Department of Chemistry, The University of Western Ontario, London, Ontario, Canada.
Analyst. 2012 Oct 21;137(20):4766-73. doi: 10.1039/c2an35548e.
Capillary electrophoresis (CE) is not only an effective separation technique, but can also serve as a sample preparation tool for enrichment and purification at sub-microliter sample volumes. Our approach is based on the use of a discontinuous buffer system consisting of an acid and a base (acetate and ammonium). Proteins and/or peptides with isoelectric points between the pH values of these two buffers will become stacked at the neutralization reaction boundary (NRB). To understand the mechanism of the NRB formation and the electrophoretic migration of various ions during the enrichment, we performed experiments using myoglobin and mesityl oxide to reveal the ion migration patterns at the buffer junction, and utilized Simul 5 to computer simulate the process. The simulated results closely resembled the experimental data, and together, they effectively revealed the characteristics of the discontinuous buffers. Importantly, the discovery allowed the manipulation of NRB behaviours by controlling the discontinuous buffer composition. To illustrate this, the removal of urea as an unwanted background molecule from the enriched protein sample was achieved based on the acquired information.
毛细管电泳（CE）不仅是一种有效的分离技术，而且可以作为微升级样品量下的浓缩和纯化的样品制备工具。我们的方法基于使用由酸和碱（乙酸盐和氨）组成的不连续缓冲系统。等电点在这两种缓冲液 pH 值之间的蛋白质和/或肽将在中和反应边界（NRB）处堆积。为了了解 NRB 形成的机制以及浓缩过程中各种离子的电泳迁移，我们使用肌红蛋白和间二甲苯氧化物进行了实验，以揭示缓冲液交界处的离子迁移模式，并使用 Simul 5 进行计算机模拟。模拟结果与实验数据非常吻合，它们共同有效地揭示了不连续缓冲液的特性。重要的是，通过控制不连续缓冲液的组成，可以操纵 NRB 的行为。例如，基于所获得的信息，可以从富集的蛋白质样品中去除脲作为不需要的背景分子。
