Quantitative profiling of the protein coronas that form around nanoparticles
Quantitative profiling of the protein coronas that form around nanoparticles
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DOI：
10.1038/nprot.2014.139
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发表时间：
2014-09-01
期刊：
影响因子：
14.8
通讯作者：
Stauber, Roland H.
中科院分区：
文献类型：
--
作者：
Docter, Dominic;Distler, Ute;Stauber, Roland H.
纳米颗粒在生物技术和生物医学中的应用正在稳步增加。在生物体液中，蛋白质与形成蛋白质冠状结构的纳米颗粒结合，这对纳米颗粒的生物特性产生了至关重要的影响。由于电晕对体外和/或体内纳米颗粒应用的影响，我们开发了一种方法来获得在不同纳米颗粒上形成的时间分辨蛋白质电晕轮廓。在血浆或类似的生物流体中孵育后，或注射到小鼠体内后，第一个分析步骤是通过蔗糖垫沉淀纳米颗粒-蛋白质复合体，从而能够分析早期电晕形成时间点。接下来，用凝胶电泳法将电晕轮廓可视化，并在胰酶消化后使用无标记液体色谱-高分辨质谱仪进行定量分析。与其他方法相比，我们建立的方法允许研究人员获得定性和定量的高分辨率日冕信号。该方法可以很容易地扩展到各种纳米材料的蛋白质冠层的研究(例如，我们将该方法应用于不同的二氧化硅纳米颗粒(SiNPs)和聚苯乙烯纳米颗粒(PSNPs))。根据样本数量的不同，从纳米颗粒-蛋白质复合体回收到数据评估的协议大约需要8-12天的时间才能完成。
Nanoparticle applications in biotechnology and biomedicine are steadily increasing. In biological fluids, proteins bind to nanoparticles that form the protein corona, crucially affecting the nanoparticles' biological identity. As the corona affects in vitro and/or in vivo nanoparticle applications, we developed a method to obtain time-resolved protein corona profiles formed on various nanoparticles. After incubation in plasma or a similar biofluid, or after injection into a mouse, the first analytical step is sedimentation of the nanoparticle-protein complexes through a sucrose cushion, thereby allowing analysis of early corona formation time points. Next, corona profiles are visualized by gel electrophoresis and quantitatively analyzed after tryptic digestion using label-free liquid chromatography-high-resolution mass spectrometry. In contrast to other approaches, our established methodology allows the researcher to obtain qualitative and quantitative high-resolution corona signatures. The protocol can be readily extended to the investigation of protein coronas from various nanomaterials (as an example, we applied this protocol to different silica nanoparticles (SiNPs) and polystyrene nanoparticles (PSNPs)). Depending on the number of samples, the protocol from nanoparticle-protein complex recovery to data evaluation takes similar to 8-12 d to complete.