基于开关型有序介孔材料的蛋白质组富集方法研究

Sample preparation has been playing an important role in the analysis of a complex proteome sample for archiving the global profiling and identification of proteins and peptides qualitatively and quantitatively. Mesoporous materials has demonstrated as the superior enrichment adsorbents in sample enrichment due to the desirable characteristics of high surface area, large pore volume, tunable mesoporous channels with well defined pore-size distribution, controllable wall composition, as well as modifiable surface properties. However, the traditional extraction or enrichment approaches are actually the “open” enrichment processes since the captured targeted molecules exposed to the rising systems, complex matrixes and varying environments, which could cause sample loss, the partial desorption or the possible contamination of the adsorbed analytes when the captured samples undergo the consequent washing or rising solvents and procedures..The aim of this proposal is to develop a novel strategy for the sample extraction/ enrichment of proteome and peptidome from complex biological samples, especially for the extraction/enrichment of protein and/or peptides with low concentrations or abundances, by constructing the pore “open/close” switchable mesoporous materials. In this work, the switchable “open/close” molecule-pairs will be first designed and investigated by screening and synthesizing the “open/close” switchable molecule-pairs; the mesopores switch with different open-pore sizes of the applied mesoporous materials will be achieved by modulating the molecular link-spaces of the “open/close” molecule-pairs and utilizing the covalent linking of these switchable molecule-pairs onto the pores of the mesoporous materials. The photo or light irradiation and/or the temperature modulation to trigger the switch responses of the “open/close” molecule-pairs on the mesoporous materials will be fully investigated by applying the lights with different “open” and “close” wavelengths or changing the temperatures before and after the enrichment or even through the washing, rising, eluting, labeling processes etc. The investigation of the adsorption, desorption, retaining as well as the size selective cut-off on the pore switchable mesoporous materials will be carried out for proteins and peptides with different physical and chemical properties including molecular weight, sizes, charges, hydrophilicity and hydrophobicity etc., to understand the switch and the adsorption/desorption performance of the “open/close” mesoporous materials. We believe the novel extraction/enrichment strategy using the “open/close” switchable mesoporous materials can be developed for the identification of proteome and peptidome with the wider coverage in concentrations and constituents due to the lowered sample loss and matrix interference attributed to the synergistic effects of the size selective and closed-pore protection against the complex matrixes and varying environments.

蛋白质组样品高度复杂，样品制备是提高蛋白质组鉴定覆盖度、检测灵敏度以及目标分子选择性分析鉴定的有效方法。针对传统孔道或表面“开放”式富集方法在蛋白质组样品处理中存在的环境干扰问题。申请人拟发展具孔道“开/关”功能材料的蛋白质组样品富集制备新方法，通过孔道开/闭状态的控制完成复杂生物样品中目标组分的抗干扰捕获。本项目将通过开关分子的合成和修饰，筛选和发展开/闭状态可逆切换的开关分子，掌握开关分子在外界刺激下的开闭状态的切换响应规律；研究不同基质有序孔道介孔结构材料的功能化修饰以及相应开关分子在材料上的偶联，构建孔道开闭可逆切换的“开/关”型介孔富集材料；掌握介孔材料孔道的开启和闭合控制规律，建立不同孔径大小、孔道结构介孔材料上孔道的开闭方法；研究不同物理化学性质蛋白质、多肽等生物分子在“开/关”型介孔富集材料上的吸附、保留、截留和洗脱规律，建立基于孔道开关功能材料的蛋白质组富集新方法。

生物样品极其复杂，高丰度蛋白质的存在严重干扰了低丰度蛋白质和多肽的定性定量分析，发展针对血液、唾液等生物复杂样品的内源性多肽和低丰度修饰肽段的样品制备方法是提高蛋白质和多肽定性定量分析的重要环节。本项目主要基于材料与生物分子相互作用关系，设计、合成和构建具有特异性表界面的纳米孔道材料，研究不同物理化学性质蛋白质、多肽等生物分子在多孔富集材料上的吸附、保留、截留和洗脱规律，建立基于多孔材料的多肽/蛋白质富集新方法。1) 发展基于锆基金属骨架材料的反相-亲水混合模式富集方法，用于多肽组的高覆盖性鉴定分析，两种模式表现出良好的正交性和互补性，可从3 L人唾液样品中鉴定到1255条非冗余肽段，较单一的HILIC或者RP模式覆盖度分别提高38%和60%; 2) 针对磷酸化多肽分析存在选择性低和覆盖度差的问题，设计并合成了双金属中心锆基金属有机骨架材料（DZMOF），自身的锆氧团簇中心和固定化的锆离子中心对单和多磷酸化多肽表现出高金属亲和性能。DZMOF在磷酸化多肽富集中，展现出了高灵敏度、高抗干扰、高选择性和高回收率的特点; 3) 针对多磷酸化多肽分析存在单磷酸化多肽干扰且缺乏高效富集材料的问题，设计并合成了1,6-二磷酸果糖（FDP）分子修饰的DZMOF（DZMOF-FDP），实现了干扰多肽的有效屏蔽和多磷酸化多肽的高选择性富集，多磷酸化多肽选择性达70.1%。4) 针对内源性糖基化多肽的富集效率低以及缺乏兼具亲水基底和亲水表面位点的富集材料等问题，发展了基于双亲水金属有机骨架材料的内源性糖基化多肽富集方法, 从5 μL人血浆中总共高效鉴定到380条内源性糖基化多肽以及180个非冗余糖基化位点。5) 设计并制备了FDP为结构单元的生物相容性金属有机骨架材料（Fru-MOF），其具有超高的水稳定性和生物催化活性。Fru-MOF成功实现了二硫键的抗干扰高效催化，催产素环化效率达94.5%。6）针对糖链富集效率低的问题，发展出基于超声辅助制备无金属基质残留的多孔石墨化纳米碳棒和3D石墨烯材料的血清N-糖链高效富集新方法，从而实现糖链的高选择性富集。