Institute of Neuroscience and the Collaborative Innovation Center for Brain Science, Chongqing Medical University, Chongqing 400016, China.
Nanoscale. 2018 Jul 9;10(26):12413-12423. doi: 10.1039/c8nr01077c.
For specific applications, gold nanoparticles (GNPs) are commonly functionalized with various biological ligands, including amino-free ligands such as amino acids, peptides, proteins, and nucleic acids. Upon entering a biological fluid, the protein corona that forms around GNPs can conceal the targeting ligands and sterically hinder the functional properties. The protein corona is routinely prepared by standard centrifugation or sucrose cushion centrifugation. However, such methodologies are not applicable to the exclusive analysis of a ligand-binding protein corona. In this study, we first proposed a lock-in strategy based on a combination of rapid crosslinking and stringent washing. Cysteine was used as a model of amino-free ligands and attached to GNPs. After corona formation in the human plasma, GNP cysteine and corona proteins were quickly fixed by 5 s of crosslinking with 7.5% formaldehyde. After stringent washing using SDS buffer with sonication, the cysteine-bound proteins were effectively separated from unbound proteins. Qualitative and quantitative analyses using a mass spectrometry-based proteomics approach indicated that the protein composition of the cysteine-binding corona from the new method was significantly different from the composition of the whole corona from the two conventional methods. Furthermore, network and formaldehyde-linked site analyses of cysteine-binding proteins provided useful information toward a better knowledge of the behavior of protein-ligand and protein-protein interactions. Collectively, our new strategy has the capability to particularly characterize the protein composition of a cysteine-binding corona. The presented methodology in principal provides a generic way to analyze a nanoparticle corona bound to amino-free ligands and has the potential to decipher corona-masked ligand functions.
对于特定的应用，金纳米粒子（GNPs）通常通过各种生物配体进行功能化，包括无氨基配体，如氨基酸、肽、蛋白质和核酸。进入生物流体后，GNPs 周围形成的蛋白质冠可以掩盖靶向配体并阻碍其功能特性。通常通过标准离心或蔗糖垫离心来制备蛋白质冠。然而，这种方法不适用于对配体结合蛋白冠的专门分析。在这项研究中，我们首先提出了一种基于快速交联和严格洗涤相结合的锁定策略。半胱氨酸被用作无氨基配体的模型，并与 GNPs 结合。在人血浆中形成冠后，用 7.5%甲醛交联 5 s 快速固定 GNP 半胱氨酸和冠蛋白。用 SDS 缓冲液超声严格洗涤后，将半胱氨酸结合的蛋白质从未结合的蛋白质中有效分离。基于质谱的蛋白质组学方法的定性和定量分析表明，与两种传统方法的全冠相比，新方法中半胱氨酸结合冠的蛋白质组成明显不同。此外，对半胱氨酸结合蛋白的网络和甲醛连接位点分析提供了有关蛋白质-配体和蛋白质-蛋白质相互作用行为的有用信息。总的来说，我们的新策略能够特别表征半胱氨酸结合冠的蛋白质组成。所提出的方法原则上提供了一种分析与无氨基配体结合的纳米颗粒冠的通用方法，并且有可能揭示被冠掩盖的配体功能。
