基于FTICR MS的慢性疾病特异性免疫球蛋白G的结构表征方法研究及其应用

Monoclonal antibody has become one important diagnostic and therapeutic agent, and so far no techniques can deeply characterize its structures. In this study, taken monoclonal antibody as model molecule, its structure in the level of intact molecule, heavy chain, light chain, Fa fragment and Fab fragment will be characterized using an ultrahigh resolution and ultrahigh accuracy Fourier transform ion cyclotron resonance mass spectrometry (FTICR MS). Our goal is to probe the relationship between its structure and efficacy and to establish the platforms of the structural characterization for developing new type drug of immunotherapy and understanding its immunotherapeutic mechanism. Immunoglobulin G is main antibody in serum and the change in its structure is closely related to its bioactivity, disease genesis and progression. Based on our previous data, we will characterize intact IgG, its heavy and light chains, its Fc and Fab from sera of patients with gastric, lung, and breast cancer, their corresponding begin diseases as well as healthy controls using the above technique platform. Finally, the relationship between IgG structure changes, disease genesis and procession will be established, and novel biomarkers for early warning of cancer will be obtained, and also evidences for understanding immune regulation mechanism, developing immunotherapy drugs and probing the prognosis for immunotherapy will be provided.

慢性疾病特异性抗体已成为慢病的诊断及其免疫治疗的主要药物，但如何获得慢病特异性抗体及其准确结构是医药领域面临的挑战。本研究以单克隆抗体为分子模型，利用超高分辨的傅立叶变换离子回旋共振质谱仪分别在完整抗体分子、重链、轻链、Fc区和Fab区水平上表征其氨基酸序列及其糖基化修饰，探究抗体结构与疗效之间的关系。以本课题组在血液中发现的慢病特异性抗体（免疫球蛋白G，IgG）为研究对象，应用该技术平台研究高发的肺癌、胃癌、乳腺癌及其对应良性疾病和健康人血液中IgG结构与不同病理状态之间的关系，建立血液IgG结构变化与疾病发生之间的关系，为恶性疾病的个体化早期预警提供新型诊断标志物，为在分子水平上认识不同慢病发生过程中IgG的多面性和开发个体化的单克隆抗体药物提供理论依据。

应用超高分辨傅立叶变换离子回旋共振质谱技术结合自主研发的完整糖肽富集分离技术，建立了完整蛋白及糖基化修饰表征技术平台；研发的环境友好且价格低廉的完整糖肽分离富集技术可避免唾液酸化糖肽中的唾液酸残基丢失，使获得的蛋白糖基化修饰形式与实际样本中的结构保持一致。获得了基于疾病特异IgG糖基化修饰区分肺癌与肺部良性疾病的个体化标志物；获得了基于疾病特异炎症相关触珠蛋白糖基化修饰区分肺癌与肺部良性疾病的个体化标志物；获得了基于疾病特异IgG糖基化修饰区分胃癌与胃部良性疾病的个体化标志物；获得了基于疾病特异IgG糖基化修饰区分不同病理类型乳腺癌的个体化标志物；发现了汉族人群新型的糖化血红蛋白修饰形式。获得的个体化标志物的诊断精度、特异性和敏感性高出目前临床使用的标志物2倍以上。血液中疾病特异蛋白个体化糖基化修饰标志物为慢病的个体化早期诊断提供了新型标志物，为在分子水平上认识不同慢病中IgG的多态性和表征个体化的单克隆抗体药物提供了新型的技术平台。