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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。
A new method for determining the absolute quantity of protein was developed based on QantCats coupled with 18O isotope labeling and multiplex reaction mass spectrometry. The recombinant protein QconCAT was characterized by SDS-PAGE. The purity of the recombinant protein was above 99% and the relative molecular mass was about 63.4 kDa. The peptide fragments after QconCAT recombinant protein digestion were analyzed by mass spectrometry and processed by pFind and pLabel software to verify the target peptide. The QconCAT recombinant protein efficiency and 18O labeling efficiency were also investigated, and the QconCAT protein binding 18O labeling-isotope dilution-multiplex reaction mass spectrometry method was also evaluated. The experimental results showed that the relative standard deviation (RSD) was less than 20% and the accuracy was relatively high when using this method to determine the absolute content of the peptides in the selected protein of Tengchong thermophilic bacteria (TTE), indicating that this method can be used in Absolute Quantification of Proteins in Complex Biological Specimens. More importantly, the proposed method not only solves the problem of expensive reagents for re-labeling of cell-culture amino acid stable isotope labeling (SILAC) technology, but also provides a new method for quantitative proteomics.