Li Jiabin, Zhou Lianqi, Wang Huanhuan, Yan Hui, Li Nannan, Zhai Rui, Jiao Fenglong, Hao Feiran, Jin Zuyao, Tian Fang, Peng Bo, Zhang Yangjun, Qian Xiaohong
State Key Laboratory of Proteomics, Beijing Proteome Research Center, Beijing Institute of Radiation Medicine, No. 33 Life Science Park Road, Changping District, Beijing 102206, P. R. China.
Analyst. 2015 Feb 21;140(4):1281-90. doi: 10.1039/c4an02092h.
A key step in the workflow of bottom-up proteomics is the proteolysis of proteins into peptides with trypsin. In addition, enzyme-catalytic (18)O labeled peptides as internal standards coupled with multiple reaction monitoring mass spectrometry (MRM MS) for the absolute quantitation of the target proteome is commonly used for its convenient operation and low cost. However, long digestion and labeling times, incomplete digestion and (18)O to (16)O back exchange limit its application, therefore, we developed a rapid and efficient digestion method based on a high ratio of trypsin to protein. In addition, after separation of the digested samples using pipette tips packed with reversed-phase packing materials in house, the trypsin can be separated, collected and reused at least four times. Based on this approach, a novel protein quantification method using (18)O-labeled QconCAT peptides as internal standards combined with MRM MS for the absolute quantitation of a target proteome is established. Experimental results showed that the novel method had high digestion and (18)O labeling efficiencies, and no (18)O to (16)O back-exchange occurred. A linear range covering 2 orders of magnitude and a limit of quantification (LOQ) as low as 5 fmol were achieved with an RSD below 10%. Then, the quantitative method is used for the absolute quantitation of drug metabolizing enzymes in human liver microsomes. The results are in good agreement with the previously reported data, which demonstrates that the novel method can be used for absolute quantitative analyses of target proteomes in complex biological samples.
自下而上蛋白质组学工作流程中的关键步骤是用胰蛋白酶将蛋白质水解成肽段。此外，酶催化的（18）O标记肽作为内标，结合多反应监测质谱（MRM MS）用于目标蛋白质组的绝对定量，因其操作简便、成本低而被广泛应用。然而，长时间的消化和标记时间、不完全消化以及（18）O到（16）O的回交换限制了其应用，因此，我们开发了一种基于高胰蛋白酶与蛋白质比例的快速高效消化方法。此外，在使用内部填充反相填料的移液器吸头分离消化后的样品后，胰蛋白酶可以被分离、收集并至少重复使用四次。基于此方法，建立了一种以（18）O标记的QconCAT肽为内标，结合MRM MS用于目标蛋白质组绝对定量的新型蛋白质定量方法。实验结果表明，该新方法具有较高的消化和（18）O标记效率，且未发生（18）O到（16）O的回交换。实现了2个数量级的线性范围和低至5 fmol的定量限（LOQ），相对标准偏差（RSD）低于10%。然后，将该定量方法用于人肝微粒体中药物代谢酶的绝对定量。结果与先前报道的数据高度一致，这表明该新方法可用于复杂生物样品中目标蛋白质组的绝对定量分析。
