Department of Chemistry, The University of Western Ontario, London, Ontario, Canada.
J Chromatogr A. 2011 Aug 19;1218(33):5705-11. doi: 10.1016/j.chroma.2011.06.054. Epub 2011 Jun 22.
Traditional CE sample stacking is ineffective for samples containing a high concentration of salt and/or buffer. We recently reported the use of a discontinuous buffer system for protein enrichment that was applicable to samples containing millimolar concentrations of salt. In this paper, the technique was investigated for samples containing unwanted buffering ions, including TRIS, MES, and phosphate, which are commonly used in biological sample preparation. Using myoglobin as a model protein, the results demonstrated that background buffering ions can be effectively removed or separated from the enriched protein. The key is to use either the acid or the base of the discontinuous buffers to adjust the pH of the sample, such that the net charge of the unwanted buffering ions is near-zero. The successful isolation and enrichment of myoglobin from up to 100 mM TRIS and 50 mM MES was demonstrated. The enrichment factors remained at approximately 200. Removal of phosphate was more challenging because its net charge was anionic in both the acid and the base of the discontinuous buffers. The enrichment was only achievable up to 30 mM of sodium phosphate, the enrichment factors observed were significantly lower, below 50, and the process was delayed due to the higher ionic strength resulted from phosphate. The migration of phosphate during enrichment was studied using a UV-absorbing analogue, phenyl phosphate. In addition, Simul 5.0 was used to simulate the discontinuous buffers in the absence and presence of TRIS and phosphate. The stimulated TRIS and phosphate concentration profiles were generally in agreement with the experimental results. The simulation also provided a better understanding on the effect of phosphate on the formation of the pH junction.
传统的 CE 样品堆积对于含有高浓度盐和/或缓冲液的样品效果不佳。我们最近报道了一种用于蛋白质富集的不连续缓冲系统，该系统适用于含有毫摩尔浓度盐的样品。在本文中，研究了该技术在含有不需要的缓冲离子的样品中的应用，包括 TRIS、MES 和磷酸盐，这些离子通常用于生物样品制备。使用肌红蛋白作为模型蛋白，结果表明可以有效地去除或分离背景缓冲离子从富集的蛋白质中。关键是使用不连续缓冲液的酸或碱来调节样品的 pH 值，使得不需要的缓冲离子的净电荷接近零。成功地从高达 100mM TRIS 和 50mM MES 中分离和富集肌红蛋白。富集因子保持在大约 200。由于磷酸盐在不连续缓冲液的酸和碱中的净电荷均为阴离子，因此去除磷酸盐更具挑战性。仅能在 30mM 的磷酸钠下实现富集，观察到的富集因子明显较低，低于 50，并且由于磷酸盐导致的离子强度增加，过程会延迟。使用紫外吸收类似物苯膦酸研究了在富集过程中磷酸盐的迁移。此外，还使用 Simul 5.0 模拟了不存在和存在 TRIS 和磷酸盐时的不连续缓冲液。模拟的 TRIS 和磷酸盐浓度分布与实验结果基本一致。模拟还提供了对磷酸盐对 pH 接头形成的影响的更好理解。
