光谱学与光谱分析, 2014, 34 (12): 3301, 网络出版: 2014-12-08  

荧光标记-高效液相色谱测定多肽(PC2~PC6)的研究

Study on Fluorescence labeling and Determination of Polypeptide(PC2~PC6) by High Performance Liquid Chromatography
作者单位
1 国家海洋局第一海洋研究所海洋生态研究中心, 山东 青岛266061
2 中国海洋大学化学化工学院, 山东 青岛266100
3 中国地质大学(北京)海洋学院, 北京100083
摘要
基于多肽(Polypeptides PC2~PC6)中富有巯基(-SH)官能团, 其与单溴二胺(Monobromobimanes mBBr)能够发生缩合反应, 生成具有荧光信号的多肽衍生物;通过优化色谱分离条件, 建立了高效液相色谱(荧光检测器)测定多肽的方法;试验中利用质谱鉴定了PCs与mBBr缩合反应的比例关系。 结果显示, 通过比较PCs标记前后化合物的质谱图, PCs与mBBr反应比例关系为1∶1, 稳定性好;高效液相色谱-荧光法测定多肽化合物, 五种化合物间分离度较好, 出峰时间集中在16.6~22.0 min间;PC2, PC3, PC4, PC5和PC6线性相关性系数>0.999 1, 方法定量限分别为0.3, 0.05, 0.3, 0.5和0.8 mg·L-1, 回收率范围为83.0 %~102.0%;重线性较好, RSD<2.0%, 该方法实现了多肽类化合物快速、 准确定量分析。
Abstract
This study was based on the thiol groups(-SH) of PC2~PC6, which could be reacted with the Monobromobimane(mBBr), in order to get polypeptide derivatives with fluorescent signal. A new method was developed for measuring the Polypeptides by high performance liquid chromatography with fluorescence detector, then the chromatographic conditions of HPLC was optimized;meawhile the reaction proportion of PCs and mBBr was identified by Trap-MS. The results showed that, the reaction proportion of PCs and mBBr was 1∶1, the polypeptide derivatives had good stability;the five compounds separation was better, and the peak time focused on the 16.6~22.0 min;the linear correlation coefficient of PC2, PC3, PC4, PC5 and PC6 was >0.999 1, and the limits of quantification were 0.3, 0.05, 0.3, 0.5 and 0.8 mg·L-1 respectively, the recovery rate was 83.0%~102.0%;the method was reproducible, RSD<2%, this method for measuring the peptide compounds was rapid and accurate.

李景喜, 高丽洁, 曹为, 郑立, 陈军辉, 徐秀丽, 王小如. 荧光标记-高效液相色谱测定多肽(PC2~PC6)的研究[J]. 光谱学与光谱分析, 2014, 34(12): 3301. LI Jing-xi, GAO Li-jie, CAO Wei, ZHENG Li, CHEN Jun-hui, XU Xiu-li, WANG Xiao-ru. Study on Fluorescence labeling and Determination of Polypeptide(PC2~PC6) by High Performance Liquid Chromatography[J]. Spectroscopy and Spectral Analysis, 2014, 34(12): 3301.

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