Phosphoamino acid analysis

The freeze-dried peptide is hydrolyzed in 6 N HCl at 110°C for 1 h, HCl is removed by evaporation, and the residue is dissolved in H2O, mixed with unlabeled phosphoamino acid markers, and electrophoresed on the Kodak cellulose sheets (Erdodi et al., 1987). The phosphoamino acids are identified by ninhydrin staining and autoradiography (Rg. 2). The procedure is only qualitative, because of the incomplete hydrolysis of the phosphopeptide and the partial destruction of the liberated phosphoserine and phosphothreonine. 

FIGURE 2 Phosphoamino add analysis of individual phospho-peptides (A-F) from p-labeled LC20. Pj, Ser-P, and Thr-P are labeled inorganic phosphate, serine-phosphate, and threonine-phosphate, respectively, and Pep-P is the unhydrolyzed P-labeled phosphate. From Erdddi et al. (1988a, Rg. 5, p. 587). 

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Figure 3 Phosphoamino acid Analysis of endogenous substrates in prothylakoids, thylakoids and in PS II particles and in lysine-rich histone phosphorylated by these membranes. Figures in the profile spots are cpm X 10". ...

As the protein dissolves completely in performic acid, the sample can be split after step 4, if it is desired to carry out both proteolytic digestion (Section C) and phosphoamino acid analysis (Sections H and I) on the protein. As many fewer counts per minute are required for phosphoamino acid analysis, one-tenth to one-fifth of the sample should be enough for this purpose. 

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. 

Twenty to 100 P cpm in a phosphoprotein or peptide sample is enough to obtain a reproducible phosphoamino acid analysis. Keep in mind that only 20% of the radioactivity will actually be recovered as free phosphoamino acids. 

Van der Geer, P., Luo, K., Sefton, B. M.,and Hunter, T. (1993) Phosphopeptide mapping and phosphoamino acid analysis on cellulose thin-layer plates. In Protein Phosphorylation A Practical Approach (D. G. Hardie, ed.), pp. 31-59. IRL Press, Oxford. 

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