同源异型框蛋白Msx1的两个新的磷酸化位点的鉴定

Identification of Two Novel Phosphorylated Sites of Homeoprotein Msx1

同源异型框蛋白Msx1的同系物Msx2中已鉴定出磷酸化修饰位点,并且发挥着重要的生物学功能。但是对Msx1的磷酸化修饰情况还不清楚,为了对Msx1的磷酸化修饰情况进行研究。利用生物信息学的方法对Msx1的磷酸化修饰位点进行预测,进一步利用免疫沉淀和质谱技术对其进行具体实验验证。C2C12细胞中的Msx1蛋白复合物经胰蛋白酶消化后,通过高效液相色谱和质谱技术进行分离和分析。结果发现两个新的磷酸化修饰位点,分别是152位的丝氨酸和160位的丝氨酸,而且两个磷酸化位点在人和小鼠中是高度保守的。进一步,制备针对这两个磷酸化位点的特异性磷酸化抗体。磷酸化位点的鉴定以及特异性抗体的获得为进一步研究Msx1的生物学功能提供了良好的条件。

Some phosphorylated sites in the homolog Msx2 of the homeoprotein Msx1 has been identified,which plays a crucial biological role. However,the phosphorylated sites in Msx1 are not known yet,thus in order to examine the Msx1 phosphorylated status, we applied bioinformatics analysis to predict phosphorylation sites in Msx1,further used immunoprecipitation and mass spectrometry to experimentally verify the phosphorylation sites in Msx1. Tryptic digests of Msx1 protein complexes purified from C2C12 cells were separated and analyzed by nLC-MS-MS. Two novel phosphorylation sites（Ser152 and Ser160）were identified,moreover,these two sites were highly conserved in mouse and human. Furthermore,the specifically-phosphorylated antibody was prepared targeting these two phosphorylation sites.