ANALYSIS OF PHOSPHORYL HYDROXYAMINO ACIDS PRESENT IN HYDROLYZED CELL-EXTRACTS USING DABSYL DERIVATIZATION

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.