Shimura K, Zhi W, Matsumoto H, Kasai K
Faculty of Pharmaceutical Sciences, Teikyo University, Sagamiko, Kanagawa, Japan.
Anal Chem. 2000 Oct 1;72(19):4747-57. doi: 10.1021/ac000387o.
To evaluate the accuracy ofisoelectric point determination by capillary isoelectric focusing, the pI values of nine proteins and a peptide, the pI values of which had been determined by other methods and ranging pI 3.55-9.60, were determined by capillary isoelectric focusing by cofocusing of recently developed peptide pI markers ranging 3.38-10.17, and the consistency of the pI values was examined. Isoelectric focusing was carried out in neutral polymer-coated capillaries, and the pH gradient was mobilized by pressure toward the cathode, to detect samples with absorption at 280 nm at a fixed detection point. Carrier ampholytes from two different suppliers and in different pH ranges were used. The sharp peaks of the highly pure peptide pI markers greatly facilitated the unambiguous identification of the peaks. When a carrier ampholyte ranging over the acidic side was used, the detection of acidic pI samples was anomalously delayed. This could be partly mitigated by reducing the viscosity of the anode solution in comparison with the pH gradient formed in the capillary. Since the detection times vs the pH relationships were not linear in most cases, the use of a linear calibration line over an entire pH gradient would be erroneous. Instead, the pI values of samples were calculated by assuming a linear relation for pH against detection time between two flanking marker peptides. Close agreement between the pI values, determined by capillary isoelectric focusing, and the reference values of the samples was observed within an average difference range of 0.04-0.08 pH unit with a sample consumption of 10-100 ng within 30-60 min. Some carrier ampholytes were preferentially more effective at either the acidic or the basic side of the pH gradient. For confirmation of the completion of focusing, the use of two different focusing times is recommended.
为评估毛细管等电聚焦法测定等电点的准确性，通过与最近开发的等电点范围为3.38 - 10.17的肽类等电点标记物共聚焦，利用毛细管等电聚焦法测定了9种蛋白质和1种肽的等电点值，这些蛋白质和肽的等电点值已通过其他方法测定，范围为3.55 - 9.60，并检验了等电点值的一致性。等电聚焦在中性聚合物涂层毛细管中进行，通过向阴极施加压力使pH梯度移动，在固定检测点检测280 nm处有吸收的样品。使用了来自两个不同供应商、不同pH范围的载体两性电解质。高纯度肽类等电点标记物的尖锐峰极大地促进了峰的明确识别。当使用在酸性侧范围的载体两性电解质时，酸性等电点样品的检测异常延迟。与毛细管中形成的pH梯度相比，通过降低阳极溶液的粘度可部分缓解这种情况。由于在大多数情况下检测时间与pH的关系不是线性的，在整个pH梯度上使用线性校准线会产生错误。相反，通过假设两个侧翼标记肽之间pH与检测时间的线性关系来计算样品的等电点值。在30 - 60分钟内，样品消耗量为10 - 100 ng时，观察到毛细管等电聚焦法测定的等电点值与样品参考值之间的密切一致性，平均差异范围为0.04 - 0.08个pH单位。一些载体两性电解质在pH梯度的酸性或碱性侧优先更有效。为确认聚焦完成，建议使用两个不同的聚焦时间。
