Shihabi Z K, Friedberg M
Pathology Department, Bowman Gray School of Medicine, Wake Forest University, Winston-Salem, NC 27157, USA.
J Chromatogr A. 1998 May 20;807(1):129-33. doi: 10.1016/s0021-9673(98)00055-7.
Stacking methods are very important in overcoming the poor detection limits in capillary electrophoresis. Human insulin, a polypeptide, was concentrated on the capillary (stacked) based on three different and simple treatment methods to the sample: dilute buffers, high salt content, and acetonitrile (66%) were added to the sample to induce stacking. A dilute buffer in the sample caused a limited stacking, while acetonitrile treatment and high salt content in the sample caused much greater (approximately 20-fold) stacking. High salt concentration in the sample caused stacking presumably by a transient isotachophoretic method. In addition to stacking, the acetonitrile treatment removed the excess proteins in the sample. Insulin did not denature or precipitate in 66% acetonitrile as confirmed by high-performance liquid chromatography (HPLC) and immunoassays. Acetonitrile treatment enabled one-third of the capillary to be loaded with sample thus increasing the detection signal greatly. The insulin peak after acetonitrile treatment and separation by capillary electrophoresis (CE) was confirmed by HPLC and by CE fraction collection followed by immunoassay. Based on acetonitrile treatment, insulin detection in pancreatic tissue homogenates is shown to be feasible.
堆积方法在克服毛细管电泳中较差的检测限方面非常重要。人胰岛素是一种多肽，基于对样品的三种不同且简单的处理方法，即向样品中添加稀释缓冲液、高盐含量和乙腈（66%）以诱导堆积，从而在毛细管上进行浓缩。样品中的稀释缓冲液导致有限的堆积，而乙腈处理和样品中的高盐含量导致大得多（约20倍）的堆积。样品中的高盐浓度可能通过瞬态等速电泳方法引起堆积。除了堆积外，乙腈处理还去除了样品中多余的蛋白质。通过高效液相色谱（HPLC）和免疫测定证实，胰岛素在66%乙腈中不会变性或沉淀。乙腈处理使毛细管的三分之一能够加载样品，从而大大增加了检测信号。通过HPLC以及通过毛细管电泳（CE）分离后进行CE馏分收集并随后进行免疫测定，证实了乙腈处理和分离后的胰岛素峰。基于乙腈处理，在胰腺组织匀浆中检测胰岛素被证明是可行的。
