Separation of diethylpyrocarbonate-modified histidine-containing peptides using strong cation exchange chromatography

Abstract

This work focuses on the separation of diethylpyrocarbonate-modified histidine-containing peptides by strong cation exchange (SCX) chromatography prior to mass spectrometric analysis. The decrease in positive charge of diethylpyrocarbonate-treated imidazole in histidine, masked by the incorporation of the carbethoxyl group, provides us with an opportunity to use SCX for separating diethylpyrocarbonate-modified histidine-containing peptides from the unmodified ones. In this report, the diethylpyrocarbonate-modified histidine-containing peptide is eluted faster than the untreated peptide. Furthermore, the carbethoxy group can be effectively removed to generate the unmodified imidazole in the histidine residue by neutral hydroxylamine.