蛋白质磷酸化是生物体内非常重要的翻译后修饰作用，在细胞信号转导、基因表达、细胞周期等生理活动中起着关键的调节作用。目前蛋白质磷酸化的检测主要包括放射自显影、抗体免疫反应和质谱检测，但只能鉴定细胞中蛋白质是否磷酸化。目前还不能在原位条件下分析磷酸化后蛋白质不同位点的构象和动态特性变化。申请人前期发展了19F标记非天然氨基酸方法实现原位条件下蛋白质核磁共振信号检测。申请人将进一步在稳态强磁场实验装置的支撑下：（1）应用19F-tfmF定点标记方法实现原位条件下磷酸化蛋白主链和侧链核磁共振化学位移、弛豫分析，（2）质谱和固体核磁相结合，特别是结合磷脂盘方法分析细菌跨膜酪氨酸激酶(ETK)在磷酸化后不同位点的构象变化和动态特性响应。相关方法的发展，将对丝氨酸、苏氨酸等其他蛋白质磷酸化检测和磷酸化后蛋白结构和动态特性变化提供新的研究方法，有助于进一步理解蛋白质磷酸化在细胞活动中的重要作用机制。

Protein phosphorylation is an essential cellular post-translational modification, which is playing critical roles in signal transduction, gene transcription regulation, cell cycles. Currently, protein phosphorylation can be detected using immuno-antibody reaction and mass spectrometry, providing only qualitative information of protein phosphorylation. Previously, the applicant has achieved in situ protein nuclear magnetic resonance signal detection using 19F labeled unnatural amino acids. Then, using the solid state NMR instrument in stable high magnetic field facility, the applicant will: (1) developed the in situ site-specific conformation and dynamic study methods using site-specific labeled 19F-tfmF, (2) analyzing conformational and dynamic variations or responses at different sites of E. coli tyrosine kinase (ETK) after protein phosphorylation, using the combinational methods of mass spectrometry, solid sate NMR and lipodisq techniques. The method developments will be helpful for further serine, thronine phosphorylation detection, consequent conformational and dynamic variations and for molecular mechanism illustration of protein phosphorylation in essential cellular processes.