Analysis of the phosphoamino acid content of phosphoproteins

A method has been developed for the analysis of phosphoserine, phosphothreonine and phosphotyrosine in 32P-phosphoprotein hydrolysates. The hydrolysates are treated with dabsyl reagent (28.8 mM) for 10 min at 70 degrees C. After a clean-up using a disposable C18 column, the covalently modified phosphoamino acids are separated on silica TLC aluminum sheets using a one-dimensional solvent system. The method is straightforward and permits the simultaneous analysis of numerous samples. Very clean chromatograms are obtained enabling the unambiguous identification of the well separated dabsylated phosphoamino acids with autoradiography. The phosphoamino acids can be quantified by simply cutting out the relevant spots from the aluminum sheets followed by 32P-quantification using liquid scintillation spectrometry.