SUCCESSFUL AND RAPID VERIFICATION OF THE PRESENCE OF A PHOSPHATE GROUP IN SYNTHETIC PHOSPHOPEPTIDES USING THE CONDITIONS OF STANDARD DABS-CL AMINO-ACID-ANALYSIS

The increased importance of phosphopeptides, and the currently used po st-assembly phosphorylation protocol in synthetic peptide laboratories , requires a rapid and sensitive method to verify the presence of the phosphate group in synthetic phosphopeptides. A reversed-phase high-pe rformance liquid chromatography protocol has been developed to verify the success of the phosphorylation reaction in synthetic phosphopeptid es after hydrolysis and derivatization with 4-dimethylamino-azobenzene -4'-sulphonyl chloride. Phosphoamino acid standards and model phosphop eptides were used to study the optimal elution and hydrolysis conditio ns of phosphoamino acids. A 1.5-hour, gas-phase acidic hydrolysis cond ition liberated the phosphoamino acids from the phosphopeptides, and s till did not destroy them. After hydrolysis, the dabsylated free phosp hoamino acids were baseline separated from the other acidic amino acid s and were eluted from the reversed-phase column in the following orde r: phosphoserine, phosphothreonine, and phosphotyrosine. The utility o f the approach was demonstrated by the phosphoamino acid analysis of s everal synthetic phosphopeptides, in which the amino acid environment of the phosphorylated serine or tyrosine was different. This method ma y not only be applicable for phosphopeptides, but also for verifying t he presence of phosphate groups in phosphoproteins.