Sample Stacking Provides Three Orders of Magnitude Sensitivity Enhancement in SDS Capillary Gel Electrophoresis of Adeno-Associated Virus Capsid Proteins
Sample Stacking Provides Three Orders of Magnitude Sensitivity Enhancement in SDS Capillary Gel Electrophoresis of Adeno-Associated Virus Capsid Proteins
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DOI：
10.1021/acs.analchem.6b02933
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发表时间：
2017-03-21
影响因子：
7.4
通讯作者：
Meagher, Michael M.
中科院分区：
文献类型：
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作者：
Zhang, Chao-Xuan;Meagher, Michael M.
基于大小的蛋白质分析，利用只有25纳克的总蛋白质已实现十二烷基硫酸钠毛细管凝胶电泳（SDS CGE）与头柱场放大样品堆积作为在线样品预浓缩技术。该方法已被用作SDS-PAGE的替代品，用于不同血清型和转基因的腺相关病毒（AAV）治疗产品的纯度分析。使用214 nm处的方便UV吸光度检测实现0.2 ng/mL（3.3 pM）衣壳蛋白的检测限，相当于自动进样器小瓶中加载的20 pg蛋白（330阿托摩尔）。对于纯度分析，仅将25 ng总AAV衣壳蛋白（4.3空白飞摩尔病毒颗粒）加载到自动进样器小瓶中。灵敏度与银染SDS-PAGE相当。纯度测定的RSD为0.0- 0.8%，与使用0.1-0.5 mg蛋白质的常规SDS CGE相当。与传统的SDS CGE相比，新方法的灵敏度提高了3个数量级。该方法既具有传统SDS CGE的优点（节省人力、易于自动化、便于定量），又具有银染SDS-PAGE的高灵敏度。样品堆积SDS CGE技术可用于其他类型蛋白质的基于尺寸的分析。当蛋白质量或浓度不足以进行常规SDS CGE或SDS-PAGE测定时，它特别有用。
Size-based protein analysis utilizing only 25 ng of total proteins has been realized by sodium dodecyl sulfate capillary gel electrophoresis (SDS CGE) with head-column field-amplified sample stacking as an online sample preconcentration technique. This method has been used as a replacement of SDS-PAGE for purity analysis of adeno-associated virus, (AAV) therapeutic products of different serotypes and transgenes. A limit of detection of 0.2 ng/mL (3.3 pM) capsid proteins was achieved with convenient UV absorbance detection at 214 nm, equivalent to 20 pg of protein (330 attomole) loaded in the autosampler vial. For purity analysis, only 25 ng of total AAV capsid proteins (4.3 Blank femtomole virus particles) were loaded to the autosampler vial. The sensitivity is comparable to silver-stained SDS-PAGE. The RSD of purity measurement was 0.0-0.8%, comparable to conventional SDS CGE utilizing 0.1-0.5 mg proteins. The new method provided 3 orders of magnitude sensitivity enhancement as compared to conventional SDS CGE. It shares all the advantages of conventional SDS CGE (labor-saving, easy automation, and convenient quantitation) and also the high sensitivity of silver stained SDS-PAGE. The sample stacking SDS CGE technique can be adopted for size-based analysis of other types of proteins. It is especially useful when protein quantity, or concentration is not sufficient for regular SDS CGE or SDS-PAGE assay.