ANALYSIS OF THE PHOSPHOAMINO ACID CONTENT OF PHOSPHOPROTEINS

A method has been developed for the analysis of phosphoserine, phospho threonine and phosphotyrosine in P-32-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 cova lently modified phosphoamino acids are separated on silica TLC aluminu m sheets using a one-dimensional solvent system. The method is straigh tforward and permits the simultaneous analysis of numerous samples. Ve ry clean chromatograms are obtained enabling the unambiguous identific ation of the well separated dabsylated phosphoamino acids with autorad iography. The phosphoamino acids can be quantified by simply cutting o ut the relevant spots from the aluminum sheets followed by P-32-quanti fication using liquid scintillation spectrometry.