Successful and Rapid Verification of the Presence of a Phosphate Group in Synthetic Phosphopeptides Using the Conditions of Standard Dabs-Cl Amino Acid Analysis

Abstract

The increased importance of phosphopeptides, and the currently used post-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-performance liquid chromatography protocol has been developed to verify the success of the phosphorylation reaction in synthetic phosphopeptides after hydrolysis and derivatization with 4-dimethylamino-azobenzene-4′-sulphonyl chloride. Phosphoamino acid standards and model phosphopeptides were used to study the optimal elution and hydrolysis conditions of phosphoamino acids. A 1.5-hour, gas-phase acidic hydrolysis condition liberated the phosphoamino acids from the phosphopeptides, and still did not destroy them. After hydrolysis, the dabsylated free phosphoamino acids were baseline separated from the other acidic amino acids and were eluted from the reversed-phase column in the following order: phosphoserine, phosphothreonine, and phosphotyrosine. The utility of the approach was demonstrated by the phosphoamino acid analysis of several synthetic phosphopeptides, in which the amino acid environment of the phosphorylated serine or tyrosine was different. This method may not only be applicable for phosphopeptides, but also for verifying the presence of phosphate groups in phosphoproteins.