Determination of dityrosine, phosphotyrosine, phosphothreonine, and phosphoserine by high-performance liquid chromatography.

4'-Dimethylaminoazobenzene-4-sulfonyl chloride is a chromophoric reagent commonly used to detect amino acids at picomole levels. This article describes a single-column reverse-phase high-performance liquid chromatography system which allows the resolution and analysis of the dabsyl chloride derivatives of several modified amino acids. Highly derivatized C18 (22 and 31%) columns from Phenomenex are run at pH 8.1 to separate dityrosine, the phosphorylated amino acids (o-phosphoserine, o-phosphothreonine, and o-phosphotyrosine), and the 17 other amino acids normally present in protein hydrolysates. In order to gain additional sensitivity and to verify the presence of dityrosine, the dityrosine and lysine peaks are collected and run at pH 4.1 on the same columns. Our experience indicates that, with the described setup, the lower limit for accurate and reproducible detection is near 1 pmol. This method has been applied to the analysis of dityrosine in uv-irradiated calmodulin and cardiac troponin C and to the detection of phosphorylation sites in several polypeptides.