Pa. Dewitte et al., ANALYSIS OF PHOSPHORYL HYDROXYAMINO ACIDS PRESENT IN HYDROLYZED CELL-EXTRACTS USING DABSYL DERIVATIZATION, Analytical biochemistry, 226(1), 1995, pp. 1-9
We have developed a new method for the quantification of phosphoserine
, phosphothreonine, and phosphotyrosine as dabsyl derivatives in acid-
hydrolyzed extracts of P-32-labeled A431 cells. In the first step the
phosphoamino acids are concentrated using a disposable anion-exchange
column. Subsequently, the phosphoamino acid fraction is treated with d
absyl reagent (28.8 mM) for 10 min at 70 degrees C. After cleanup with
a second anion-exchange column followed by separation on a disposable
C18 column, the covalently modified phosphoamino acids are separated
on silica TLC sheets using a one-dimensional solvent system. The major
advantages of this method are the complete separation of dabsylated P
-Ser, P-Thr, and P-Tyr on silica aluminum sheets in a very reproducibl
e way without the interference of P-32 contaminants originating from h
ydrolyzed cell extracts. Very clean chromatograms are obtained, enabli
ng the fast and unambiguous quantification of the phosphoamino acids b
y simply cutting out the relevant spots from the aluminum sheets. A hi
gh sensitivity is achieved by the removal of the amino acids before de
rivatization of the sample. This allows the use of relatively low amou
nts of [P-32]orthophosphate to load up the cells. Most important, the
method allows the simultaneous analysis of dozens of samples within 1
day, making it a very convenient technique for routine analysis of the
phosphorylation state of cultured cells. Consequently the method is w
ell suited to implementation in large screenings for inhibitors of pro
tein kinases, e.g., PTK inhibitors, in whole-cell-studies. (C) 1995 Ac
ademic Press, Inc.