Detection of Nonderivatized Peptides in Capillary Electrophoresis Using Quenched Phosphorescence
作者:Jacobus Kuijt、Raymond van Teylingen、Tom Nijbacker、Freek Ariese、Udo A. Th. Brinkman、Cees Gooijer
DOI:10.1021/ac0105282
日期:2001.11.1
A capillary electrophoresis detection technique for (small) peptides is presented, i.e. quenched phosphorescence, a method that is generally applicable and does not require chemical derivatization. For this purpose, a novel phosphorophore, 1-bromo-4-naphthalenesulfonic acid (BrNS), was synthesized. BrNS has sufficient water solubility and provides strong phosphorescence at room temperature over a wide pH range. The detection is based on the dynamic quenching of the BrNS phosphorescence background signal by electron transfer from the amino group of the peptides at pH 9.5−10. For the di- and tripeptides Val-Tyr-Val, Val-Gly-Gly, Ala-Ser, Gly-Asn, Gly-Ala, and Gly-Tyr, detection limits in the range of 5−20 μg/L were obtained. The novel technique is even a good alternative for the (limited) group of peptides containing tyrosine and, thus, exhibiting native fluorescence as well as strong UV absorption: using Gly-Tyr, Val-Tyr-Val, methionine enkephalin, and human angiotensin II as test compounds, quenched phosphorescence detection was found to compare favorably with absorption detection at 190- and 266-nm laser-induced fluorescence detection, as performed with a recently developed, small-size, quadrupled Nd:YAG laser.
介绍了一种用于(小)肽的毛细管电泳检测技术,即淬灭磷光法,这是一种普遍适用且不需要化学衍生化的方法。为此,合成了一种新型磷光体1-溴-4-萘磺酸(BrNS)。BrNS具有足够的水溶性,并在室温下在广泛的pH范围内提供强磷光信号。检测基于在pH 9.5−10时,肽的氨基与BrNS磷光背景信号之间的电子转移动态淬灭。对二肽和三肽Val-Tyr-Val、Val-Gly-Gly、Ala-Ser、Gly-Asn、Gly-Ala和Gly-Tyr的检测限在5−20 μg/L范围内。该新技术甚至是一个很好的替代方案,适用于包含酪氨酸的(有限)肽,因此展现出本征荧光以及强紫外吸收:使用Gly-Tyr、Val-Tyr-Val、甲硫氨酸内啡肽和人血管紧张素II作为测试化合物时,发现淬灭磷光检测与在190 nm和266 nm激光诱导荧光检测下的吸收检测具有良好的比较效果,后者是使用最近开发的小型四倍Nd:YAG激光器进行的。