纳米粒子“蛋白质环冠”的蛋白质组学新方法研究

Nanomaterials possess the distinct properties as compared with the corresponding bulk form of the same materials due to the particle size at the nanometer scale, which are revolutionizing many areas of biotechnology in life science. Unfortunately, the understanding of biological effects of nanomaterials is relatively weak because of the absence of the interaction of the nanoscale objects with living systems, despite the remarkable rapid development of nanotechnology. In a real biological system with fluids containing proteins, proteins would tend to associate with nanoparticles via various interactions between nanoparticles and proteins. The adsorption of proteins on the surface of nanoparticles, leading to the formation of so called "protein corona" that largely de?nes the biological identity of the particle, would lead to the real in vivo biological responses of nanoparticles. Thus, the knowledge of rates, af?nities, and stoichiometries of protein association with, and dissociation from, nanoparticles is a crucial issue to understand the nature of the particle surface "seen" by the biological functional machinery of cells. Here, we are planning to develop new methods and technologies including the controllable and selective elution and enzymatic digestion to identify the presenting protein corona on the surface of nanomaterials in different biological model systems including cellular incubation media, serum, plasma etc. Since the attractive porous nanoparticles with broad potentials in drug and gene delivery systems and diagnosis systems, we will develop new methods such as the selective enzymatic digestion approaches to discriminate the real protein corona on porous nanomaterials from the interferences of dissociated proteins from the nanopores of a nanomaterial. Moreover, to identify the "seeable" proteins on the outer layer of protein corona of nanomaterials, we are planning to establish new methods to qualitatively and quantitatively determine the out layer proteins via the isotopic labeling and/or the protein cross linking. We believe that the development of methods on the identification of protein corona on nanomaterial would greatly help the deep understanding of the association and dissociation of proteins on the surface of nanomaterial as well as the knowledge of the real biological response of a biological system toward nanomaterials.

纳米材料表面蛋白质复合物- - "蛋白质环冠"的定性定量分析对于纳米材料/蛋白质复合物的形成机制、纳米材料的生物行为、纳米材料的生物相容性和纳米材料的表面改性等方面的研究具有重要意义。本项目拟开展针对纳米材料表面"蛋白质环冠"的选择性洗脱、酶解、化学标记、共价交联等方面的研究工作，建立起纳米颗粒上"蛋白质环冠"的定性定量分析新方法新技术。主要研究纳米材料表面"蛋白质环冠"的多步骤串联洗脱和酶解规律，发展多孔纳米材料表面"蛋白质环冠"的固定化酶反应器的选择性酶解技术，建立基于蛋白质交联反应、化学标记反应的"蛋白质环冠"的分析鉴定方法，为系统地分析鉴定纳米材料上蛋白质复合物的形成及其生物学效应研究提供新方法支持。

纳米材料的生物表面/界面行为是生物纳米材料生物安全应用的关键。项目研究目标是针对纳米材料表面的蛋白质复合物的定性定量分析需求，建立起高效、高选择性的纳米材料/蛋白质复合物的分析表征新技术和新方法。.项目主要完成了：纳米材料表面蛋白质复合物的动态演变研究；纳米材料表面蛋白质复合物的固定化酶高效选择性酶解技术研究；纳米材料表面吸附蛋白受水分子影响的分子微观机理研究；基于交联质谱技术的纳米材料表面蛋白复合物的原位表征研究；纳米材料表界面顽固蛋白复合物的研究；纳米材料表面多肽复合物的研究等方面的研究工作：..建立纳米粒子表面蛋白质复合物或蛋白质环冠的定性、定量蛋白质组学分析方法，开展了不同粒径的磁性纳米粒子表面蛋白质复合物的研究，发现粒径对于纳米粒子表面蛋白复合物的种类和丰度的影响。..建立纳米材料表面蛋白质复合物的高效选择性酶解方法，用于介孔材料表面形成的蛋白质环冠的分析，实现了介孔纳米粒子表面蛋白质环冠的快速高效分析，同时去除了孔道内部吸附的蛋白质分子的影响。..利用化学交联剂处理纳米材料表面形成的蛋白质复合物，建立了一种纳米材料表面蛋白复合物的交联质谱原位表征方法，提供纳米材料表面蛋白复合物中的构像和相互作用信息。..建立了生物-纳米界面蛋白质复合物表征方法，发现界面蛋白质复合物能够抵挡强烈洗脱，必须在电场辅助的条件下才能从二氧化硅纳米材料表面脱离。..项目从蛋白和多肽角度考察了纳米材料表面的生物复合物。发现纳米材料生物复合物中不仅有蛋白分子也有内源性肽，是蛋白质-多肽复合物。..基于分子动力学模拟，研究了一种由β蛋白和石墨烯堆叠组装形成的生物纳米界面，发现水分子控制的蛋白石墨烯的组装过程。..研究了蛋白质环冠对于纳米粒子细胞毒性的影响，发现蛋白质环冠可改变材料的细胞毒性。.主要研究成果为：主要以通讯作者形式发表SCI研究论文16篇，申请和授权发明专利共23件，培养博士研究生4名。