色谱

色谱 ›› 2012, Vol. 30 ›› Issue (03): 239-244.DOI: 10.3724/SP.J.1123.2012.01035

• 研究论文 • 上一篇    下一篇

氨基酸-稳定同位素稀释质谱法用于合成肽段准确含量分析

王雪颖1,2, 秦伟捷2, 钱小红2, 张养军2*   

  1. 1. 沈阳药科大学, 辽宁 沈阳 110015; 2. 蛋白质组学国家重点实验室, 北京蛋白质组研究中心, 军事医学科学院放射与辐射医学研究所, 北京 102206
  • 收稿日期:2012-01-23 修回日期:2012-02-24 出版日期:2012-03-28 发布日期:2012-05-02
  • 通讯作者: 张养军,博士,研究方向为蛋白质组新方法新技术.
  • 基金资助:

    国家重大科学计划项目(Nos. 2012CB910603, 2010CB912704)、国家重大科学仪器设备开发专项项目(Nos. 2011YQ030139, 2011YQ06008408, 2011YQ09000504)和国家自然科学基金项目(Nos. 20735005, 20905077).

Accurate quantification of synthetic peptides by amino acid-stable isotope dilution mass spectrometry

WANG Xueying1,2, QIN Weijie2, QIAN Xiaohong2, ZHANG Yangjun2*   

  1. 1. Shenyang Pharmaceutical University, Shenyang 110015, China; 2. State Key Laboratory of Proteomics, Beijing Proteome Research Center, Beijing Institute of Radiation Medicine, Beijing 102206, China
  • Received:2012-01-23 Revised:2012-02-24 Online:2012-03-28 Published:2012-05-02

PDF

331

被引次数

6

摘要： 建立了氨基酸同位素稀释液相色谱-串联质谱法准确测定合成肽段绝对含量的方法。实验中对合成肽段的纯度进行了表征,色谱纯度表征结果为99%以上,质谱纯度为90%以上。在肽段溶液中加入13C标记的氨基酸后进行酸溶液水解时间的优化,水解后的氨基酸直接经液相色谱分离和质谱检测,结果表明肽段中的被测氨基酸在150 ℃、6 mol/L HCl溶液水解4～6 h就可以达到水解平衡。每个肽段选择两个或两个以上的被测氨基酸,测得随机选择的5种合成肽段的绝对含量为62.07%～88.18%,测定结果的相对标准偏差小于8%,相对误差小于5%,均满足定量要求。除常用的被测氨基酸苯丙氨酸、缬氨酸、异亮氨酸外,还考察了选择赖氨酸和精氨酸作为被测氨基酸的可行性,实验结果表明增加精氨酸为被测氨基酸是可行的,从而进一步增加了方法的普适性。该方法的建立避免了色谱法定量时氨基酸衍生化处理带来的副反应影响及操作繁琐等问题,提高了肽段含量测定的准确度和精密度,为肽段含量的准确测定提供了一种新的方法。

关键词: 氨基酸分析, 合成肽段, 同位素稀释质谱法, 液相色谱

Abstract: An approach for quantification of peptides by hydrolysis followed by reversed-phase liquid chromatography (RPLC)-stable isotope dilution mass spectrometry (MS) is presented. The purities of all the determined peptides were greater than 99% by using RPLC and over 90% detected by MS, indicating that the method was workable. The hydrolysis conditions optimized were 4~6 h with 6 mol/L HCl at 150 ℃. Two or more amino acids were chosen to ensure the accuracy of the determined results. The content range of peptides was determined to be 62.07%~88.18% with the relative standard deviations less than 8% and relative errors less than 5%. Besides Phe, Val and Ile which were commonly used for the analysis of peptide contents, Arg as another amino acid for peptide quantification can be chosen to enhance the popularity of the method. In a word, application of this method for direct determination of peptide content can avoid the side effects in the derivatization of amino acids and the tedious operation in liquid chromatography. This will improve the precision and accuracy of the method and provide an alternative for peptide quantification.

Key words: amino acid analysis, isotope dilution coupled to mass spectrometry, synthetic peptide, liquid chromatography （LC）