Phosphoamino acid analysis hydrolysis

Phosphoamino acid analysis is accomplished by partial hydrolysis of the purified P-labeled protein either eluted from an SDS gel (Sections A and B) or when bound to Immobilon-P (Section D). Incubation of proteins or peptides in concentrated acid or base results in hydrolysis of peptide bonds and release of peptides and, eventually, individual amino acids. Phosphodiester bonds, however, are also unstable under these reaction conditions and dephosphorylation of the phosphoamino acids and release of free [ PJphosphate also occur. As a consequence hydrolysis times are critical. Hydrolysis of membrane-bound phosphoproteins is recommended except in cases where the protein of interest transfers to Immobilon-P poorly. 

Alkaline hydrolysis with barium, sodium, or lithium hydroxides (0.2-4 M) at 110°C for 18-70 h126-291 requires special reaction vessels and handling. Reaction mixtures are neutralized after hydrolysis and barium ions have to be removed by precipitation as their carbonate or sulfate salts prior to analysis which leads to loss of hydrolysate. Correspondingly, peptide contents are difficult to perform by this procedure. Preferred conditions for alkaline hydrolysis are 4M LiOH at 145 °C for 4-8 h where >95% of tryptophan is recovered 291 An additional inconvenience of the alkaline hydrolysis procedure is the dilution effect in the neutralization step and thus the difficult application to the analyzer if micro-scale analysis is to be performed. The main advantage is the good recovery of tryptophan and of acid-labile amino acid derivatives such as tyrosine-0-sulfate1261 (Section 6.6) as well as partial recovery of phosphoamino acids, particularly of threonine- and tyrosine-O-phosphate (Section 6.5). 

Kamps, M.P., Sefton, B.M (1989) Acid and Base Hydrolysis of Phosphoproteins Bound to Immo-bilon Facilitates the Analysis of Phosphoamino Acids in Gel-Fractionated Proteins, Anal. Biochem. 176, 22-27. 

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