Controlling the separation of native proteins with temperature in thermal gel transient isotachophoresis
Controlling the separation of native proteins with temperature in thermal gel transient isotachophoresis
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DOI：
10.1007/s00216-022-04331-w
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发表时间：
2022-09-23
影响因子：
4.3
通讯作者：
Linz，Thomas H.
中科院分区：
文献类型：
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作者：
Thanthri，Shakila H. Peli;Linz，Thomas H.
聚丙烯酰胺凝胶电泳（PAGE）是生物化学研究实验室中用于表征蛋白质样品的普遍技术。尽管其流行，PAGE是相对缓慢的，并提供有限的分离分辨率，特别是对天然蛋白质。本报告描述了一种微流控热凝胶瞬时等速电泳（TG-tITP）方法的发展，以快速分离天然蛋白质的高分辨率。热凝胶被用作分离基质，因为它们具有响应于温度而改变粘度的独特能力。将蛋白质添加到热凝胶中并加载到微流体装置中。进行电解质优化以实现稳健的tITP以等速电泳预浓缩蛋白质，然后电泳分离它们。通过时间和距离收集电泳图，以使小型和大型蛋白质能够在单个分析中进行测量。温度的影响进行了评估，发现表现出显着的效果上的分离。然后采用温度梯度随时间改变热凝胶粘度，以最大化蛋白质之间的分离分辨率。本文的结果证明了梯度TG-tITP如何实现天然蛋白质的快速、高性能分离。该分析提供了宽的质量范围（6-464 kDa），分辨率比非变性PAGE高两倍，同时所需的蛋白加载量减少15，000倍，分析时间缩短5倍。图形摘要
Polyacrylamide gel electrophoresis (PAGE) is a ubiquitous technique used in biochemical research laboratories to characterize protein samples. Despite its popularity, PAGE is relatively slow and provides limited separation resolution, especially for native proteins. This report describes the development of a microfluidic thermal gel transient isotachophoresis (TG-tITP) method to rapidly separate native proteins with high resolution. Thermal gels were employed as a separations matrix because of their unique ability to change viscosity in response to temperature. Proteins were added into thermal gel and loaded into a microfluidic device. Electrolyte optimization was conducted to achieve robust tITP to isotachophoretically preconcentrate proteins and then electrophoretically separate them. Electropherograms were collected through both time and distance to enable both small and large proteins to be measured within a single analysis. The effects of temperature were evaluated and found to exhibit a pronounced effect on the separation. Temperature gradients were then employed to alter thermal gel viscosity over time to maximize separation resolution between proteins. The results herein demonstrate how gradient TG-tITP achieves rapid, high-performance separations of native proteins. This analysis provided a wide mass range (6–464 kDa) with two-fold higher resolution than native PAGE while requiring 15,000-fold less protein loading and providing five-fold faster analysis times.Graphical abstract