Department of Electrical Engineering and Computer Science, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139, USA.
Anal Chem. 2010 Mar 15;82(6):2317-25. doi: 10.1021/ac9025219.
In this paper, we are evaluating the strategy of sorting peptides/proteins based on the charge to mass without resorting to ampholytes and/or isoelectric focusing, using a single- and two-step free-flow zone electrophoresis. We developed a simple fabrication method to create a salt bridge for free-flow zone electrophoresis in PDMS chips by surface printing a hydrophobic layer on a glass substrate. Since the surface-printed hydrophobic layer prevents plasma bonding between the PDMS chip and the substrate, an electrical junction gap can be created for free-flow zone electrophoresis. With this device, we demonstrated a separation of positive and negative peptides and proteins at a given pH in standard buffer systems and validated the sorting result with LC/MS. Furthermore, we coupled two sorting steps via off-chip titration and isolated peptides within specific pI ranges from sample mixtures, where the pI range was simply set by the pH values of the buffer solutions. This free-flow zone electrophoresis sorting device, with its simplicity of fabrication, and a sorting resolution of 0.5 pH unit, can potentially be a high-throughput sample fractionation tool for targeted proteomics using LC/MS.
在本文中，我们评估了一种基于电荷与质量而非两性电解质和/或等电聚焦来对肽/蛋白质进行分类的策略，使用了单步和两步自由流区电泳。我们开发了一种简单的制造方法，通过在玻璃基底上表面印刷疏油层来为 PDMS 芯片中的自由流区电泳创建盐桥。由于表面印刷的疏油层防止了 PDMS 芯片和基底之间的等离子键合，因此可以为自由流区电泳创建电气连接间隙。使用该装置，我们在标准缓冲系统中在给定 pH 值下展示了正、负肽和蛋白质的分离，并通过 LC/MS 验证了分类结果。此外，我们通过离片滴定将两个分类步骤耦合，并从样品混合物中分离出特定等电点范围内的肽，其中等电点范围仅由缓冲溶液的 pH 值确定。这种自由流区电泳分类装置具有制造简单、分类分辨率为 0.5 pH 单位的特点，有望成为用于 LC/MS 的靶向蛋白质组学的高通量样品分级工具。
