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目的:比较鼻咽癌(nasopharyngeal carcinoma,NPC)组织与正常鼻咽上皮组织磷酸化蛋白质组差异,筛选差异磷酸化蛋白质,为揭示NPC的发病机制提供依据。方法:采用双向凝胶电泳技术(2-DE)分离NPC组织与正常鼻咽上皮组织的总蛋白质,蛋白质转膜后与抗酪氨酸磷酸化抗体进行Western印迹分析,图像分析识别差异磷酸化蛋白质点,电喷雾-四极杆-串联质谱(ESI-Q-TOF MS/MS)鉴定差异的酪氨酸磷酸化蛋白质,采用NetPhos软件预测蛋白质的酪氨酸磷酸化位点,并采用生物信息学方法对差异磷酸化蛋白质的功能和亚细胞定位进行分析。采用Western印迹检测差异蛋白质(phosphatidylethanolamine-binding protein 1)在正常鼻咽黏膜组织和鼻咽癌组织的总蛋白质中的磷酸化水平。结果:建立了NPC组织与正常鼻咽上皮组织的酪氨酸磷酸化蛋白质表达谱,识别了25个差异酪氨酸磷酸化蛋白质,共鉴定了13个差异酪氨酸磷酸化蛋白质,其中7个蛋白质的酪氨酸磷酸化水平在NPC组织中增强,而6个蛋白质的酪氨酸磷酸化水平降低。NetPhos软件预测和生物信息学分析结果显示,13个差异蛋白质均存在酪氨酸磷酸化位点,其功能涉及物质代谢、细胞结构、细胞防御以及信号转导。与正常鼻咽黏膜组织相比较,差异蛋白phosphatidylethanolamine-binding protein 1在鼻咽癌组织中磷酸化表达水平下调。结论:鉴定了13个可能与NPC发病相关的酪氨酸磷酸化蛋白质,为揭示NPC发病机制提供了科学论依据。
OBJECTIVE: To compare the phosphoproteome difference between nasopharyngeal carcinoma (NPC) tissues and normal nasopharyngeal epithelial tissues, and to screen for differentially phosphorylated proteins to provide basis for revealing the pathogenesis of NPC. Methods: The total protein of NPC tissues and normal nasopharyngeal epithelial tissues was separated by 2-DE gel electrophoresis (2-DE). Western blot analysis was performed after transfection of protein and anti-tyrosine phosphorylation antibody. Image analysis identified differential phosphorylated protein Electrophoresis-quadrupole-tandem mass spectrometry (ESI-Q-TOF MS / MS) was used to identify the tyrosine phosphorylated proteins. NetPhos software was used to predict the tyrosine phosphorylation sites of proteins. Bioinformatics Methods The function and subcellular location of differentially phosphorylated proteins were analyzed. Western blotting was used to detect the level of phosphorylation of phosphatidylethanolamine-binding protein 1 in the total protein of normal nasopharyngeal mucosa and nasopharyngeal carcinoma tissues. Results: The expression patterns of tyrosine phosphorylated proteins in NPC tissues and normal nasopharyngeal epithelial tissues were established. Twenty-five differential tyrosine phosphorylated proteins were identified, of which 13 differential tyrosine phosphorylated proteins were identified, of which 7 The level of tyrosine phosphorylation of protein is enhanced in NPC tissues while the level of tyrosine phosphorylation of 6 proteins is decreased. NetPhos software prediction and bioinformatics analysis showed that all 13 differential proteins have tyrosine phosphorylation sites, and their functions are related to metabolism, cell structure, cell defense and signal transduction. Phosphorylation of phosphatidylethanolamine-binding protein 1 was down-regulated in NPC compared with normal nasopharyngeal mucosa. CONCLUSION: Thirteen tyrosine phosphorylated proteins that may be associated with the pathogenesis of NPC were identified, providing a scientific basis for revealing the pathogenesis of NPC.