糖蛋白标志物分离鉴定新技术新方法研究

Many clinically used cancer biomarkers are glycoproteins, due to the fact that glycosylation is closely related to carcinogenesis. Glycoproteins usually have many isoforms along with complex glycans, bringing great challenges to bioseparation and analysis. Therefore, it is urgent to develop new technologies for the accurate identification of glycoprotein biomarkers. In this project, blood and saliva samples are selected as targets for research. Through developing novel and high resolution two-dimensional difference gel electrophoresis, glycoproteins enriched by lectins will be separated and analyzed. Thus new technology will be established for the discovery of glycoprotein biomarkers. Antibody immunoprecipitation is employed to enrich target glycoproteins. Different isoforms of target glycoprotein with various glycans will be separated and characterized. Combining the developed new technologies, the project will find glycoprotein biomarkers for gastric cancer and lung cancer. The values of this project are as follows. Firstly, novel two-dimensional gel electrophoresis will benefit proteomics studies. Secondly, new technology will overcome the shortcoming of routine method that cannot separate the isoforms of glycoproteins. Thirdly, the newly developed technology of glycan structure characterization could lead to the precise identification of biomarker. Fourthly, newly developed technologies will contribute to the development of cancer molecular diagnostics. Through the study of this project, it is expected to solve the key issues in the separation and identification of glycoprotein biomarkers, which will ultimately improve the specificity and sensitivity of cancer detection. The achievement of this project will provide key technology support to the discovery of novel glycoprotein biomarkers.

临床上的癌症标志物多是糖蛋白，因为癌症与蛋白质糖基化密切相关。由于糖蛋白存在诸多亚型和复杂糖修饰，给分离分析带来巨大挑战，迫切需要发展新技术对其开展精准研究。本项目选择血液和唾液作为研究对象，通过发展新型高分辨双向差异凝胶电泳技术，对凝集素富集的糖蛋白进行分离分析，建立糖蛋白标志物发现新技术；选择目标糖蛋白进行抗体免疫富集，结合双向凝胶电泳建立糖蛋白亚型和糖链结构分离鉴定新技术；将发展的新技术用于发现胃癌和肺癌的糖蛋白标志物。本研究意义在于：第一，新型双向凝胶电泳技术将促进蛋白质组学研究；第二，新技术将弥补常规方法对糖蛋白亚型难以区分的缺点；第三，糖蛋白糖链结构分离鉴定新技术有助于实现标志物的精准定性；第四，新技术的应用将推动癌症分子诊断的发展。通过项目研究，有望解决糖蛋白标志物分离鉴定困难的现状，提高其诊断癌症的特异性和灵敏度，为糖蛋白标志物的发展提供技术支持。

糖蛋白标志物的临床诊断准确性较低，部分原因是由于糖蛋白标志物存在不同亚型和复杂的糖链修饰结构。针对这一关键科学问题，本项目构建了具有自主知识产权的新型高分辨双向凝胶电泳技术，对目标糖蛋白标志物进行了异构体分离及其糖链解析，建立了基于亲和色谱富集、新型双向凝胶电泳分离和液相色谱质谱分析的目标糖蛋白结构解析新方法。采用该方法，本项目定性比较了肺癌组和正常对照组唾液样本中AZGP1的异构体及其糖链结构差异，揭示了肺癌特征的AZGP1糖链组成和糖链修饰。通过定量蛋白质组学技术，系统表征和定量分析了肺癌组和正常对照组唾液蛋白糖基化位点和对应糖蛋白的差异，全面定性和定量分析了肺癌组织和肺癌细胞中糖蛋白的糖基化位点、完整糖肽和糖蛋白组的差异；揭示了肺癌治疗耐药相关的血液糖蛋白标志物；揭示了用于胃癌诊断的唾液糖蛋白标志物；对血液和唾液中的蛋白质组、细胞外囊泡、唾液微生物组、唾液磷酸化蛋白质组、细胞膜蛋白、细胞核酸结合蛋白等进行了分离分析，为肺癌和胃癌发现了有价值的糖蛋白标志物。通过本项目的实施，解决了糖蛋白标志物分离和糖链结构鉴定面临的困境，提高了目标糖蛋白标志物诊断癌症的特异性和灵敏度，为糖蛋白标志物的发展提供了新技术和新策略。项目执行期间，课题负责人共培养博士3名、硕士4名。项目原创性成果主要以论文形式发表，目前已发表标注基金号的SCI论文18篇，其中包括分析化学领域权威期刊Anal. Chem., Anal. Chim. Acta (4篇), Microchim. Acta，生化研究方法领域权威期刊Mol. Cell. Proteomics，J. Proteome Res. (2篇), 化学多学科领域主流期刊Chem. Comm.等。已经发表并标注基金号的中文核心期刊学术论文2篇，申请中国发明专利3项，获得授权专利5项。各项指标均超额完成任务。