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In recent years, antibodies specific for phosphopeptides have become a major tool for assessing the phosphorylation of specific serine, threonine, and tyrosine sites on proteins (1,2). Typically, a phosphopeptide-specific antibody is used for the qualitative detection of phosphorylation at a site through immunoblotting the protein of interest. Here we describe a relatively simple method by which a phosphopeptidespecific antibody can be used to estimate the stoichiometry of phosphorylation at its corresponding site. By contrast, other methods for the estimation of the stoichiometry of phosphorylation at a site are considerably more demanding and complicated (3). The stoichiometry of phosphorylation at a particular site is defined as Pt*/Pt, where Pt* is the total moles of protein phosphorylated on the site and Pt is the total moles of protein. To determine this ratio, the phosphopeptidespecific antibody is used to immunoprecipitate only the phosphorylated form of the protein. The overall efficiency of immunoprecipitation of the protein is measured by immunoblotting aliquots of the starting solution and the immunoprecipitate with an antibody against a nonphosphorylated epitope in the protein and comparing the signals of the latter with those of the former. This efficiency is P*p/Pt, where P*p is the moles of immunoprecipitated phosphorylated protein. If all the phosphorylated form of the protein were immunoprecipitated, then P*p would equal P*t, and the stoichiometry of phosphorylation would be equal to the overall efficiency of immunoprecipitation. However, usually only a fraction of the phosphorylated form is immunoprecipitated, and, consequently, a correction must be made. To do so, the efficiency of immunoprecipitation of only the phosphorylated protein is measured by immunoblotting aliquots of the starting solution and the immunoprecipitate with the phosphopeptidespecific antibody and comparing the signals of the latter with those of the former. This efficiency is P*p/P*t. Thus, the stoichiometry of phosphorylation (P*t/Pt) is equal to the ratio of the overall efficiency of immunoprecipitation (P*p/Pt) to the efficiency of immunoprecipitation of the phosphorylated form (P*p/P*t). The protein is prepared for the immunoprecipitation with the phosphopeptide-specific antibody by denaturation with SDS and reduction with dithiothreitol. This procedure should expose the phosphopeptide site in the protein for reaction with the antibody. In addition, if the native protein exists as an oligomer, it converts it into the individual subunits, and thus the nonphosphorylated subunits do not coimmunoprecipitate with phosphorylated ones. The immunoprecipitation is performed after addition of sufficient nonionic detergent to incorporate the SDS into micelles so that the antibody is not denatured. A useful modification of the method would be to cap the sulfhydryl groups with N-ethylmaleimide to ensure that no disulfide bonds reform. This was not done in the present study because the protein examined contains a cysteine two residues from a phosphorylation site. As an example of this method, we examined the phosphorylation of the serine kinase Akt1 on two sites, Thr 308 and Ser 473. Phosphorylation of these sites results in activation of this kinase (4). The required reagents were purchased as follows: active and inactive recombinant Akt1 (Upstate, Charlottesville, VA, USA); antibodies against the Akt1 phospho Thr 308 and phospho Ser 473 peptides (Cell Signaling Technology, Beverly, MA, USA); and a monoclonal antibody against the nonphosphorylated PH domain of Akt1 (Upstate). Stock solutions of 500 ng/mL Akt1 were prepared in 2% SDS, 10 mM dithiothreitol, 150 mM NaCl, 50 mM HEPES, pH 7.4, with 1 mg/mL ovalbumin as carrier, and held at 100°C for 5 min. For immunoprecipitations, 200 μL of this solution were mixed with 1.125 mL 1.7% nonaethyleneglycol dodecyl ether (Sigma, St. Louis, MO, USA), 150 mM NaCl, 50 mM HEPES, pH 7.4 (buffer A), and either 1 μg antibody against phospho Thr 308 or 200 ng antibody against phospho Ser 473 were added. After 2 h at 4°C, the immune complexes were collected by mixing for a further 2 h with 20 μL protein ASepharose. The beads were then washed four times with buffer A, and Proteomic Technologies

Stoichiometry of Site-Specific Protein Phosphorylation Estimated with Phosphopeptide-Specific Antibodies