色谱

色谱 ›› 2013, Vol. 31 ›› Issue (6): 522-530.DOI: 10.3724/SP.J.1123.2013.03057

• 特别策划：定量蛋白质组学专栏 • 上一篇    下一篇

18O同位素标记定量肽段串联体蛋白质结合同位素稀释-多反应监测质谱的蛋白质绝对定量新方法

李楠楠1,2, 周廉淇2, 毛心丽2, 张姣2, 卫军营2, 林虹君2, 李佳斌2, 田芳2, 张养军2, 钱小红2   

  1. 1. 广西医科大学, 广西 南宁 530021;
    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:2013-03-31 修回日期:2013-05-03 出版日期:2013-06-28 发布日期:2013-06-06
  • 通讯作者: 张养军
  • 基金资助:

    国家重大科学计划项目(2012CB910603,2010CB912704);国家重大科学仪器设备开发专项项目(2011YQ030139,2011YQ06008408,2011YQ09000504,2012YQ12004407);国家高技术研究发展计划(2012AA020202);国家自然科学基金重点项目(20735005,21275159,31100591).

A novel method for absolute protein quantification using 18O isotope labeled concatamers of Q peptides combined with isotope dilution-multiple reaction monitoring mass spectrometry

LI Nannan1,2, ZHOU Lianqi2, MAO Xinli2, ZHANG Jiao2, WEI Junying2, LIN Hongjun2, LI Jiabin2, TIAN Fang2, ZHANG Yangjun2, QIAN Xiaohong2   

  1. 1. Guangxi Medical University, Nanning 530021, China;
    2. State Key Laboratory of Proteomics, Beijing Proteome Research Center, Beijing Institute of Radiation Medicine, Beijing 102206, China
  • Received:2013-03-31 Revised:2013-05-03 Online:2013-06-28 Published:2013-06-06
  • Contact: 10.3724/SP.J.1123.2013.03057

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摘要：

建立了定量肽段串联体蛋白质(concatamers of Q peptides, QconCATs)结合18O同位素标记-多反应监测质谱的蛋白质绝对定量新方法。首先对QconCAT重组蛋白质进行了纯度表征,十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)表征结果表明重组蛋白质的纯度在99%以上,相对分子质量约为63.4 kDa。对QconCAT重组蛋白质酶切后的肽段混合物进行质谱分析,并经pFind和pLabel软件处理,验证了目标肽段。还考察了QconCAT重组蛋白质的酶切效率和18O标记效率,并对QconCAT蛋白质结合18O标记-同位素稀释-多反应监测质谱方法进行了评价。实验结果表明,采用该方法对腾冲嗜热厌氧菌(Thermoanaerobacter tengcongensis, TTE)中选定蛋白质的肽段进行绝对含量测定时,相对标准偏差小于20%,准确度较高,说明该方法可用于复杂生物样本中蛋白质的绝对定量。更重要的是所建方法不仅解决了细胞培养氨基酸稳定同位素标记(SILAC)技术的重标试剂价格昂贵的问题,也为定量蛋白质组学提供了一种新的方法。

关键词: 18O标记, 蛋白质组学, 定量肽段串联体蛋白质, 多反应监测质谱, 同位素稀释

Abstract:

A method of concatamers of Q peptides (QconCATs) protein labeled with 18O-multiple reaction monitoring mass spectrometry for absolute quantification of proteins is established. The purity of the QconCAT recombinant protein was characterized by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), and its purity was above 99%. The relative molecular mass was approximately 63.4 kDa. The peptides digested from the QconCAT recombinant protein and the extract of Thermoanaerobacter tengcongensis (TTE) were analyzed by mass spectrometry. The raw data were processed by pFind and pLabel softwares. The results showed that the efficiencies of protein digestion and the 18O labeling efficiency were able to meet the need of the protein quantification. The performance of the method was evaluated. The absolute contents of the selected proteins in TTE were determined with the relative standard deviations of less than 20% and the accuracy is high. The method not only avoid using the expensive reagent of stable isotope labeling with amino acids in cell culture (SILAC), but also provides an alternative way for the accurately absolute quantification of proteins in biological samples for quantitative proteomic research.

Key words: 18O labeling, concatamers of Q peptides (QconCATs), isotope dilution, multiple reaction monitoring mass spectrometry, proteomics

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