionic phosphoric group in the analyte. On the contrary, the hydrophobic interaction became predominant upon increasing the temperature. The results showed that both the electrostatic and the hydrophobic interactions became controllable with a temperature change during the chromatographic process. Dual pH- and temperature-responsive chromatography would be very useful for biomacromolecules separation and purification.