Problems with phosphoamino acid analysis using alkaline hydrolysis

Although a seemingly simple concept, sample volume and the reaction vessel size are important considerations when undertaking an alkaline hydrolysis of a phosphoprotein, particularly if the phosphoamino acid of interest is either phosphohistidine or phosphotyrosine. It should be noted that the experiments conducted in this article used large concentrations of both phosphotyrosine and the most stable form of phosphohistidine (8), which highlights the problems that may be faced during phosphoamino acid analysis of a protein sample that contains only small amounts of either phosphoamino acid. Although not tested, it is likely that similar hydrolysis effects may occur for phospholysine. If the reaction volume is to be kept to a minimum and the alkaline digestion is from either a membrane blot or in solution, then the use of a mineral oil overlay should be considered to prevent concentration of the alkali and hydrolysis of the phosphate moiety.