CICS-UBI-Health Sciences Research Centre, University of Beira Interior, 6201-506, Covilhã, Portugal.
Chemistry Department, Faculty of Sciences, University of Beira Interior, 6201-001, Covilhã, Portugal.
Anal Bioanal Chem. 2019 Aug;411(20):5115-5126. doi: 10.1007/s00216-019-01887-y. Epub 2019 May 31.
Despite technological advances, two-dimensional electrophoresis (2DE) of biological fluids, such as vitreous, remains a major challenge. In this study, artificial neural network was applied to optimize the recovery of vitreous proteins and its detection by 2DE analysis through the combination of several solubilizing agents (CHAPS, Genapol, DTT, IPG buffer), temperature, and total voltage. The highest protein recovery (94.9% ± 4.5) was achieved using 4% (w/v) CHAPS, 0.1% (v/v) Genapol, 20 mM DTT, and 2% (v/v) IPG buffer. Two iterations were required to achieve an optimized response (580 spots) using 4% (w/v) CHAPS, 0.2% (v/v) Genapol, 60 mM DTT, and 0.5% (v/v) IPG buffer at 35 kVh and 25 °C, representing a 2.4-fold improvement over the standard initial conditions of the experimental design. The analysis of depleted vitreous using the optimized protocol resulted in an additional 1.3-fold increment in protein detection over the optimal output, with an average of 761 spots detected in vitreous from different vitreoretinopathies. Our results clearly indicate the importance of combining the appropriate amount of solubilizing agents with a suitable control of the temperature and voltage to obtain high-quality gels. The high-throughput of this model provides an effective starting point for the optimization of 2DE protocols. This experimental design can be adapted to other types of matrices. Graphical abstract.
尽管技术有所进步，但生物体液（如玻璃体）的二维电泳（2DE）仍然是一个主要挑战。在这项研究中，通过将几种溶解剂（CHAPS、Genapol、DTT、IPG 缓冲液）、温度和总电压结合使用，人工神经网络被应用于优化玻璃体蛋白的回收及其通过 2DE 分析的检测。使用 4%（w/v）CHAPS、0.1%（v/v）Genapol、20 mM DTT 和 2%（v/v）IPG 缓冲液可实现最高的蛋白质回收率（94.9%±4.5）。使用 4%（w/v）CHAPS、0.2%（v/v）Genapol、60 mM DTT 和 0.5%（v/v）IPG 缓冲液在 35 kVh 和 25°C 下需要进行两次迭代，以实现优化的响应（580 个斑点），与实验设计的标准初始条件相比提高了 2.4 倍。使用优化的方案对耗尽的玻璃体进行分析，与最佳输出相比，蛋白质检测增加了 1.3 倍，在不同的玻璃体视网膜病变的玻璃体中平均检测到 761 个斑点。我们的结果清楚地表明，将适量的溶解剂与适当的温度和电压控制相结合对于获得高质量凝胶至关重要。该模型的高通量为 2DE 方案的优化提供了有效的起点。这种实验设计可以适用于其他类型的基质。
