Method for Protein Phosphorylation Analysis by Use of Antibodies Specific to Phosphorylation Motifs

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The special aim in this study is to develop "phosphorylation motif"-specific monoclonal antibody for analysis of protein phosphorylation. Antibody specific to phosphorylation site modified by protein kinase is an essential tool for the study of protein phosphorylation. Phosphopeptide containing phosphoserine is usually used as epitope for antibody production. However phosphoserine is liable to phosphatase during circulation, resulting in antibody specific to unphosphorylated peptide. We tried to produce phosphopeptide-specific antibodies for the Ser6 and Se9 sites of tumor suppressor protein p53. When we used regular phosphopeptides as epitope to immunize rabbits. Although an antibody against Ser9 phosphorylation site was produced, no production of a Ser6 phosphorylation-specific antibody was observed. To overcome this problem, the stable phosphoserine derivative L-2-amino4-phosphono-4, 4-difluorobutanoic acid (F_2Pab) was synthesized to incorporate into peptides for immunization. Using F_2Pab-containing peptide, we have successfully obtained the Ser6 phosphorylation-specific antibody. This result demonstrates that the method using F_2Pab is effective for phosphorylation-specific antibody production. Second, we have developed the method to produce phosphorylation motif-specific monoclonal antibody. We selected the ATM phosphorylation motif that consists the sequence of Ser(P)-Gln. The monoclonal antibody for the motif only recognized phosphopeptides containing the Ser(P)-Gln sequence and did not react to phosphopeptides with different phosphorylation motifs. During the process of antibody production, we also found that lymphnode cells with short term immunization produced the specific antibody more effectively than spleen cells with regular term immunization.