新型纳米材料整体柱及固载酶和蛋白质组学分析

A new monolith functionalized with nanomaterials was designed and synthesized as a support for enzyme immobilization, which was then used for protein digestion for application of proteomic analysis. A generic porous monolithic support was first prepared by in-situ polymerization, followed by further functionalization with nanomaterials, which serve as intermediate ligands to immobilize enzyme via the reversible affinity interactions. The desired enzymatic bioreactor was applied for protein digestion. Rapid and high throughput proteomics analysis was achieved by integration of on-line separation and identification with HPLC-MS and MALDI-TOF. Porous polymer monoliths have high chemical and mechanical properties, contain large through-pores and exhibit high permeability to flow. These properties enable mass transport to the active site of the enzyme and substrate by fast convection since all the liquid phase must flow through the pores of the support. The chosen nanomaterials have high specific surface area to volume ratio and good biocompatibility, which is helpful in maintaining the native conformation and high activity of the biocatalyst. The combination of nanomaterials and porous polymer monolith could significantly increase the immobilization amount of enzyme, improve the enzymatic stability, operational life, and enzymatic rate and efficiency. The affinity supramolecular interaction mechanism between nanomaterials and enzyme was investigated using molecular simulation, NMR, and circular dichroism (CD).The use of affinity interaction facilitates regeneration of the activity of the bioreactor simply by removing the denatured enzyme utilizing suitable agents, and subsequently recharging the bioreactor with a fresh enzyme. This will significantly reduce the production cost and provide new methods for proteomics analysis.

研究一种新型的纳米材料整体柱，应用于酶的固定化，进行酶解反应，实现对蛋白质组学的快速高通量分析。首先合成整体柱，通过对整体柱进行功能化修饰，键合纳米材料；再以纳米材料为中间配体，通过纳米材料与酶的亲和超分子识别作用对酶进行固定化，设计合成固载酶生物微反应器，对蛋白质进行酶解反应，通过多维色谱质谱联用和 MALDI-TOF-MS对酶切反应后的肽片断进行快速分离与高通量鉴定。充分结合有机高聚物整体柱良好的化学机械稳定性、通透性以及快速的对流传质性能，和纳米材料的高比表面积、良好的生物相容性，从而显著提高酶的固载量、使用寿命、催化速率和效率。结合分子模拟方法和核磁、圆二色谱等分析测试手段探讨纳米材料与酶的超分子识别作用机理，研究纳米材料对酶的稳定性、使用寿命和催化效率的影响。酶固定化亲和作用力可逆，洗脱失活酶后键合新酶，可以实现酶微反应器的再生利用，大大降低成本，为蛋白质组学分析提供了新方法。

开发新型的固定化酶载体和固定化酶方法，对于实现蛋白质组学的快速高通量分析具有重要的意义。本项目开发了基于Au-S的可逆亲和超分子识别作用，应用于酶的固定化。设计合成了多种类型的可再生纳米材料整体柱固载酶生物微反应器，充分结合有机高聚物整体柱快速的对流传质性能和纳米材料的高比表面积、良好的生物相容性，显著提高了酶的固载量、使用寿命、催化速率和效率。在43 s内即可实现目标蛋白质的快速酶解，结合HPLC和MALDI-TOF-MS成功实现了蛋白质组学分析。通过Au与巯基的可逆亲和作用，实现了固载酶体系的再生利用。同时利用分子动力学模拟，结合核磁、圆二色谱等分析测试手段成功研究了纳米材料与酶的超分子识别作用机理。综上所述，本项目为实现蛋白质组学的快速分析提供了新思路和新方法。项目资助相关研究成果发表SCI收录论文17篇，申请国家发明专利5项。项目培养博士研究生3名，培养硕士研究生3名，参加9次国际国内会议介绍相关成果。项目投入经费25万元，支出22.7162万元，各项目支出基本与预算相符，剩余经费2.2838万元，剩余经费计划用于本项目研究后续支出。