Department of Chemistry, Wayne State University, Detroit, Michigan 48202-3489, United States.
Anal Chem. 2020 May 5;92(9):6741-6747. doi: 10.1021/acs.analchem.0c00876. Epub 2020 Apr 14.
Proteins must maintain proper folding conformations and express the correct post-translational modifications (PTMs) to exhibit appropriate biological activity. However, assessing protein folding and PTMs is difficult because routine polyacrylamide gel electrophoresis (PAGE) methods lack the separation resolution necessary to identify variants of a single protein. Additionally, standard PAGE denatures proteins prior to analysis precluding determinations of folding states or PTMs. To overcome these limitations, a microfluidic thermal gel electrophoresis platform was developed to provide high-sensitivity, high-resolution analyses of native protein variants. A thermally reversible gel was utilized as a separation matrix while in its solid state (30 °C). This thermal gel provided sufficient separation resolution to identify three variants of a fluorescently labeled model protein. To increase detection sensitivity, analyte preconcentration was conducted in parallel with the separation. Continuous analyte enrichment afforded detection limits of 500 fg of protein (250 pM) while simultaneous baseline separation resolution was achieved between variants. The effects of temperature on thermal gel electrophoresis were also characterized. The unique temperature-dependent outcomes illustrated how method performance can be tuned through a thermal dimension. Ultimately, the high detection sensitivity and separation resolution provided by thermal gel electrophoresis enabled rapid screening of native protein variants.
蛋白质必须保持适当的折叠构象，并表达正确的翻译后修饰（PTMs），以表现出适当的生物活性。然而，评估蛋白质折叠和 PTMs 是困难的，因为常规的聚丙烯酰胺凝胶电泳（PAGE）方法缺乏分离分辨率，无法识别单一蛋白质的变体。此外，标准的 PAGE 在分析前使蛋白质变性，从而排除了对折叠状态或 PTMs 的测定。为了克服这些限制，开发了一种微流控热凝胶电泳平台，以提供对天然蛋白质变体的高灵敏度、高分辨率分析。在其固态（30°C）下，使用热可逆凝胶作为分离基质。这种热凝胶提供了足够的分离分辨率，可识别荧光标记模型蛋白的三种变体。为了提高检测灵敏度，在分离的同时进行了分析物的预浓缩。连续的分析物富集提供了 500fg 蛋白质（250pM）的检测限，同时在变体之间实现了同时基线分离分辨率。还对热凝胶电泳中的温度效应进行了表征。独特的温度依赖性结果说明了如何通过热维度来调整方法性能。最终，热凝胶电泳提供的高检测灵敏度和分离分辨率使快速筛选天然蛋白质变体成为可能。
