Pa. Dewitte et al., ANALYSIS OF THE PHOSPHOAMINO ACID CONTENT OF PHOSPHOPROTEINS, Journal of pharmaceutical and biomedical analysis, 14(8-10), 1996, pp. 1063-1067
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.