基于高密度配体功能化石墨烯的蛋白质高通量分离富集研究

An effective sample preparation method with high selectivity, large capacity and high-throughput ability is one of the key factors for achieving accurate identification and quantitative analysis of protein species in proteomics. The development of new materials and devices for protein separation needs long-term investigations and efforts to achieve the above aims. In the present project, functionalized graphene composites with high density ligands are combined with novel high-throughput extraction device, so that facilitating new approaches for highly selective and high-throughput separation and enrichment of protein species with large capacity. Herein, graphene oxide nanosheets are selected as substrate, several appropriate modifiers, i.e., polyhedral oligomeric silsesquioxane, polypeptide, as well as modified polysaccharide, are employed as multi-substituted ligands, and then three kind of novel functionalized graphene composites with high density ligands are prepared. Furthermore, we try to investigate the adsorption behaviors of specific protein species onto these composites, compare their protein adsorption performances with those of graphene modified by mono-substituted ligands, and thus expound the mechanism of selective adsorption of protein onto the composites in order to improve protein theory of separation and purification. Moreover, after optimization of the parameters during the adsorption and desorption processes, several novel approaches are established that perform highly selective separation and purification of target proteins with large capacity from biological samples of complex matrixes. Based on the 96-blade device used in microextraction field, we design a new device for high-throughput separation of proteins, and employ functionalized graphene composites as testing coatings for evaluation of the high-throughput separation performance of this device in protein analysis. Based on this project, we aim to develop novel functionalized graphene composites and high-throughput separation device to provide sufficient theoretical and technical supports for protein separation and purification.

高选择性、高容量和高通量的样品预处理方法是实现蛋白质组学准确鉴定和精确定量的关键之一。而开发新型分离介质和分离装置是蛋白质分离领域长期探索的科学问题。本项目以高密度配体功能化石墨烯材料和新型高通量分离装置为基础，开发蛋白质高选择性、高容量和高通量分离富集新方法。以氧化石墨烯为基底，选择多面体低聚倍半硅氧烷、聚多肽和改性多糖为三类多取代基修饰配体，制备新型高密度配体功能化石墨烯复合材料。研究蛋白质在复合材料表面的吸附行为，并与单取代配体修饰石墨烯材料比较，阐述提高蛋白质选择性吸附机理，丰富蛋白质分离纯化理论。优化蛋白质吸附洗脱条件，建立复杂生物样品中蛋白质高选择性分离纯化新方法。借鉴96-blade微萃取装置的设计思路，开发蛋白质高通量分离新装置，并以功能化石墨烯材料为涂层，考察蛋白质高通量分离分析方法的各项性能。为蛋白质组学分析提供有效的蛋白质分离分析理论支持和样品前处理技术支持。

针对基于固相分离介质在复杂生物样品中的蛋白质分离富集应用过程中存在选择性差、吸附容量低等问题，本项目制备了多种高密度功能化的石墨烯复合材料，建立了蛋白质高选择和高容量分离富集新方法，并将其应用于复杂生物样品（人全血、细菌裂解液等）中目标蛋白质的高选择性分离纯化。本项目首先以磁性石墨烯为基底，选择聚赖氨酸和羧甲基纤维素作为高密度配体修饰剂，再通过共价键合在复合材料表面接枝金属亲和基团，制备了高密度金属亲和功能化的磁性石墨烯复合材料。以富组氨酸蛋白（血红蛋白）为目标蛋白质，考察了其在复合材料表面的吸附选择性和吸附容量。结果表明与未经高密度配体修饰剂功能化的石墨烯材料相比，聚赖氨酸和羧甲基纤维素功能化的复合材料中，金属亲和基团的接枝密度分别提高了1.2倍和1.7倍，相应的血红蛋白吸附容量分别提高了1.5倍和1.8倍。与此同时，非目标蛋白质的吸附得到有效抑制，显著提高了对富组氨酸蛋白的选择性。两种复合材料在人全血及细菌裂解液中血红蛋白和组氨酸标签重组蛋白的分离纯化结果表明其可用于复杂生物样品中富组氨酸蛋白的高选择性分离纯化，有望在重组蛋白药物高效分离纯化领域得到广泛应用。其次本项目还开发了聚合离子液体和沸石型咪唑酯有机骨架材料功能化的石墨烯复合材料，分别通过提高复合材料表面正电荷密度和调控石墨烯与金属有机骨架材料比例的方式，实现了人全血样品中血清白蛋白和血红蛋白高选择性分离纯化的目标。本研究为实现复杂基体样品中蛋白质高选择和高容量样品预处理提供了新思路。此外，本项目还开发了交联有机-无机杂化材料和阴离子掺杂金属有机骨架材料并将其应用于有机染料的高选择性吸附和去除，可为印染废水处理提供新途径。