Corona Isolation Method Matters: Capillary Electrophoresis Mass Spectrometry Based Comparison of Protein Corona Compositions Following On-Particle versus In-Solution or In-Gel Digestion
Corona Isolation Method Matters: Capillary Electrophoresis Mass Spectrometry Based Comparison of Protein Corona Compositions Following On-Particle versus In-Solution or In-Gel Digestion
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DOI：
10.3390/nano9060898
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发表时间：
2019-06-01
期刊：
影响因子：
5.3
通讯作者：
Lindner, Herbert H.
中科院分区：
文献类型：
--
作者：
Faserl, Klaus;Chetwynd, Andrew J.;Lindner, Herbert H.
对纳米材料蛋白质冠在驱动纳米材料吸收进入细胞和生物体并对细胞和生物体产生影响方面的作用的理解增加，以及随后对冠表征的需求，导致了过去十年中用于分离和分析组成蛋白质的方法的蓬勃发展。然而，尽管迄今为止有超过700项日冕研究，但人们对哪种方法能提供最精确和最全面的日冕表征知之甚少。随着冠形成的建模及其与生物影响的相关性的重要性越来越高，现在是时候适当地表征和验证用于确定蛋白质冠的分离方法。目前的工作介绍了毛细管电泳与电喷雾电离质谱（CESI-MS）作为一种新的方法蛋白质冠表征和开发的颗粒上的胰蛋白酶消化方法，比较肽溶解溶液和表征蛋白质的回收率从纳米材料表面。将CESI-MS与用于电晕分析的金标准nano-LC-MS进行比较，并保持高度的再现性，同时将通量提高>3倍。将颗粒上消化与蛋白质冠的溶液内消化和凝胶内消化进行比较。有趣的是，在不同的方法中，发现一系列不同的蛋白质类别或多或少地被回收。当使用表面活性剂溶解肽时，在较低浓度下检测到载脂蛋白，而免疫球蛋白通常对纳米材料具有高亲和力，因此使用颗粒上消化显示出较低的回收率。使用6种纳米材料验证了优化的颗粒上消化，并证明能够回收超过97%的蛋白质冠。这些都是设计电晕研究和模拟电晕形成和影响时需要考虑的重要因素，突出了纳米材料电晕分析方法全面验证的重要性。
Increased understanding of the role of the nanomaterial protein corona in driving nanomaterial uptake into, and impacts on, cells and organisms, and the consequent need for characterization of the corona, has led to a flourishing of methods for isolation and analysis of the constituent proteins over the past decade. However, despite over 700 corona studies to date, very little is understood in terms of which methods provide the most precise and comprehensive characterization of the corona. With the increasing importance of the modeling of corona formation and its correlation with biological impacts, it is timely to properly characterize and validate the isolation approaches used to determine the protein corona. The current work introduces Capillary Electrophoresis with Electro Spray Ionization Mass Spectrometry (CESI-MS) as a novel method for protein corona characterizations and develops an on-particle tryptic digestion method, comparing peptide solubilization solutions and characterizing the recovery of proteins from the nanomaterial surface. The CESI-MS was compared to the gold standard nano-LC-MS for corona analysis and maintained a high degree of reproducibility, while increasing throughput by >3-fold. The on-particle digestion is compared to an in-solution digestion and an in-gel digestion of the protein corona. Interestingly, a range of different protein classes were found to be recovered to greater or lesser extents among the different methods. Apolipoproteins were detected at lower concentrations when a surfactant was used to solubilize peptides, whereas immunoglobulins in general have a high affinity for nanomaterials, and thus show lower recovery using on-particle digestion. The optimized on-particle digestion was validated using 6 nanomaterials and proved capable of recovering in excess of 97% of the protein corona. These are important factors to consider when designing corona studies and modeling corona formation and impacts, highlighting the significance of a comprehensive validation of nanomaterial corona analysis methods.