High-performance liquid chromatography of acid-stable and acid-labile phosphoamino acids

A high-performance liquid chromatographic system has been developed which permits the separation of both acid-stable and acid-labile phosphoamino acids. An anion-exchange resin and two buffers of different ionic strength and near neutral pH are used. A low-ionic-strength buffer is used for the separation of N-omega-phosphoarginine and N-epsilon-phospholysine, while the higher-ionic-strength buffer permits the clear separation of tau-phosphohistidine, omicron-phosphoserine and omicron-phosphothreonine. An in-stream fluorometric detection system using omicron-phthalaldehyde permits the rapid analysis of samples containing as little as 25 pmoles of phosphoamino acid. This method has been applied to the detection of tau-phosphohistidine from alkaline digests of chemically phosphorylated calf thymus histone 4 and bovine myelin basic protein.