应用原初态生物质谱及毛细管电泳技术解读蛋白质变性及凝集机理

Protein denaturation/aggregation is a phenomenon that is frequently encountered in many fields of chemistry, biology, pharmaceutics, etc. It attracts significant interests because of its pivotal role in human diseases as well as its effect on the economics and safety in both academic and bio-pharmaceutical sectors. Due to the high degree of complexity, heterogeneity and transient nature displayed by the denatured and aggregating protein systems, the task of characterizing them at the molecular level using conventional techniques remains extremely challenging, since their resolution and general applicability are rarely sufficient. In this proposal we will take the full advantage of electrospray ionization mass spectrometry (in terms of high resolution, accuracy, sensitivity, operational flexibility, etc.) and capillary electrophoresis (in terms of the separating power provided by its multiple modes), in combination with the techniques including hydrogen/deuterium exchange, and structural analysis strategies including top-down and middle-down, to develop an arsenal of analytical methods that are competent to capture both covalent and non-covalent interactions. These proposed methods are conceived to characterize the denatured or aggregating protein species, which are induced by heat, pH or oxidation, at multiple levels such as covalent modification, conformational change, and oligomerization state. Furthermore, we will apply these methods and complementary tools available in this field to the in-depth study of denaturation/aggregation of therapeutic proteins to decipher the underlying mechanisms, where the determination of correlation between conformation and aggregation, the driving force of aggregation, and inter-protomer binding interfaces will be highlighted. With these efforts we seek to contribute to both academia and industry by exploring the structural factors that stabilize proteins.

蛋白质的变性/凝集是化学、生物、药学等诸多领域内的常见现象，与科研成本、药物安全性和人类疾病均密切相关。由于蛋白质变性/凝集体系成分复杂且有失稳定，传统分析方法因分辨率和通用性不尽人意而难以在分子层面上对其实现精确表征。本项目拟利用电喷雾质谱在分辨率、准确度、灵敏度、灵活度等方面的优势，结合毛细管电泳技术中多种分离模式的高效分离能力，整合氢/氘交换等辅助技术和top-down、middle-down等结构分析策略，开发一系列能表征蛋白质物种共价及非共价作用的分析方法，用于在共价修饰、构象变化和凝集程度等多个层面上对由热、pH、氧化等应力引起的蛋白质变性/凝集产物进行全面表征；并以这些方法为主体、以业界相关的成熟方法为补充，从蛋白质结构-凝集关联性、凝集驱动力和凝集界面等几个角度，深入理解治疗性蛋白的变性/凝集机理，参与解决学术界和工业界共同关注的蛋白质稳定性问题。

蛋白质的变性/凝集是化学、生物、药学等诸多领域内的常见现象，与科研成本、药物安全性和人类疾病均密切相关。由于蛋白质变性/凝集体系成分复杂且有失稳定，传统分析方法因分辨率和通用性不尽人意而难以在分子层面上对其实现精确表征。本项目针对学术界与生物药物产业共同关注的天然蛋白质及人工重组蛋白质（含蛋白质药物）的变性及凝集现象，以生物质谱为核心工具，辅以毛细管电泳、色谱等前端分离技术，结合非变性质谱、top-down分析策略和氢/氘交换反应原理，通过在仪器设备、分析方法、数据处理等层面的创新，发展相关联的5种分析方法，并将其应用于对处于应激等条件下发生变性及凝集过程的蛋白质体系中，通过实时表征翻译后修饰方式和位点变化、构象变化和结合界面变化，深入解读蛋白质变性及凝集过程中伴随的结构演变、生物物理学性质演变及影响因素。研究成果发表于Anal. Chem.、Analyst等期刊。这些技术可作为表征蛋白质多层级结构、区分共存构象、动态监测蛋白质高级结构演变等研究目标的有力工具，提供独到的结构和动态学信息，并有望在生物物理学界及生物制药产业中获得广泛应用。